Anca G Radu

Sakina Torch

Florence Fauvelle

Karin Pernet-Gallay

Renaud Blervaque

Anthony Lucas

Véronique Delmas

Uwe Schlattner

Laurence Lafanechère

Pierre Hainaut

Nicolas Tricaud

Véronique Pingault

Nadège Bondurand

Nabeel Bardeesy

Lionel Larue

Chantal Thibert
email: chantal.thibert@univ-grenoble-alpes.fr

Marc Billaud
email: marc.billaud@lyon.unicancer.fr

Marie Mével

Mylène Pezet

Laura Attardi

LKB1 controls cell fate of the neural crest lineage through pyruvate-alanine transamination

Keywords: neural crest cells, LKB1/AMPK, glial lineage, pyruvate-alanine transamination neural crest cells, glial differentiation, LKB1/AMPK, oxidative DNA damages, p53 signaling, lysosomal homeostasis

Energy metabolism represents all chemical reactions generating adenosine triphosphate, a molecule providing the energy required for most metabolic and cell processes that are essential to sustain life. Main pathways contributing to energy metabolism are glycolysis, citric acid cycle and oxidative phosphorylation. Recent "omics" explorations allowing collective characterization and quantification of chemical reactions, emphasized that metabolic pathways control cell fate and tissue homeostasis. In particular, mapping of energy metabolic signalings that are activated in early stem cells has begun to shed light on the respective contribution of glycolysis versus oxidative phosphorylation in the maintenance of pluripotency and cell fate commitment. Moreover, energy metabolism controls the balance between proliferation and quiescence by regulating metabolic enzymes called transaminases and consequently amino acid levels.

Amino acids are critical build up units required for proteinogenesis. Besides, amino acids act as metabolic regulators of several enzymes and/or metabolic fluxes either locally in the cell they were biosynthesized or through paracrine action. Amino acids also regulate signaling pathways such as the mammalian target of rapamycin mTOR which coordinates nutrient availability to cell growth through the regulation of most anabolic and catabolic processes. Interestingly, mTOR activation specifically takes place at the lysosomal surface and amino acids are known for years to regulate lysosomal proteolysis. Such regulations could be critical to limit premature aging and promote individuals healthspan and longevity. However, the role of metabolic remodeling and in particular amino acids contribution in cell fate during development remains poorly understood. Therefore, my PhD project aimed at characterizing the underlying mechanisms that coordinately regulate energy metabolism and cell fate.

In the lab where I did my PhD, one model generated to study this coordinated regulation was based on neural crest stem cells.

Neural Crest Cells (NCC) are multipotent cells delaminating from the neural tube and migrating along stereotypical routes to colonize embryonic territories. In their target organs, NCC give rise to a broad range of derivatives including neurons and glial cells of the peripheral nerves and enteric nervous system, most of the head skeleton and melanocytes. Disruption of neural crest formation is causative of human diseases called neurocristopathies or ribosomopathies, which combine various symptoms derived from neural crest dysfunction such as skin pigmentation defects, craniofacial malformations, enteric defects and exceptionally peripheral neuropathies. NCC fate and stemness maintenance are controlled by transcriptional PREFACE 4 | P a g e and epigenetic inputs driven by numerous transcription factors and chromatin remodelers.

However, the metabolic processes underlying NCC development remain poorly understood.

Uncovering connections between metabolic responses and cell signaling pathways has recently encounter an impressive increase. One important signaling pathway regulating energy metabolism is controlled by the tumor suppressor Liver Kinase B1 (LKB1) mainly through its downstream substrate 5' Adenosine MonoPhosphate (AMP)-activated protein kinase (AMPK) and subsequent inhibition of mTOR. The tumor suppressor p53 signaling pathway is also intimately connected to energy metabolism especially upon cellular stress. Furthermore, p53 dosage has been shown to be crucial for neural crest cells development.

During my PhD, I demonstrated that LKB1 is essential for the formation of several NCCderived structures in vertebrates such as the face, pigmented cells, the enteric nervous system and peripheral nerves. I have uncovered that metabolic regulations exerted by LKB1 during NCC fate act through the regulation of amino acid levels and subsequent mTOR limitation. In particular, we identified that LKB1 controls pyruvate-alanine cycling during glial differentiation.

Interestingly, this metabolic regulation is also conserved in Sertoli cells that share similar metabolic functions with glial cells. Moreover, we identified signaling crosstalk between LKB1 and p53 pathways. These signaling could converge especially through lysosome homeostasis thereby potentially impacting cellular aging. Altogether, our findings suggest that deregulations of LKB1 signaling could be involved in a broader range of human pathologies than suspected so far.

As energy metabolism has been the guideline of my PhD project, the bibliographic chapters of the introduction of this manuscript open with a short and general presentation of how the energy metabolic network impacts cell functions during development and diseases and how it is regulated, with a focus on transaminases and amino acids. Next, I briefly describe neural crest cell development and human disorders associated with defects of neural crest formation. The third chapter concerns some knowledge about signaling pathways of the two metabolic regulators LKB1 and p53, as well as their roles during development. The last chapter is about the importance of LKB1/AMPK and mTOR signaling in connection with lysosomal homeostasis, p53 hyperactivation, aberrant lysosome formation and premature aging.

Then, for the results chapter, a brief paragraph addresses the main questions asked during my PhD followed by three chapters describing my experimental contributions. These results sections include three articles (one published, one submitted and one in preparation) and three additional projects in progress.

A general discussion expands our results in a broader context and proposes some perspectives to my PhD work. Figure 5: Metabolite shuttles between cells 14 | P a g e A. Glial cells regulate neuronal mitochondrial respiration and ATP production via lactate shuttle. Glial cells are also involved in recycling of synaptic glutamate through glutamate-glutamine cycling (Magistertti and Allaman, 2015). B. Sertoli cells as glial cells are more glycolytic cells and regulate germ cell mitochondrial respiration and ATP production via lactate or alanine shuttles through MCT (Rato et al., 2012). C. Paneth cells control Intestinal stem cell function through lactate shuttle which increases ssmitochondrial respiration, ROS production and p38 activity in intestinal stem cells (CBC) (Adapted from Rodriguez-Colman et al., 2017). D. Tumor microenvironment regulates tumor cell energy and lipid production through amino acids and most notably alanine shuttle (Adapted from Kamphorst and Gottlieb, 2016). GLUT, glucose transporter; LDH, lactate dehydrogenase; MCT, monocarboxylate transporters; GS, glutamine synthetase; GLS, glutaminase; EAAT, excitatory amino acid transporters; ALAT, alanine aminotransferase. p-p38, phospho-p38, ROS, reactive oxygen species.

Table 2: Representative examples of amino acids as signaling molecules for cell proliferation, differentiation and survival * glucose transporters responsible for glucose uptake into cells (Kawauchi et al.,

ENERGY METABOLISM

Definition of energy metabolism and historical context

Intermediary metabolism regroups all the life-sustaining chemical transformations that take place within cells and tissues. These chemical reactions are usually divided in two categories: catabolic reactions that are required to recycle molecules and refurnish the pool of energy and anabolic reactions necessary for biosynthesis of molecules and which consume energy. Over a century ago, Theodor Schwann, a German physiologist who defined the cell as the basic unit of animal structures and discovered Schwann cells in the peripheral nervous system, also coined the term metabolism which comes from the Greek metabole (to change) [START_REF] Schwann | Microscopical researches into the accordance in the structure and growth of animals and plants[END_REF].

Until the mid-20th century, many research groups in University science and medical departments were working on enzymes and metabolic pathways. But this research field was overtaken by the revolutions in genomics and cell biology as well as the idea that metabolism was more like a housekeeping process. Recently, there was a strong renewed interest in energy metabolism based (i) on our understanding that cell needs and functions are intimately associated to their metabolic activities and (ii) on the development of new technologies to measure metabolic processes in vivo in particular with new tools allowing the integration of data at the "omics" scale. This recrudescence of research on metabolism has enabled the recent awareness that energy metabolism is finely tuned during development and deregulations of these processes are responsible of multiple diseases [START_REF] Metallo | Understanding metabolic regulation and its influence on cell physiology[END_REF].

Energy metabolism is the process of generating energy (adenosine triphosphate: ATP) from nutrients. It comprises a set of interconnected pathways that function in the presence or absence of oxygen. Anaerobic metabolism, also called glycolysis, converts one glucose into 2 pyruvates and produces 2 ATP (Figure 1). The reverse process is called gluconeogenesis.

Pyruvate can be transformed into lactate and possibly exported or transported to the mitochondria where it is converted into Acetyl CoA by the citric acid cycle (TCA, also called Krebs cycle) while producing 2 ATP per glucose (Figure 1). Finally, aerobic metabolism, the mitochondrial oxidative phosphorylation (OXPHOS), also produces ATP and this is the most efficient system. In fine, combining glycolysis, TCA and OXPHOS produces up to 38 ATP molecules from one glucose molecule (Figure 1).

Figure 2: Amino acids

There are twenty-one amino acids grouped according to their side chains. In yellow are amino acids with non polar uncharged side chains, in blue are amino acids with polar uncharged side chains, in green are amino acids with electrically positive charged side chains and in purple with electrically negative charged side chains. There are eleven non-essential amino acids (synthesized by the body), encircled in red. The others are essential amino acids (obtained from nutrition), except for selenocysteine which is a rare, non-standard amino acid (modified from http://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/24-proteins/amino-acids.html).

Energy metabolic reprograming

Energy metabolism of the cells is very plastic and it is adjusted multiple times and by different manners during normal development. For example, during the first two days of mouse embryonic development, embryos can do more oxidative phosphorylation than glycolysis.

When the blastocyst is formed, the oxygen consumption increases even more. But after blastocyst formation, cells undergo a metabolic switch leading to a large increase in their glycolytic capacities [START_REF] Leese | Metabolism of the preimplantation embryo: 40 years on[END_REF][START_REF] Leese | History of oocyte and embryo metabolism[END_REF]. Therefore, embryonic stem cells (ESC), which are pluripotent cells derived from the inner cell mass of blastocyst, are producing energy to maintain their rapid proliferation and self-renewal mostly by glycolysis [START_REF] Varum | Energy Metabolism in Human Pluripotent Stem Cells and Their Differentiated Counterparts[END_REF].

The metabolic balance between oxidative phosphorylation and glycolysis is therefore critical for the self-renewal status of the cells [START_REF] Chandel | Metabolic regulation of stem cell function in tissue homeostasis and organismal ageing[END_REF] but also for their differentiation as blocking glycolysis induced myogenic differentiation whereas inhibiting oxidative phosphorylation impaired muscle stem cells differentiation [START_REF] Bracha | Carbon metabolismmediated myogenic differentiation[END_REF][START_REF] Chung | Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells[END_REF]. Thus, mapping the metabolic pathways that are activated in early stem cells has begun to shed light on the respective contribution of glycolysis versus oxidative phosphorylation in the maintenance of pluripotency and cell fate commitment (Ochocki and Simon, 2013).

Deregulations of energy metabolism are associated to numerous human pathologies such as developmental diseases, metabolic disorders (e. g. diabetes) and cancers. In particular, Otto Warburg suggested in 1926 that energy metabolism is a key regulator of tumor formation [START_REF] Koppenol | Otto Warburg's contributions to current concepts of cancer metabolism[END_REF]. He showed that cancer cells switched from oxidative phosphorylation to glycolysis, even in presence of oxygen [START_REF] Warburg | On the Origin of Cancer Cells[END_REF]. Since, energy metabolic reprograming has been suggested as a key hallmark of cancer progression [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF][START_REF] Ward | Metabolic reprogramming: a cancer hallmark even warburg did not anticipate[END_REF]. It has been shown in many different situations that cancer cells adapted their energy metabolism to the tumor microenvironment in order to fulfill the bioenergetic and biosynthetic needs for rapid cell proliferation [START_REF] Biswas | Metabolic Reprogramming of Immune Cells in Cancer Progression[END_REF].

Therefore, there are striking similarities in metabolic reprograming contributing to development or tumorigenesis. Abundant work described how metabolic adjustments modulate the supply of precursor metabolites for building blocks of the cells such as nucleotides, lipids and amino acids depending on cell needs. Likewise, external stimuli and/or signaling pathways involved in these metabolic adaptations are more and more documented.

In particular, the mTOR signaling pathway, composed of mTORC1 and mTORC2 complexes, is a major regulator of cell growth as it governs most anabolic and catabolic processes depending on nutrients availability from the environment (see chapter III.A.1.2.1) [START_REF] Laplante | mTOR signaling in growth control and disease[END_REF][START_REF] Sabatini | Twenty-five years of mTOR: Uncovering the link from nutrients to growth[END_REF]. Interestingly, intermediary metabolites also modify metabolic fluxes through regulation of metabolic pathways. This is the case for amino acids, the basic structural units of proteins, now known to control non-proteinogenic processes (mTOR activity B. Amino acids degradation. Glucogenic amino acids are the amino acids whose degradation form metabolic intermediates give rise to glucose via gluconeogenesis. Amino acids which are forming ketone bodies such as acetoacetate and are not involved in gluconeogenesis are named ketogenic. All amino acids are glucogenic, except leucine and lysine which are only ketogenic.

being one such example) involved in various cell fate such as self-renewal, differentiation and growth [START_REF] Ito | Metabolic requirements for the maintenance of self-renewing stem cells[END_REF][START_REF] Meijer | Amino acids as regulators and components of nonproteinogenic pathways[END_REF][START_REF] Ryall | Metabolic Reprogramming of Stem Cell Epigenetics[END_REF].

Amino acids acting as metabolic regulators

There are 21 amino acids mainly classified depending on their electric charge or the presence of a hydrophobic chain. Apart from selenocysteine, 9 amino acids are obtained from nutrition and represent essential amino acids whereas the 11 others are synthetized by the body and are therefore called non-essential amino acids (Figure 2). Many enzymes are required for amino acid biogenesis and catabolism. Several non-essential amino acids such as serine, alanine, aspartate and glutamate are synthesized from metabolites that derive from glycolysis or mitochondrial citric acid cycle (Figure 3A). Others are generated by direct or indirect biochemical reactions from another amino acid. Among enzymes required for amino acid biosynthesis, transaminases, also known as aminotransferases, are the enzymes catalyzing a transamination reaction between an amino acid and an a-keto acid and a very recently growing literature supports their essential contribution to cell behavior (Table 1).

The first paragraph below describes the capacity of amino acids to monitor intrinsic energy metabolism. The second one is focused on amino acids as paracrine factors to regulate metabolism of neighboring cells, or even of distant organs via their transport through the bloodstream. Finally, in the last paragraph I present the role of amino acids in development and tumorigenesis. The following paragraphs briefly illustrate these 3 roles for amino acids based on specific and not exhaustive examples, with a special focus on transaminases.

Amino acids regulate intrinsic metabolism

As previously mentioned, amino acids are not only essential for protein synthesis but also for metabolic regulations either by modulating levels of metabolites and thus controlling metabolic fluxes or through their direct interaction with proteins such as metabolic enzymes or signaling proteins thereby modifying their activities.

Amino acids catabolism is essential to produce metabolites involved in several metabolic processes such as gluconeogenesis, TCA cycle as well as folate and methionine cycles (Figure 3B). Amino acids also regulate these processes indirectly by controlling different cytosolic/mitochondrial shuttles like the malate/aspartate one, which is important for mitochondrial NADH formation and therefore for mitochondrial respiration and ATP production [START_REF] Lanoue | Evidence for electrogenic aspartate transport in rat liver mitochondria[END_REF][START_REF] Lu | Role of the Malate-Aspartate Shuttle on the Metabolic Response to Myocardial Ischemia[END_REF][START_REF] Meijer | The metabolic significance of anion transport in mitochondria[END_REF][START_REF] Stein | The dynamic regulation of NAD metabolism in mitochondria[END_REF].

Furthermore, amino acids dependent shuttles control other mechanisms such as lactate oxidation and pyruvate transport [START_REF] Kane | Lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work[END_REF][START_REF] Stein | The dynamic regulation of NAD metabolism in mitochondria[END_REF] (Figure 4). Moreover, in some cases amino acids act as compensatory mechanism. For example, alanine is essential for hepatic glucose production when the pyruvate transporter from cytosol to mitochondria is Aminotransferases or transaminases are enzymes able to transfer nitrogenous groups between an amino acid and an α-keto acid. This list of aminotransferases is not exhaustive, there are 101 enzymes presented on ExPASy database. Data obtained from GeneCards Human Gene database.

deficient. Indeed, in this situation alanine is preferentially generated from cytosolic pyruvate via Alanine aminotransferase1 (ALAT1) and translocates into mitochondria. There, ALAT2, the mitochondrial transaminase, retransforms alanine into pyruvate which then supplies the TCA cycle [START_REF] Mccommis | Loss of Mitochondrial Pyruvate Carrier 2 in Liver Leads to Defects in Gluconeogenesis and Compensation via Pyruvate-Alanine Cycling[END_REF].

Another metabolic regulation exerted by amino acids is through the control of metabolic enzymes such as Pyruvate Kinases. These kinases catalyze the last reaction of glycolysis and therefore control pyruvate levels. It has been shown that alanine and ATP are allosteric inhibitors of two isoforms of the pyruvate kinases (PK) family (PKM2 and PKL) whereas they are not affecting PKM1 isoform [START_REF] Ibsen | Inhibition of chicken pyruvate kinases by amino acids[END_REF][START_REF] Llorente | Regulation of Liver Pyruvate Kinase and the Phosphoenolpyruvate Crossroads[END_REF][START_REF] Van Veelen | Alanine inhibition of pyruvate kinase in gliomas and meningiomas. A diagnostic tool in surgery for gliomas?[END_REF]. Inhibition of pyruvate kinases is thereby essential to control the balance between glycolysis and the reverse metabolic pathway that is gluconeogenesis.

Amino acids also directly regulate mTOR pathway which controls cell metabolism such as amino acid biosynthesis, glucose homeostasis and lipid metabolism [START_REF] Kim | Regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis[END_REF][START_REF] Um | Absence of S6K1 protects against age-and diet-induced obesity while enhancing insulin sensitivity[END_REF]. Recently it has been shown that mTOR signaling is activated at the lysosomal surface in a dependent manner of several amino acid concentrations such as leucine, glutamine and arginine (see also Chapter IV.2).

Altogether these data underline the role of amino acids as metabolic regulators by controlling metabolic fluxes, metabolic enzyme activities and the signaling mTOR pathway. Lactate (LAC) is transported to mitochondria where it is oxidized to pyruvate by lactate dehydrogenase (LDH). Amino acids such as glutamate (Glu) and aspartate (Asp) regulate NAD + regeneration via different cycles such as malate (Mal)asparate cycles. MDH, malate dehydrogenease; AAT asparate-aminotransferase; PDH, pyruvate dehydrogenase; αKG, alpha-ketoglutarate; OAA, oxaloacetate [START_REF] Kane | Lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work[END_REF].

Paracrine and systemic activities of amino acids

Besides their capacity to regulate intrinsic metabolism, amino acids also act through a paracrine activity as signal molecules in cell-cell communication to regulate tissue homeostasis and organ functions. Furthermore, metabolites and in particular amino acids are secreted and enter the bloodstream thus having also systemic activities.

One example to illustrate this metabolic communication between cells is the glutamateglutamine cycle that takes place in the nervous system between neurons and their adjacent glial cells. Glial cell metabolism is important for neuronal survival as glial cells uptake the glutamate secreted by neurons to convert it into glutamine to sustain neurotransmission. This mechanism requires ATP because glutamate transport into glial cells as well as its transformation into glutamine are ATP dependent processes (Figure 5 A). Therefore, to produce energy, glial cells uptake glucose and synthesize ATP but also lactate which is secreted to fuel neuronal mitochondria for TCA cycle. Hence, neurons are doing oxidative phosphorylation whereas glial cells are glycolytic. Lactate regulates also the expression of genes involved in synaptic plasticity [START_REF] Magistretti | A cellular perspective on brain energy metabolism and functional imaging[END_REF]. In Drosophila, glial cells not only secrete lactate to fuel neuronal mitochondria but also alanine [START_REF] Trevisiol | Brain Energy Metabolism: Conserved Functions of Glycolytic Glial Cells[END_REF][START_REF] Volkenhoff | Glial Glycolysis Is Essential for Neuronal Survival in Drosophila[END_REF].

Similarly to glial cells, Sertoli cells are also glycolytic cells which produce alanine and lactate from pyruvate and export them to germ cells by specific monocarboxylate transporters (MCTs) (Figure 5 B). This supply has an antiapoptotic effect on germ cells and is essential for their maturation [START_REF] Oliveira | Sertoli Cell Metabolism and Spermatogenesis[END_REF][START_REF] Rato | Metabolic regulation is important for spermatogenesis[END_REF].

Another example of metabolic communication with epithelial cells is the dialogue between Paneth cells and intestinal epithelial stem cells. Paneth cells are glycolytic cells that provide lactate to intestinal stem cells to sustain their enhanced mitochondrial oxidative phosphorylation (OXPHOS). Increased OXPHOS promotes mitochondrial reactive oxygen spaces (ROS) production which activates p38 MAP kinase, a regulator of intestinal stem cells function (Figure 2B) [START_REF] Rodríguez-Colman | Interplay between metabolic identities in the intestinal crypt supports stem cell function[END_REF].

Furthermore, paracrine regulation of energy metabolism through amino acids is also observed in tumor cells. Indeed, cancer cell metabolism is regulated by the tumor microenvironment, notably by the stromal cells (Figure 5 B). It has been shown recently that stroma-associated pancreatic stellate cells increase the OXPHOS of pancreatic ductal adenocarcinoma through the secretion of non-essential amino acids such as alanine [START_REF] Sousa | Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion[END_REF].

Moreover, amino acids have systemic activities. It has been suggested more than 45 years ago that alanine and lactate are synthesized by different tissues and released in plasma.

For example, alanine and lactate are released by muscle cells into the blood circulation and subsequently enter into the liver where they sustain gluconeogenesis and promote urea cycle (Figure 6A) [START_REF] Felig | The glucose-alanine cycle[END_REF][START_REF] Felig | Alanine: key role in gluconeogenesis[END_REF]. Consoli et al. suggested that muscle is the major source of plasma alanine and lactate in the fasting state in humans [START_REF] Consoli | Contribution of liver and skeletal muscle to alanine and lactate metabolism in humans[END_REF].

Recently, scientists emphasized that lactate is much more abundant than glucose in circulating blood. Systemic lactate is a primary source of carbon for TCA cycle in all tissues except brain and muscle in fasted or fed mice as well as in tumors (Figure 6B) [START_REF] Faubert | Lactate Metabolism in Human Lung Tumors[END_REF][START_REF] Hensley | Metabolic heterogeneity in human lung tumors[END_REF][START_REF] Hui | Glucose feeds the TCA cycle via circulating lactate[END_REF].

Collectively, these data demonstrate that the behavior of a cell and its energy metabolism depends in part on amino acids levels either intrinsically or locally available from the microenvironment. These regulations play an important role during both development and tumorigenesis.

Amino acids as regulators of cell fate

During the past years, it has been shown that metabolic pathways control cell fate and self-renewal partly through epigenetic reprograming [START_REF] Ryall | Metabolic Reprogramming of Stem Cell Epigenetics[END_REF]. In particular, amino acid metabolism (synthesis and catabolism, Figure 3 A and B) regulates metabolic pathways and thereby modulates epigenetic changes necessary for stem cell properties (Table 2) [START_REF] Kilberg | Influence of Amino Acid Metabolism on Embryonic Stem Cell Function and Differentiation[END_REF][START_REF] Shyh-Chang | Stem cell metabolism in tissue development and aging[END_REF].

For example, threonine and glycine catabolism are essential for self-renewal and differentiation of embryonic stem cells by regulating methionine cycle. Methionine is transformed into S-Adenosyl Methionine (SAM), which is a methyl donor for numerous reactions [START_REF] Locasale | Serine, glycine and one-carbon units: cancer metabolism in full circle[END_REF]. Threonine and glycine degradation are essential for SAM activation and trimethylation of lysine 4 on histone 3 (H3K4). H3K4 trimethylation is associated with open chromatin and has been shown to be crucial for maintaining the pluripotency and proliferation of embryonic stem cells (Shyh- [START_REF] Shyh-Chang | Stem cell metabolism in tissue development and aging[END_REF][START_REF] Wang | Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition[END_REF][START_REF] Wang | Dependence of Mouse Embryonic Stem Cells on Threonine Catabolism[END_REF].

Amino acid metabolism is not only a regulator of prenatal stem cells development but also of their postnatal development, including adult stem cell commitment (Table 2). One example is glutamine catabolism which is crucial for hematopoietic stem cell specification. The differentiation of hematopoietic stem cell into erythroid rather than myeloid lineage needs glutamine utilization for nucleotide biosynthesis. Thus, glutamine synthetase which transforms glutamate into glutamine is highly expressed during erythropoiesis (Oburoglu et al., 2014).

So far, it is hard to point out a specific mechanism because amino acids involved in cell commitment seems to be stem cell type specific.

Interestingly, proliferating cells generate aspartate and alanine via transaminases, by using glutamine-derived nitrogen whereas in quiescent cells, transaminase expression and glutamine consumption are decreased leading to reduced levels of aspartate and alanine production (Figure 7 and Table 2). A. Glucose-alanine cycle (Cahill cycle) and glucose -lactate cycle (Cori cycle) between liver and muscle are important for hepatic gluconeogenesis and urea cycle (Berg JM et al., 2002). NH2; amino functional group. B. Circulating lactate as major TCA substrate in tissues and tumors (Adapted from Sheng Hui et al., 2017).

Glutamate metabolism is different in proliferated cells compared to quiescent cells. Proliferative cells are characterized by increased transaminase expression, increased NEAA synthesis and low GLUD expression.

In opposition, GLUD is induced in quiescent cells and transaminases and NEAA expression are decreased. Glu, glutamate; GLUD, glutamate dehydrogenase; NEAA, non-essential amino acids; αKG, αKetoglutarate. (Coloff et al., 2016). These differences partly result of mTOR activity which increases transaminases expression and decreases glutamate deshydrogenase expression, favoring non-essential amino acids production (Coloff et al., 2016).

As cancer cells are highly proliferative, it is possible to limit their growth by regulating amino acid levels [START_REF] Lorincz | Response of malignancy to phenylalanine restriction. A preliminary report on a new concept of managing malignant disease[END_REF][START_REF] Lorincz | Tumor response to phenylalanine-tyrosinelimited diets[END_REF]. Coloff et al. showed that most highly proliferative tumors depend on glutamine anaplerosis and generate non-essential amino acids such as alanine, serine and aspartate via transaminases (Coloff et al., 2016).

Several other studies also suggest that alanine is accumulating in cancer cells, its concentration being directly proportional with tumor malignancy (Table 2) [START_REF] Cornel | Characterization of human prostate cancer, benign prostatic hyperplasia and normal prostate by in vitro 1H and 31P magnetic resonance spectroscopy[END_REF][START_REF] Tessem | Evaluation of Lactate and Alanine as Metabolic Biomarkers of Prostate Cancer Using 1H HR-MAS Spectroscopy of Biopsy Tissues[END_REF][START_REF] Ziegler | High glycolytic activity in rat glioma demonstrated in vivo by correlation peak 1H magnetic resonance imaging[END_REF].

Strikingly, alanine is also accumulated in human Hereditary Sensory and Autonomic

Neuropathy type 1 (HSAN1) and metabolic syndromes associated with peripheral neuropathies [START_REF] Hla | Sphingolipid signaling in metabolic disorders[END_REF]. In these pathologies alanine is responsible for deoxysphinganine formation, a neurotoxic sphingolipid (see Table 2 and Discussion Figure 2).

Thus, amino acids are acting as signaling components to regulate stem cell renewal and differentiation as well as to increase proliferation and tumor growth. They also control neuronal survival through deoxysphingolipids formation. Therefore, knowing how the levels of non-essential amino acids are regulated and how they act in cells represent a first step for prevention of developmental diseases or cancer therapy. Moreover it could allow novel approaches to reverse pathological phenotypes associated with deoxysphingolipids.

Neural crest cells formation

Neural crest cells (NCC) are embryonic multipotent cells specific to vertebrates, discovered by Wilhelm His in chick embryos in 1868 [START_REF] Hall | The neural crest and neural crest cells: discovery and significance for theories of embryonic organization[END_REF]. NCC originate during early embryogenesis from the neural plate border (Figure 8 A). NCC delaminate through an epithelial to mesenchymal transition from the dorsal part of the neural tube (Figure 8 C) [START_REF] Duband | Epithelium-mesenchyme transition during neural crest development[END_REF]. Then NCC migrate throughout the embryo to reach their target organs where they differentiate into multiple cell types (Figure 8 D, E) [START_REF] Acloque | Epithelialmesenchymal transitions: the importance of changing cell state in development and disease[END_REF][START_REF] Simões-Costa | Establishing neural crest identity: a gene regulatory recipe[END_REF]. There are five NCC subpopulations depending on their localization along the anterior-posterior axis [START_REF] Burns | Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia[END_REF]Douarin and Kalcheim, 1999;[START_REF] Dupin | Neural crest progenitors and stem cells: From early development to adulthood[END_REF][START_REF] Lumb | Transcriptome profiling reveals expression signatures of cranial neural crest cells arising from different axial levels[END_REF]:

-Cranial NCC arise anterior to somite 5 and give rise to melanocytes and head structures such as bone, cartilage and tendons.

-Cardiac NCC arise from somites 1 to 3 and give rise to melanocytes, vascular smooth muscles lining of the outflow tract and contribute to the aortic-pulmonary septum.

-Vagal NCC arise between somites 1-7 and form melanocytes as well as neurons and glia of the enteric nervous system.

-Trunk NCC arise posterior to somite 4 and differentiate into neurons of the sensory and sympathetic systems as well as into Schwann cells and melanocytes.

-Sacral NCC arise posterior to somite 28 and generate melanocytes, enteric neurons and glial cells.

Thus, not all NCC give rise to all derivatives since they have fate restriction depending on their anterior-posterior position along the neural tube. Melanocytes represent an exception since they are generated by all NCC subpopulations [START_REF] Cichorek | Skin melanocytes: biology and development[END_REF][START_REF] Gilbert | The Neural Crest[END_REF]. An other exception concerns Schwann Cell Precursors (SCP) generated by cranial, vagal and truncal NCC subpopulations (see paragraph 2.3). During embryogenesis SCP differentiate into immature Schwann cells which constitute the first step of the Schwann cell lineage. Very recently it has been shown that SCP persist through adulthood and conserve the multipotency and high plasticity of NCC, being therefore able to generate several cell types during both embryogenesis and adulthood [START_REF] Gargiulo | Psychiatry and Neuroscience Update[END_REF] (see paragraph 2.3).

In the past 150 years, studies of NCC were mostly based on various animal models (mouse, chick, quail-chick chimeras, frog, zebrafish) in order to understand NCC origin and destination, induction and specification, polarized migration, stem cell properties and functions upon differentiation into specific cell types [START_REF] Theveneau | Neural crest migration: interplay between chemorepellents, chemoattractants, contact inhibition, epithelial-mesenchymal transition, and collective cell migration[END_REF][START_REF] Barriga | Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo[END_REF][START_REF] Dupin | The neural crest, A multifaceted structure of the vertebrates[END_REF]Milet and Monsoro-Burq, 2012).

Based on quail-chick chimeras, Le Douarin lab identified NCC contribution to head, heart and other organs (Le [START_REF] Le Douarin | Neural crest cell plasticity and its limits[END_REF].

Single cell tracing of migratory NCC in mice has been obtained using the so-called Confetti model. This inducible multicolor Cre-reporter allowed to characterize the multipotency of trunk NCC in vivo [START_REF] Baggiolini | Premigratory and migratory neural crest cells are multipotent in vivo[END_REF][START_REF] Zurkirchen | Quo vadis: tracing the fate of neural crest cells[END_REF] and to analyze facial vertebrate shaping [START_REF] Kaucka | Analysis of neural crest-derived clones reveals novel aspects of facial development[END_REF]. It has been demonstrated that while original NCC are totipotent, when they divide, they become tripotent and then bipotent progenitors before being committed to a specific cell type (Le [START_REF] Le Douarin | Neural crest cell plasticity and its limits[END_REF]. Besides, Bronner lab and others have shown that progressive development of neural crest is regulated by a neural crest gene regulatory network. This network is composed of a multisteps cascade of genes activation [START_REF] Simões-Costa | Establishing neural crest identity: a gene regulatory recipe[END_REF]. The first step corresponds to inductive signals such as Wnt, Bone Morphogenetic Protein (BMP), Notch and Fibroblast Growth Factor (FGF), which induce the formation of neural plate border. Then a second set of genes are activated such as Paired box (Pax), Zinc finger (Zic) and Homeobox (Msx) genes. These neural plate border genes in a third step up-regulate expression of neural crest specifier genes such as Sox10, Forkhead box gene D3 (FoxD3) and Snail family transcriptional repressor 2 (Snai2).

Finally, these specifier genes activate in a fourth step downstream pathways essential for NCC migration (partly through cadherins expression and thereby their epithelial-to-mesenchymal transition) [START_REF] Dady | Cadherin interplay during neural crest segregation from the nonneural ectoderm and neural tube in the early chick embryo[END_REF][START_REF] Duband | Resolving time and space constraints during neural crest formation and delamination[END_REF] as well as for NCC differentiation and selfrenewal capacity (Simões-Costa and Bronner, 2015) (Figure 9). NCC migration is also regulated by cell-cell interaction and guidance cues [START_REF] Kulesa | Cranial Neural Crest Migration: New Rules for an Old Road[END_REF][START_REF] Scarpa | Collective cell migration in development[END_REF]. For example, NCC and placode cells, which are embryonic ectodermal cells that contribute to sensory organs, undergo a chase-and-run mechanism essential for their collective cell migration [START_REF] Theveneau | Chaseand-run between adjacent cell populations promotes directional collective migration[END_REF].

Although the cascade of molecular and cellular events that specify neural crest territories, orchestrate NCC delamination and guide their polarized migration throughout the embryo has been extensively explored, the contribution of metabolic regulations to NCC formation has received less attention to date. To our knowledge the only study that analyzed the metabolism in NCC was done by Monsoro-Burq lab. They revealed that during NCC specification pfkfb4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) which encodes a glycolytic enzyme, is upregulated and controls NCC migration, in Xenopus laevis, through the regulation of both AKT signaling and glycolysis [START_REF] Figueiredo | PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation[END_REF]. Moreover, inactivation of PFKFB4 in Xenopus laevis leads to craniofacial malformations [START_REF] Figueiredo | PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation[END_REF].

In the next paragraphs, I will further detail the origin, fate and functions of cranial, trunk and vagal NCC, the three NCC subpopulations that I studied during my PhD.

NCC subpopulations and their derivatives

Cranial neural crest cells

Cranial NCC originate from different parts of the brain and differentiate into many cell types like melanocytes, cells of cranial nerve sensory ganglia, facial bones and cartilage, inner ear, cranial vault, parafollicular and Schwann cells [START_REF] Trainor | Specification of neural crest cell formation and migration in mouse embryos[END_REF]. Cranial NCC represent the only NCC subpopulation that can differentiate into skeletal tissues. Before 19th century, it has been thought that all the skeletal structures originate exclusively from mesoderm [START_REF] Knight | Cranial Neural Crest and Development of the Head Skeleton[END_REF]. Later, tracing of cranial NCC showed that these cells directly participate to the formation of head skeleton as they give rise to premaxilla, maxilla, mandible, to a part of temporal bones, the incus and the malleus of middle ear, hyoid bone, thymus as well as cartilages such as thyroid and epiglottis [START_REF] Dupin | The neural crest, A multifaceted structure of the vertebrates[END_REF][START_REF] Lumsden | Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo[END_REF][START_REF] Santagati | Cranial neural crest and the building of the vertebrate head[END_REF][START_REF] Schilling | Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo[END_REF].

Many other experimental studies on mouse, zebrafish and amphibian confirmed the involvement of cranial NCC in craniofacial morphogenesis and showed how cranial NCC positional identity is regulated by homeodomain transcription factors (Hox and Otx2) and environmental signals such as BMP (bone morphogenetic proteins from the TGFβ family), FGF (Fibroblast growth factor) and Shh (Sonic hedgehog) [START_REF] Bhatt | Signals and Switches in Mammalian Neural Crest Cell Differentiation[END_REF][START_REF] Borchers | Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification[END_REF][START_REF] Creuzet | Patterning the neural crest derivatives during development of the vertebrate head: insights from avian studies[END_REF][START_REF] Donoghue | The origin and evolution of the neural crest[END_REF][START_REF] Minoux | Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development[END_REF][START_REF] Nissen | Zebrafish foxi one modulates cellular responses to Fgf signaling required for the integrity of ear and jaw patterning[END_REF].

Moreover, Mayor lab has recently underlined the importance of cell micro-environment for cranial NCC migration. They showed that the stiffening of the head mesoderm during early developmental stages of Xenopus laevis induces cranial NCC epithelial-to-mesenchymal transition as well as their collective migration [START_REF] Barriga | Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo[END_REF].

In conclusion, cranial NCC are multipotent stem cells essential for head formation.

These cells are key for human face diversity regarding the shape, size and individual features.

Thus, defects in cranial NCC development, migration and specification are responsible for many craniofacial malformations that I will present in more details in Chapter II.3.

Trunk, vagal and sacral neural crest cells

The trunk, vagal and sacral NCC generate sensory neurons and glial cells that are forming the peripheral nervous system (PNS) and melanocytes. PNS comprises nerves and ganglia localized outside the brain and spinal cord. Peripheral nerves are primary structures of PNS and contain axons of sensory and motor neurons, glial cells as well as blood vessels and connective tissues necessary for nutrients supply (Figure 11). The enteric nervous system (ENS) also belongs to PNS. ENS, one of the main autonomic nervous system (also named the second brain), innervates the gastrointestinal tract and governs digestive motricity [START_REF] Furness | The first brain: Species comparisons and evolutionary implications for the enteric and central nervous systems[END_REF].

Trunk NCC & peripheral nerves development

The specification of trunk NCC depends on the pathways they are following. Trunk NCC which migrate dorsolaterally differentiate into melanocytes. Trunk NCC that migrate ventrolaterally form sensory neurons of dorsal root ganglia. NCC that migrate more ventrally generate several cell types such as Schwann cells and sympathetic ganglia [START_REF] Gilbert | The Neural Crest[END_REF].

Although trunk NCC are a major source of Schwann cells, other NCC such as cranial NCC also give rise to Schwann cells [START_REF] Bronner | Development and evolution of the neural crest: an overview[END_REF][START_REF] Dupin | Neural crest progenitors and stem cells: From early development to adulthood[END_REF].

Trunk NCC participate also to ENS formation, however their participation is very minor in comparison with vagal and sacral NCC [START_REF] Heanue | Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies[END_REF][START_REF] Lake | Enteric nervous system development: migration, differentiation, and disease[END_REF].

Trunk NCC fate is controlled by the path they are following during their migration but also depends on the time course of embryo development. As my PhD subject focused on Schwann cells, I will further detail the time course of Schwann cells-derived neural crest development.

The glial cell specification process is defined by two transitional steps. Firstly, NCC differentiate into Schwann Cell Precursors (SCP) and secondly SCP differentiate into immature Schwann cells. This second step is associated with an up-regulation of glial markers: Glial Fibrillary Acidic Protein (GFAP) and S100 calcium-binding protein β (S100β) and a downregulation of N-cadherin (Figure 10) [START_REF] Balakrishnan | Temporal Analysis of Gene Expression in the Murine Schwann Cell Lineage and the Acutely Injured Postnatal Nerve[END_REF][START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF][START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF]. In mice, SCP are specified at embryonic day 12 or 13 (E12/E13) and they differentiate at E15/16 into immature Schwann cells which persist until birth (Figure 10). Thus, Schwann cell maturation and myelination occurs after birth.

When the embryonic nerves are formed, they are exclusively composed of axon bundles and SCP derived from NCC. They do not have extracellular matrix, connective tissue and blood vessels [START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF]. These neural crest-derived cells migrate along nerves through embryo tissues to reach distant target organs. Once NCC reach the developing nerves, they are specified into SCP (Adameyko et al., 2009). After E14, when SCP are already formed and the axons present in peripheral nerves are developing the first synaptic connections with their target tissues, the nerve enriched its composition with more cell types: endothelial cells and pericytes that surround the blood vessels and endoneurial fibroblasts [START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF][START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF][START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF]. At this period of time there is also development of perineurial cells which are forming a protective sheath around the nerves. Inside the nerves, axons are grouped together and are surrounded by SCP (Figure 10). The interaction between these two cell types is essential for reciprocal regulation:

-first, SCP regulate neuronal survival. They are not required for motor and sensory neurons development or for guidance of axons at their target tissues. Instead they are necessary

Adulthood

for neuronal maintenance because after axons reach their target organs, SCP loss triggers a massive neurodegeneration [START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF].

-second, neurons are also essential for SCP survival. The axon-derived factor neuregulin1 (NRG1) regulates SCP survival and induces a direct migration of these cells [START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF][START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF].

-finally, the direct contact between axons and Schwann cells is important for the differentiation of SCP into immature Schwann cells [START_REF] Adameyko | Glial versus melanocyte cell fate choice: Schwann cell precursors as a cellular origin of melanocytes[END_REF]Adameyko et al., 2009;[START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF][START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF]. NRG1 is able and sufficient to promote Schwann cells maturation, beside its capacity to regulate SCP survival and direct migration.

As mentioned, SCP are multipotent precursors. Indeed, they differentiate into immature Schwann cells as well as into fibroblasts (see paragraph 2.3, Figure 14). These two cell fates depend on the interaction of SCP with axons. SCP which are not in contact with axons are giving rise to endoneurial fibroblasts. However, when SCP interact with axons, they integrate axonal signals (such as NRG1 and Notch which inhibit their capacity to generate endoneurial fibroblasts) and differentiate into immature Schwann cells [START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF][START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF]. Thus, fibroblasts, in addition to Schwann cells and neurons, are neural crest derivatives, whereas other cells found in nerves (perineurial cells, pericytes and endothelial cells) are not.

Before birth, immature Schwann cells change their morphology and develop long cytoplasmic extensions that promote axon defasciculation. Axons with a large diameter (>1µm)

undergo radial sorting and are surrounded by one immature Schwann cell destined to become a myelinating Schwann cell (Figure 10). In this case, the axon-Schwann cell interaction is one to one. Axons with smaller diameter are intended to remain un-myelinated and are in contact with an immature Schwann cell committed to become non-myelinating Schwann cell. In this case, the axon-Schwann cell relationship is several neurons for one non-myelinating Schwann cell. Thus, the radial sorting is an embryonic process that happens before Schwann cell maturation into myelinating and non-myelinating cells and continues until 10 days after birth (P10) (Figure 10) [START_REF] Balakrishnan | Temporal Analysis of Gene Expression in the Murine Schwann Cell Lineage and the Acutely Injured Postnatal Nerve[END_REF][START_REF] Feltri | How Schwann Cells Sort Axons: New Concepts[END_REF][START_REF] Woodhoo | Development of the Schwann cell lineage: from the neural crest to the myelinated nerve[END_REF].

Moreover, the temporal molecular expression profile of each cell type in the Schwann cell lineage was characterized in different studies although these observations remain controversial. Therefore, in Figure 10 I mentioned only proteins that were common to several studies and/or that I used during my PhD for stem and glial lineage characterization (Figure 10) [START_REF] Balakrishnan | Temporal Analysis of Gene Expression in the Murine Schwann Cell Lineage and the Acutely Injured Postnatal Nerve[END_REF][START_REF] Blake | Pax genes: regulators of lineage specification and progenitor cell maintenance[END_REF][START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF]. The postnatal peripheral nerve organization is represented in Figure 11.

Thus, trunk NCC are differentiating into several cell types, including Schwann cells.

Over the recent years, lots of studies analyzed Schwann cells development from their specification to myelination and characterized the molecular profiles of each stage of their glial lineage.

Vagal and sacral NCC & Enteric nervous system

Vagal NCC specification also depends on the pathways they are following. Vagal NCC that migrate to the pharyngeal arches and the heart take the dorsolateral pathway, while the cells that take the ventral pathway are forming the enteric nervous system (ENS). Vagal NCC are the most abundant source of ENS precursors and are colonizing the rostral part of the gut and secondly the more caudal part of the gut [START_REF] Anderson | Neural Crest and the Development of the Enteric Nervous System[END_REF][START_REF] Le Douarin | The migration of neural crest cells to the wall of the digestive tract in avian embryo[END_REF][START_REF] Pomeranz | Colonization of the post-umbilical bowel by cells derived from the sacral neural crest: direct tracing of cell migration using an intercalating probe and a replication-deficient retrovirus[END_REF]. Sacral NCC form neuronal and glial populations of ENS but their contribution is less important than vagal NCC [START_REF] Burns | The sacral neural crest contributes neurons and glia to the post-umbilical gut: spatiotemporal analysis of the development of the enteric nervous system[END_REF][START_REF] Kapur | Early death of neural crest cells is responsible for total enteric aganglionosis in Sox10(Dom)/Sox10(Dom) mouse embryos[END_REF].

Indeed, these cells are mostly present in the distal part of the colon and rectum where they form up to 17% of enteric neurons [START_REF] Burns | The sacral neural crest contributes neurons and glia to the post-umbilical gut: spatiotemporal analysis of the development of the enteric nervous system[END_REF].

In human, the ENS is composed of 200-600 million neurons and glial cells which are grouped into many small ganglia distributed in 2 plexuses: the myenteric and submucosal plexuses (Figure 12) [START_REF] Furness | The first brain: Species comparisons and evolutionary implications for the enteric and central nervous systems[END_REF][START_REF] Furness | The enteric nervous system and gastrointestinal innervation: integrated local and central control[END_REF].

Contrary to the peripheral nerves, ENS is fully formed during embryonic development.

At E9-E9.5 in mice (4 weeks in humans), pre-enteric-derived NCC colonize the foregut and migrate rostro-caudally (Figure 13) [START_REF] Heanue | Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies[END_REF][START_REF] Lake | Enteric nervous system development: migration, differentiation, and disease[END_REF].

At the same time they migrate, they proliferate and start to differentiate. Indeed, they differentiate at E10 into neurons and later, at E11.5, into glial cells (the earliest enteric glial marker discovered so far is brain fatty acid-binding protein, BFABP) (Figure 13) [START_REF] Lake | Enteric nervous system development: migration, differentiation, and disease[END_REF]. Thus, in ENS development, unlike in other nervous systems like the central nervous system, glial cells and neurons proliferate at the same time they differentiate [START_REF] Heanue | Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies[END_REF]. The ENS is composed of sensory and motor neurons, interneurons and glial cells. At E13.5 (7 weeks in humans) NCC migration and contribution to myenteric plexus is complete and the pre-enteric-derived NCC undergo inward radial migration in the mucosa to form submucosal plexuses (Figure 13) [START_REF] Lake | Enteric nervous system development: migration, differentiation, and disease[END_REF].

Finally, sacral NCC colonize the gut in significant numbers only 4 days after vagal NCC had completed their migration along the entire length of the gut. These cells, as vagal NCC, contribute to the formation of both myenteric and submucosal plexuses [START_REF] Burns | The sacral neural crest contributes neurons and glia to the post-umbilical gut: spatiotemporal analysis of the development of the enteric nervous system[END_REF][START_REF] Burns | Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia[END_REF].

Enteric NCC survival, proliferation, migration and differentiation are controlled, among other signaling, by glial-derived neurotrophic factor (Gdnf) / Ret receptor [START_REF] Sasselli | The enteric nervous system[END_REF].

Gdnf is expressed in the gut mesenchyme and interacts with Ret and co-receptor Gdnf family receptor α1 (GFRα1) which are present at the surface of migrating enteric NCC. This pathway promotes cell differentiation required for the formation of ENS during normal development [START_REF] Mckeown | Exposure to GDNF Enhances the Ability of Enteric Neural Progenitors to Generate an Enteric Nervous System[END_REF][START_REF] Taraviras | Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system[END_REF]. On the contrary, Endothelin 3-endothelin B receptor (EDNRB) signaling blocks the differentiation of enteric NCC and increases the proliferation and maintenance of undifferentiated progenitors which are important for gut colonization during development [START_REF] Bondurand | Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures[END_REF][START_REF] Bondurand | Maintenance of mammalian enteric nervous system progenitors by SOX10 and endothelin 3 signalling[END_REF][START_REF] Sasselli | The enteric nervous system[END_REF].

Endothelin 3 -EDNRB signaling regulates ENS ontogeny also through its interaction with other pathways like Gdnf/Ret [START_REF] Sasselli | The enteric nervous system[END_REF], β1 integrin pathway [START_REF] Gazquez | Endothelin-3 stimulates cell adhesion and cooperates with β1-integrins during enteric nervous system ontogenesis., Endothelin-3 stimulates cell adhesion and cooperates with β1-integrins during enteric nervous system ontogenesis[END_REF] or with Sox10 [START_REF] Stanchina | Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development[END_REF] which controls the expression of Itgb1, encoding for β1 integrin [START_REF] Watanabe | Sox10 and Itgb1 interaction in enteric neural crest cell migration[END_REF].

Similarly to cranial NCC, enteric NCC undergo a collective migration regulated by complement anaphylatoxin C3a [START_REF] Broders-Bondon | N-cadherin and β1-integrins cooperate during the development of the enteric nervous system[END_REF], 2016). Complement is known for its role in immune system, as well as its chemotactic properties [START_REF] Carmona-Fontaine | Complement fragment C3a controls mutual cell attraction during collective cell migration[END_REF]. Enteric NCC express both the receptor C3aR and C3a. Secreted C3a by enteric NCC has a paracrine effect: it interacts with C3aR at the surface of neighbor enteric NCC and regulates their collective migration. Hence, C3a/C3aR co-attraction modulates gut colonization. Moreover, it has been proposed that this process is linked to N-cadherin signaling [START_REF] Broders-Bondon | Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin[END_REF].

To conclude, both vagal and sacral NCC participate to enteric nervous system formation by following specific migration pathways.

Schwann Cell Precursors

Embryonic Schwann Cell Precursors

As previously described, SCP derive from truncal and cranial NCC and give rise to immature Schwann cells at embryonic stages E15.5 [START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF]. Strikingly, it has been discovered that they also give rise to many other cell types and thereby constitute an amazing new population of embryonic and adult stem cells.

It has been first identified that SCP along nerves are also a source of melanocytes (Figure 14). The direct contact of SCP with axons, and more precisely the NRG1 signal delivered by axons, blocks the melanocytic fate and promotes immature Schwann cell development. However, expression of insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) by immature Schwann cells in absence of NRG1 signal, induces melanocytic differentiation of SCP in nerves (Adameyko et al., 2009[START_REF] Adameyko | Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest[END_REF].

This capacity of SCP to form melanocytes is evolutionary conserved: not only mammals develop nerve-derived melanocytes but also birds and bony fishes [START_REF] Petersen | Nerve-associated neural crest: peripheral glial cells generate multiple fates in the body[END_REF].

Neurons also derived from SCP. Indeed, SCP migrate along developing preganglionic nerves and differentiate into both Schwann cells and parasympathetic neurons (Dyachuk et al., 2014;[START_REF] Espinosa-Medina | Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors[END_REF].

It has long been known that SCP originate from truncal and cranial cells. Recently, it was shown that vagal NCC from somites 1 and 2 are also forming SCP which give rise to enteric neurons of the foregut. More precisely, the stomach and half of the esophageal nervous system originate from SCP which are migrating along vagus nerves [START_REF] Espinosa-Medina | Dual origin of enteric neurons in vagal Schwann cell precursors and the sympathetic neural crest[END_REF]. Interestingly, the involvement of vagus nerves into the formation of ENS was suggested in 1910 but it has been overlooked since then [START_REF] Kuntz | The development of the sympathetic nervous system in birds[END_REF].

Finally, SCP multipotency passes far beyond their ability to differentiate into melanocytes, glial cells and neurons, as recently reviewed [START_REF] Petersen | Nerve-associated neural crest: peripheral glial cells generate multiple fates in the body[END_REF].

Indeed, SCP generate (Figure 14):

-endoneurial fibroblasts during embryo development as mentioned previously [START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF] -bone marrow mesenchymal stem cells which specify hematopoietic stem cell niche functions [START_REF] Isern | The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function[END_REF] -dental mesenchymal stem cells in the murine incisor [START_REF] Kaukua | Glial origin of mesenchymal stem cells in a tooth model system[END_REF].

-chromaffin cells in the medulla of the adrenal glands in mammals [START_REF] Furlan | Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla[END_REF].

Thus, during development, embryonic SCP conserve the multipotency profile of NCC.

They travel along nerves and give rise to numerous cell types.

Adult Schwann Cell Precursors

In the last few years, it has been shown that SCP multipotency is not restricted to development. Indeed, SCP migrating across nerves also give rise to several cell types during adulthood [START_REF] Petersen | Nerve-associated neural crest: peripheral glial cells generate multiple fates in the body[END_REF]:

-adult SCP from nerves represent a cellular origin of melanocytes (Adameyko et al., 2009).

-adult SCP also differentiate into dental mesenchymal stem cells in mice incisor and promote the formation of odontoblasts and pulp cells [START_REF] Kaukua | Glial origin of mesenchymal stem cells in a tooth model system[END_REF].

-SCP invade the submucosal region of the small intestine alongside the extrinsic nerves and differentiate into neurons in the postnatal period, therefore promoting postnatal expansion of ENS (Uesaka et al., 2015).

-Sox2 positive Schwann cells, including SCP, are also important for regeneration of dermis after injury [START_REF] Johnston | Sox2-mediated regulation of adult neural crest precursors and skin repair[END_REF]. Moreover, during mouse digit tip regeneration, SCP migrate into the injured zone and induce mesenchymal cell proliferation and regeneration through paracrine secretion of oncostatin M and platelet-derived growth factor AA (PDGF) [START_REF] Johnston | Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip[END_REF].

-Finally, SCP are also essential to repair nerve injury. They differentiate into specific Schwann cells dedicated for nerve repair and form the Bungner band; these cells are necessary to direct axons inside the wounded region [START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF]Jessen et al., 2015aJessen et al., , 2015b)).

To conclude, in the last three years many studies underlined the existence of SCP into the nerves and their contribution to numerous cell types such as neurons, Schwann cells, mesenchymal stem cells and melanocytes during embryogenesis and adulthood (Figure 14). All these findings completely changed the vision about neural crest cells and peripheral nerves. We now know that peripheral nerves are structures which contain multipotent cells.

Therefore, peripheral nerves could represent a niche for adult stem cells. Exploring the molecular mechanisms underlying SCP selfrenewal and differentiation is a topic of growing interest as SCP multipotency could lead to several potential clinical applications.

Neural crest cell defects

Neural crest cells (NCC) defects lead to complex human pathologies named neurocristopathies by R. Bolande in 1974. Perturbations in NCC ontogeny are also observed in other diseases called ribosomopathies, which are human pathologies due to defects in ribosomal biogenesis (Figure 15). These pathologies can result from germline mutations (e.g.

CHARGE syndrome and Diamond blackfan anemia), from inappropiate micro-environment (e.g. Fetal alcohol syndrome), from age-related defects associating in particular somatic mutations (5q deletion syndrome).

Neurocristopathies

Neurocristopathies arise from NCC defects at different developmental stages, such as their formation, delamination, migration, differentiation and survival [START_REF] Ross | The emerging roles of ribosome biogenesis in craniofacial development[END_REF]Trainor, 2014). These disorders include common human birth defects combining several defaults such as colon aganglionosis, craniofacial malformations (cleft lip/palate, sensorineural deafness, smelling defects), heart malformations, skin/hair pigmentation defects and for some patients, demyelination [START_REF] Trainor | Neural Crest Cells: Evolution, Development and Disease[END_REF] (Table 3). Some examples of these human pathologies are listed below.

Hirschsprung's disease (HSCR) is characterized by the lack of ENS in the distal colon [START_REF] Butler Tjaden | The developmental etiology and pathogenesis of Hirschsprung disease[END_REF][START_REF] Ross | The emerging roles of ribosome biogenesis in craniofacial development[END_REF]. In 80% of HSCR patients the aganglionosis is limited to rectum and sigmoid colon and in 20% of cases the aganglionosis is found also proximally to the sigmoid colon. Only 3 to 8 % of patients have a total colonic aganglionosis [START_REF] Amiel | Hirschsprung disease, associated syndromes, and genetics: a review[END_REF][START_REF] Butler Tjaden | The developmental etiology and pathogenesis of Hirschsprung disease[END_REF][START_REF] Parisi | Hirschsprung Disease Overview[END_REF].

Several genes, which are known to be involved in NCC proliferation, migration, differentiation and survival, such as SOX10, RET, GDNF and EDNRB have been identified to regulate the etiology of HSCR (Table 3) [START_REF] Butler Tjaden | The developmental etiology and pathogenesis of Hirschsprung disease[END_REF][START_REF] Gazquez | Endothelin-3 stimulates cell adhesion and cooperates with β1-integrins during enteric nervous system ontogenesis., Endothelin-3 stimulates cell adhesion and cooperates with β1-integrins during enteric nervous system ontogenesis[END_REF][START_REF] Lecerf | An impairment of long distance SOX10 regulatory elements underlies isolated Hirschsprung disease[END_REF].

Waardenburg syndrome (WS) is divided in four types [START_REF] Arias | Genetic heterogeneity in the Waardenburg syndrome[END_REF][START_REF] Zaman | Waardenburg syndrome: more common than you think![END_REF]:

-Types I (WS1) and II (WS2) are characterized by abnormal pigmentation and sensorineural deafness due to a partial or total loss of melanocytes [START_REF] Arias | Genetic heterogeneity in the Waardenburg syndrome[END_REF][START_REF] Etchevers | Molecular bases of human neurocristopathies[END_REF][START_REF] Ross | The emerging roles of ribosome biogenesis in craniofacial development[END_REF].

-Type III (WS3) includes all of the WS1 and 2 features in association with musculoskeletal abnormalities of the upper limbs (Table 3) [START_REF] Alford | Medical Genetics in the Clinical Practice of ORL[END_REF].

-Type IV (WS4) also named Waardenburg-Shah syndrome combines Waardenburg symptoms and Hirshprung disease [START_REF] Inoue | Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations[END_REF][START_REF] Trainor | Neural Crest Cells: Evolution, Development and Disease[END_REF]. A subtype of WS4

is PCWH which includes a Peripheral demyelinating neuropathy, Central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung disease [START_REF] Inoue | Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations[END_REF][START_REF] Trainor | Neural Crest Cells: Evolution, Development and Disease[END_REF].

Thus, PCWH patients have a deficiency of melanocytes, enteric neurons, Schwann cells and oligodendrocytes (Table 3).

CHARGE syndrome is characterized by different clinical features (Table 3) related to craniofacial malformations, heart defects, blindness, deafness and balance disorders [START_REF] Harris | Epidemiology of choanal atresia with special reference to the CHARGE association[END_REF][START_REF] Issekutz | An epidemiological analysis of CHARGE syndrome: preliminary results from a Canadian study[END_REF][START_REF] Pauli | CHARGEd with neural crest defects[END_REF]. Approximately 80% of CHARGE syndrome patients present heterozygous mutations in CHD7 gene [START_REF] Aramaki | Phenotypic spectrum of CHARGE syndrome with CHD7 mutations[END_REF][START_REF] Lalani | Spectrum of CHD7 Mutations in 110 Individuals with CHARGE Syndrome and Genotype-Phenotype Correlation[END_REF][START_REF] Sanlaville | Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development[END_REF][START_REF] Vissers | Mutations in a new member of the chromodomain gene family cause CHARGE syndrome[END_REF]. CHD7 is a chromatin remodeling enzyme which uses the ATP produced by ATP hydrolysis to alter nucleosome structure in order to create DNA access [START_REF] Eberharter | ATP-dependent nucleosome remodelling: factors and functions[END_REF][START_REF] Narlikar | Cooperation between complexes that regulate chromatin structure and transcription[END_REF][START_REF] Sif | ATP-dependent nucleosome remodeling complexes: Enzymes tailored to deal with chromatin[END_REF]. It controls nucleoplasmic and nucleolar genes transcription. Therefore, CHD7 inactivation decreases the formation of 45S pre-rRNA, being therefore involved in CHARGE syndrome [START_REF] Zentner | CHD7 functions in the nucleolus as a positive regulator of ribosomal RNA biogenesis[END_REF].

Di George syndrome also known as velocardiofacial syndrome is due to chromosome 22q11.2 deletion (Van Nostrand and Attardi, 2014) which corresponds to over 35 genes [START_REF] Mcdonald-Mcginn | Chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome)[END_REF]. Due to this large number of genes involved, the phenotype is extremely variable even within families. The major phenotypic features are heart anomalies, palatal anomalies, immune deficiency and learning difficulties (Table 3) [START_REF] Mcdonald-Mcginn | Chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome)[END_REF].

Even if numerous studies characterized the underlying molecular mechanisms responsible for these pathologies, there is so far no treatment or cure for these diseases.

In order to foster our knowledge and prevention of these disorders, several mouse models reliably mimicking human neurocristopathies were generated and are presented in Table 4. Here are listed only some examples of murine models reminiscent to human neurocristopathies but there are many other models with neural crest cells defects. Di George syndrome mouse models were not included in this list because of their large number.

In bold and underlined are the mouse models that have been used in studies presented in All the data were also obtained from OMIM data base.

Ribosomopathies

Ribosomes represent a complex molecular system, containing proteins and RNA, essential for protein translation (Figure 16). They are important for polypeptide chain formation by promoting the interaction between amino acids in the order specified by messenger RNA.

Ribosomes contain two components: the small 40S ribosomal subunit and the large 60S. The 40S subunit is composed of 18S rRNA and 32 Ribosomal Protein Small subunit (RPS). The 60S subunit contains 5S, 5.8S and 28S rRNA and 47 Ribosomal Protein Large subunit (RPL).

The 40S and 60S subunits are then exported into the cytoplasm, where they assemble with mRNA to form the 80S ribosome critical for amino acids assembly [START_REF] Van Riggelen | MYC as a regulator of ribosome biogenesis and protein synthesis[END_REF][START_REF] Rodnina | Recent mechanistic insights into eukaryotic ribosomes[END_REF].

In this introduction, I focused on two of these pathologies among others presented in Table 5:

Treacher-Collins Syndrome is an autosomal dominant disease characterized by craniofacial malformations due to deficit of cranial NCC development [START_REF] Trainor | Treacher Collins syndrome: etiology, pathogenesis and prevention[END_REF]. It is hard to classify this syndrome as a neurocristopathy or ribosomopathy because it is caused by ribosomal biogenesis defects but it affects only neural crest cell derivatives (Figure 15).

In this manuscript, I have chosen to present it as a ribosomopathy. It is caused by Tcof1 mutations. Tcof1 encodes a nucleolar protein named Treacle which is essential for ribosomal RNA transcription and thereby for ribosomal biogenesis [START_REF] Ross | The emerging roles of ribosome biogenesis in craniofacial development[END_REF][START_REF] Trainor | Treacher Collins syndrome: etiology, pathogenesis and prevention[END_REF]. When its expression is lost, many cranial neural cells die by apoptosis thereby leading to craniofacial malformations. TCS patients undergo speech therapy, hearing, orthodontic and ophthalmologic aid as well as multiple reconstructive craniofacial surgeries, which are rarely fully corrective.

Diamond black anemia (DBA) is another hereditary ribosomopathy characterized by craniofacial malformations as well as anemia [START_REF] Narla | Ribosomopathies: human disorders of ribosome dysfunction[END_REF][START_REF] Ross | The emerging roles of ribosome biogenesis in craniofacial development[END_REF]. This inherited pathology is associated to mutations in different genes encoding ribosomal proteins of the large ribosome subunit (RPL15, RPL26, RPL35, RPL35a, RPLP2, RPS14) or the small ribosome subunit (RPS19) [START_REF] Ross | The emerging roles of ribosome biogenesis in craniofacial development[END_REF]. These mutations reduce the number of mature ribosomes which impact the quantity of erythroid progenitors in the bone marrow and of immature red blood cells hence leading to severe anemia. Mutations in ribosomal proteins also cause, as in TCS, deficiency of cranial NCC and thereby craniofacial defects [START_REF] Narla | Ribosomopathies: human disorders of ribosome dysfunction[END_REF][START_REF] Ross | The emerging roles of ribosome biogenesis in craniofacial development[END_REF].

In order to better understand the molecular mechanisms underlying these pathologies, a broad list of mouse models was generated (Table 6). They harbor phenotypes reminiscent of human ribosomopathies therefore allowing to explore therapeutic strategies. Their main treatment so far is surgery but is often variable and rarely entirely corrective. In some cases, surgery is not even possible. Recent prevention using antioxidant treatment as well as better understanding of Schwann Cell Precursors open new avenues for cellular therapeutic approaches (see Discussion). Surprisingly, not much is known about energy metabolism in NCC.

Table 5: Description of some human ribosomopathies

In bold and underlined are the mouse models that have been used in studies presented in Table 8 (Introduction, Chapter III.B.4) to be mated to p53 null mice in order to assess if p53 loss rescues the phenotype.

Table 6: Mouse models reminiscent to human ribosomopathies

All the data were also obtained from OMIM data base.

III. METABOLIC REGULATORS: LKB1 AND p53

Signal transduction that dictates cell fate depending on the cell environment participates to metabolism reprograming. Furthermore, metabolically sensitive posttranslational modifications of signaling proteins (such as phosphorylation or acetylation) provide a feedback mechanism to control signaling pathways. The relationship between cell signaling and metabolism is thus bidirectional [START_REF] Ward | Metabolic reprogramming: a cancer hallmark even warburg did not anticipate[END_REF].

Several signaling pathways are known to regulate energy metabolism: the growth factor essential to establish and maintain cell polarity, an important process for NCC migration and differentiation. Finally, LKB1 is also a well-known metabolic regulator. As mentioned previously, the potential contribution of metabolic regulations during NCC formation remains largely unexplored. These data prompted the lab in which I did my PhD to explore LKB1 functions during neural crest formation as a good candidate to control NCC fate potentially through both the regulation of cell polarity and energy metabolism. Therefore, the first part of this chapter will describe some characteristics of the LKB1 signaling pathway.

Another pathway which regulates different aspects of cell metabolism is p53 signaling.

Furthermore, p53 dosage is crucial for NCC formation as aberrant p53 signaling is associated with neural crest defects both in patients and animal models. Therefore, the second part of this chapter will describe p53 signaling, how it regulates energy metabolism and how p53 deregulations are involved in neural crest defects.

III.A. THE MASTER KINASE LKB1

Over the last decade, researchers have shown that LKB1 is a newly regulator of metabolism and is considered as a novel uncovered link between metabolism and growth control (Shackelford and Shaw, 2009). The master kinase LKB1 is a serine/threonine kinase encoded by the LKB1 gene, also known as STK11, which regulates lipid, carbohydrate, and energy metabolism mostly through mechanisms dependent of 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) (Hardie and Alessi, 2013;[START_REF] Srivastava | AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases[END_REF] but in some cases also independently of AMPK [START_REF] Jeppesen | LKB1 Regulates Lipid Oxidation During Exercise Independently of AMPK[END_REF].

I will start this Chapter by giving some general information about LKB1 structure, expression, localization and regulation. After that I will describe some functions of the LKB1 signaling pathway. As metabolism was the guideline of my PhD work, I will focus on metabolic functions of LKB1. I will next describe how LKB1 signaling contributes to cell polarity and embryonic development. Of note, the bibliographic data showed here will not exhaustively present the massive knowledge available about LKB1 signaling.

III.

A.1. LKB1 structure, localization and regulation.

Structure and expression of LKB1 isoforms

The human LKB1 gene, localized on chromosome 19p13.3, was discovered by Junichi Nezu of Chugai Pharmaceuticals, in 1996, in a screen used to find new kinases [START_REF] Zhao | Targeting the LKB1 tumor suppressor[END_REF]. Three isoforms for LKB1, mostly generated by alternative splicing, have been describe so far: LKB1 full-length, LKB1 Short isoform (S) and LKB1 deleted of the N-terminal domain 17).

(ΔN-LKB1) (Figure
Human LKB1 full-length is composed of 433 amino acids (436 amino acids in mice)

and has a molecular weight of 50kDa. It comprises a catalytic domain (amino acids 49 to 309) and a nuclear localization site (NLS, amino acids 38-43) (Figure 17). This isoform is ubiquitously expressed with a wide pattern of expression in embryonic and adult tissues.

During mouse and human development, LKB1 is ubiquitously expressed at the early stages of embryogenesis and with time its expression becomes restricted in specific tissues, being more pronounced in lungs, stomach, small intestine, colon, kidney, pancreas and seminiferous tubules of testes (Table 7). Later in embryonic development, prominent expression of Lkb1 was observed in central nervous system and in peripheral nervous system, in both glial cells and neurons [START_REF] Luukko | Expression of LKB1 and PTEN tumor suppressor genes during mouse embryonic development[END_REF][START_REF] Rowan | In situ analysis of LKB1/STK11 mRNA expression in human normal tissues and tumours[END_REF]. In adult tissues LKB1 expression is widespread but is predominant in epithelia, glial cells, seminiferous tubules of the testis and myocytes from skeletal muscle (Table 7) [START_REF] Conde | Specific pattern of LKB1 and phospho-acetyl-CoA carboxylase protein immunostaining in human normal tissues and lung carcinomas[END_REF][START_REF] Sanchez-Cespedes | A role for LKB1 gene in human cancer beyond the Peutz-Jeghers syndrome[END_REF].

LKB1S (48kDa, 404 amino acids) differs from full length isoform in its C-terminal domain (Figure 17). The expression pattern of LKB1S is more restricted than LKB1 full-length since LKB1S is specific to spermatids, the haploid male germ cells, in testis. Lkb1S inactivation in mice causes male infertility [START_REF] Kong | Differential regulation of spermatogenic process by Lkb1 isoforms in mouse testis[END_REF][START_REF] Towler | A novel short splice variant of the tumour suppressor LKB1 is required for spermiogenesis[END_REF].

C. Perret lab in collaboration with our team uncovered the existence of another isoform of LKB1 called ΔN-LKB1 (Figure 17) [START_REF] Dahmani | A novel LKB1 isoform enhances AMPK metabolic activity and displays oncogenic properties[END_REF]. This isoform of 42kDa is mainly expressed in heart and skeletal muscle. This protein is exclusively cytoplasmic and is catalytic inactive. However, it is capable to enhance AMPK metabolic activity because it has a dominant

CL CLEUS

positive effect on LKB1 full-length-induced AMPK activity [START_REF] Thibert | AMPK potentiation by LKB1 isoforms[END_REF]. Moreover, it has also pro-oncogenic properties which may explain why in some sporadic cancers LKB1 is inactivated and in others its expression is increased [START_REF] Dahmani | A novel LKB1 isoform enhances AMPK metabolic activity and displays oncogenic properties[END_REF].

1.2 LKB1 subcellular localization and activation.

LKB1 is localized both in the cytoplasm and nucleus as well as to the plasma membrane. In the nucleus, LKB1 is present in a heterotrimeric complex with the mouse protein 25 (MO25α) and the pseudokinase STE20 related kinase adaptor (STRADα) (Figure 18).

MO25 connects LKB1 and the C-terminal part of STRADα thereby stabilizing the interaction between LKB1 and STRADα. This association is essential for LKB1 translocation from nucleus to cytoplasm and for its kinase activity (Boudeau et al., 2003a[START_REF] Boudeau | MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm[END_REF].

LKB1 phosphorylation on serine 428 could also be important for both its cytoplasmic translocation and its kinase activity [START_REF] Xie | Phosphorylation of LKB1 at Serine 428 by Protein Kinase C-ζ Is Required for Metformin-Enhanced Activation of the AMP-Activated Protein Kinase in Endothelial Cells[END_REF] although being controversial [START_REF] Denison | Characterization of an Alternative Splice Variant of LKB1[END_REF][START_REF] Hou | SIRT1 regulates hepatocyte lipid metabolism through activating AMPactivated protein kinase[END_REF]. The C-terminal part of LKB1 contains a prenylation site (cysteine 433) necessary for the localization of LKB1 to the plasma membrane [START_REF] Houde | Investigation of LKB1 Ser431 phosphorylation and Cys433 farnesylation using mouse knockin analysis reveals an unexpected role of prenylation in regulating AMPK activity[END_REF].

LKB1 acetylation on lysine 48 regulates also its subcellular localization [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation[END_REF]. Indeed, NAD + -dependent sirtuin 1 (Sirt1) deacetylates LKB1 on lysine 48 promoting its translocation into the cytoplasm. LKB1 deacetylation increases its phosphorylation on serine 428 and threonine 336 thereby changing its subcellular localization and its kinase activity quantified by AMPK activation [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation[END_REF].

Finally, LKB1 is also transported to the lysosome membrane where it activates AMPK [START_REF] Zhang | The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism[END_REF] (see Chapter IV.2).

Regulation of LKB1 activity by post translational modifications

Several post translational modifications have been described for LKB1. These modifications include ubiquitination which leads to LKB1 degradation by the proteasome [START_REF] Gaude | Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1[END_REF]Nony et al., 2003) and prenylation, phosphorylation and acetylation which regulate LKB1 activity, subcellular localization and interaction with other proteins (Figure 19).

Below, I will focus on LKB1 phosphorylation and acetylation.

Phosphorylation

Phosphorylation is a post translational modification of a protein characterized by the addition of phosphate groups being able to alter the structural conformation of the protein and to modify its function. This post translational modification is regulated by phosphatases and kinases. LKB1 is phosphorylated by different upstream kinases such as Ataxia-telangiectasia mutated kinase (ATM), Proline-directed kinase, Protein kinase B (AKT) and Protein kinase A (PKA) (Figure 19A). The most studied phosphorylation sites in human LKB1 are serines 31, 325, 428 and threonine 363 (Alessi et al., 2006a;Baas et al., 2004a;Sapkota et al., 2002aSapkota et al., , 2002b)). LKB1 also autophosphorylates on several sites: threonine 185 and 189, serine 404 and also threonine 336 which is the major site of autophosphorylation.

Phosphorylation of LKB1 at all these sites is not involved in regulating its kinase activity or its subcellular localization (Boudeau et al., 2003a;Sapkota et al., 2002) except serine 428 which has a controversial role regarding LKB1 localization that I presented previously (cf.

paragraph 1.2). Instead, some of these sites are essential for the tumor suppressor function of LKB1 (Alessi et al., 2006a). For example, the replacement of serine 431 by an alanine (to prevent phosphorylation) or threonine 336 with glutamate (to mimic phosphorylation) inhibits the capacity of LKB1 to limit cell growth (Alessi et al., 2006a). However, not all the phosphorylation sites regulate LKB1 tumor suppressor capacity because sites as serine 31, serine 325 or threonine 366 have no effect on cell growth (Alessi et al., 2006;Boudeau et al., 2003a;Sapkota et al., 2002).

Acetylation

Acetylation is a post translational modification of a protein involving the addition of an acetyl group obtained from the metabolite acetyl-CoA. This post translational modification is regulated by the combined functions of deacetylases and acetyltransferases, themselves modulated by energy metabolism [START_REF] Wellen | A two-way street: reciprocal regulation of metabolism and signalling[END_REF]. For example, Sirtuin deacetylases are regulated by metabolic status of the cell because these enzymes are dependent on coenzyme NAD + which is involved in redox reactions and plays important roles in energy metabolism. Protein acetylation is also regulated by metabolism because it depends on Acetyl-CoA availability which is a key node in metabolism.

Our team and Y. Ido lab have shown by mass spectrometry that LKB1 is acetylated on different lysines (Figure 19 B) [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation[END_REF]. The acetylation of lysine 97 regulates LKB1 activity and function during oxidative stress [START_REF] Calamaras | Post-translational modification of serine/threonine kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxytrans-2-nonenal (HNE) at lysine residue 97 directly inhibits kinase activity[END_REF] and, as already mentioned, LKB1 acetylation on lysine 48 controls its subcellular localization. We identified the acetyltransferase GCN5 as one of the enzymes which acetylates LKB1 on lysine 48 (cf Results

Project 1.2).

GCN5 was the first histone acetyltransferase described which regulates transcriptional activity of proteins [START_REF] Brownell | An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei[END_REF]. Since, it has been shown that GCN5 also acetylates non-histone proteins. GCN5 is composed of three important domains:

à a bromodomain in the C terminal part, important for anchoring to its substrates [START_REF] Owen | The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p[END_REF]. But this domain is not the only region that is responsible for GCN5 binding to its substrates [START_REF] Carré | The histone H3 acetylase dGcn5 is a key player in Drosophila melanogaster metamorphosis[END_REF].

à an acetyltransferase central domain which contains the catalytic activity of GCN5.

à a p300/CBP Associated Factor (PCAF) homology domain. This domain is unique to animals [START_REF] Smith | Cloning of Drosophila GCN5: conserved features among metazoan GCN5 family members[END_REF] and allows the interaction of GCN5 with p300 and CBP.

GCN5 acetyltransferase regulates several cellular processes such as energy metabolism. One example is the acetylation of the transcription factor peroxisome proliferatoractivated receptor-gamma coactivator-1 alpha (PGC1α) which induces its translocation from the promoter regions of its target genes encoding gluconeogenic enzymes to nuclear foci.

Thus, GCN5 regulates glucose metabolism through transcriptional repression of PGC1α [START_REF] Lerin | GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of PGC-1α[END_REF]. Moreover, these GCN5 metabolic activities are controlled by the Sirtuin deacetylase 6 (Sirt6). Indeed, Sirt6 directly deacetylates GCN5 on lysine 549 which promotes its phosphorylation on serine 307 and threonine 735 and therefore triggers its activation.

Activated GCN5 acetylates PGC1α and limits gluconeogenesis [START_REF] Dominy | The deacetylase Sirt6 activates the acetyltransferase GCN5 and suppresses hepatic gluconeogenesis[END_REF].

III.A.2. LKB1 signaling: focus on AMPK/mTOR pathway

LKB1 is a master kinase which phosphorylates 14 kinase substrates that are members of the AMPK family. Through the regulation of its downstream kinases, LKB1 contributes to several cellular processes such as cell polarity and energy metabolism (Figure 20) [START_REF] Gan | Recent Progress on Liver Kinase B1 (LKB1): Expression, Regulation, Downstream Signaling and Cancer Suppressive Function[END_REF]Shackelford and Shaw, 2009;Shaw, 2009a). Among the 14 kinases, some of them control cell polarity, like Synapses of the Amphid Defective kinases (SAD), Microtubule Affinity

Regulating Kinases (MARK/Par-1) and Sucrose-Non-fermenting protein-Related Kinase (SNRK/NUAK) (Figure 20) [START_REF] Barnes | LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons[END_REF]Shaw, 2009a;[START_REF] Shelly | LKB1/STRAD promotes axon initiation during neuronal polarization[END_REF]. NUAK regulates neurons maturation through mitochondria immobilization (Courchet et al., 2013) and is also expressed during early chick development being important for vertebrate head formation [START_REF] Bekri | Analysis of NUAK1 and NUAK2 expression during early chick development reveals specific patterns in the developing head[END_REF]. Other kinases like AMPK and Salt-Inducible Kinases (SIK) control cell growth and metabolism (Figure 20) [START_REF] Patel | The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver[END_REF]Shaw, 2009b).

During my PhD, as my objective was to analyze the energy metabolism, I focused on LKB1-AMPK pathway, AMPK being an energy sensor that activates catabolic processes and inhibits anabolic ones. Therefore, in this manuscript I will present data mostly about the AMPK substrate which is, so far, the only LKB1 substrate sensing the cellular energy status.

AMPK activation

AMPK is a serine/threonine kinase which consists of three subunits: a catalytic subunit (AMPKα) and two regulatory subunits (AMPKβ and AMPKɣ) (Figure 21). Each subunit is represented by several isoforms: α1, α2, β1, β2, ɣ1, ɣ2, ɣ3 [START_REF] Viollet | AMPK: Lessons from transgenic and knockout animals[END_REF].

AMPK phosphorylation

AMPKα is the catalytic subunit. It contains, in its N terminal part, the kinase domain (Figure 21) [START_REF] Crute | Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase[END_REF] and the threonine 172 site which can be phosphorylated by several kinases: LKB1, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) and LKB1 is a master kinase which regulates 14 kinase substrates which in turn phosphorylate other proteins, being therefore implicated in many cellular processes such as cell polarity, energy metabolism, cell proliferation, differentiation, etc. (Shackelford and Shaw, 2009).

Figure 21: AMPK structure and regulation by LKB1

AMPK is a heterotrimeric complex composed of α, β and ɣ subunits. α-subunit contains Thr172 which is phosphorylated by LKB1. Activated AMPK increases catabolic processes (ATP production) and decreases anabolic processes (ATP consomption). AID, auto-inhibitory domain; CBM, carbohydrate-binding module; CBS, cystathionine-β-synthase (Adapted from Herzing and Shaw, 2017).

TAK1 (Transforming growth factor beta-activated kinase 1) [START_REF] Hawley | Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase[END_REF][START_REF] Momcilovic | Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro[END_REF][START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress[END_REF][START_REF] Tamás | Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes[END_REF]The master kinase LKB1 is the major activator of AMPK in many cell types that we and others used as cellular models such as mouse embryonic fibroblast cells (MEF), human embryonic cells (HEK-293), mouse glial cells (S16 and glial differentiated JoMa1), mouse Sertoli cells (TM4) and kidney monkey cells (COS7). Indeed, LKB1 inactivation in these cell lines leads to a nearly complete loss of AMPK phosphorylation on threonine 172, in response to different stimuli [START_REF] Hawley | Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade[END_REF]Tanwar et al., 2012a;[START_REF] Woods | LKB1 is the upstream kinase in the AMP-activated protein kinase cascade[END_REF]; our studies -cf Results. Projects 1.2, 2.1 and 3.1).

AMPK regulation

AMPK is an enertgy sensor activated by AMP and ADP (less than AMP) and inhibited by ATP [START_REF] Hardie | AMPK: a nutrient and energy sensor that maintains energy homeostasis[END_REF] The sensitivity to AMP/ATP or ADP/ATP ratio is due to its ɣ subunit (Figure 21). This subunit contains four tandem sequence repeats named cystathionine β synthase (CBS). These domains allow AMPK to reversibly bind AMP or ATP and the binding choice depends on their concentration [START_REF] Xiao | Structural basis for AMP binding to mammalian AMP-activated protein kinase[END_REF]. In other words, if AMP is at high concentration, it competes with ATP to bind to AMPKɣ subunit. Even if mammalian AMPK have four CBS domains only CBS 1, 3 and 4 can reversibly bind AMP or ATP (Figure 21). In normal conditions, AMPK is inactive because the ATP concentration is high and one molecule of ATP binds each CBS domain. In stress conditions, ATP is depleted and therefore the concentration of AMP is higher and can replace ATP from CBS motifs. First, AMP replaces ATP from CBS3 and protects the threonine 172 site to be dephosphorylated. This replacement increased AMPK activity 100 times. Secondly, if the energetic stress is even higher, the ATP from CBS1 is also replaced by AMP which activates AMPK by an allosteric regulation [START_REF] Xiao | Structural basis for AMP binding to mammalian AMP-activated protein kinase[END_REF].

This second replacement increases AMPK activity 10 times more than the first activation. Thus, in stress conditions, AMP activates AMPK by two complementary mechanisms: by protecting threonine 172 from dephosphorylation and by an allosteric activation. To restore normal ATP levels, AMPK inhibits anabolic processes which consume energy, such as triglyceride and protein synthesis and induces catabolic processes which produce energy, like fatty acid oxidation or mitochondrial biogenesis [START_REF] Ruderman | AMPK, insulin resistance, and the metabolic syndrome[END_REF].

Beside upstream kinases and AMP/ATP ratio, there are others regulators of AMPK activity (Figure 22). Among these regulators, we can find hormones (adiponectine, leptin, Il-6), natural compounds (rooibos, resveratrol, α-lipoic acid, berberine, AICAR) and pharmacological drugs which are indirect (biguanides, thiazolidinediones) or direct (A-769662, PT1) activators [START_REF] Coughlan | AMPK activation: a therapeutic target for type 2 diabetes?[END_REF][START_REF] Fogarty | Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer[END_REF]. Indirect drugs activate AMPK by increasing the cellular AMP/ATP ratio. Drugs activating directly AMPK act as AMP, preventing threonine AICAR (5-AminoImidazole-4-Carboxamide Ribonucleotide) was the first AMPK-activator molecule discovered in vivo and in cellulo [START_REF] Corton | 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?[END_REF][START_REF] Sullivan | Characterisation of 5'-AMP-activated protein kinase in human liver using specific peptide substrates and the effects of 5'-AMP analogues on enzyme activity[END_REF]. AICAR is a natural intermediate in purine nucleotide synthesis and thus a precursor of several purine monophosphates including ZMP, IMP and AMP (Figure 22) [START_REF] Mikanagi | Purine and Pyrimidine Metabolism in Man VI: Part A: Clinical and Molecular Biology[END_REF]. AICAR mode of action is actually ambiguous since it has been shown to act:

-directly on AMPK as an AMP analogue: AICAR is taken up into cells through the adenosine transporter and is metabolized by adenosine kinase in ZMP. ZMP synthesis from extracellular AICAR is faster than its subsequent metabolism. As a consequence, ZMP accumulates and mimics the effects of AMP by binding AMPK CBS domains. Hence, ZMP activates AMPK (Figure 22) either by triggering an increase of threonine 172 phosphorylation or by allosteric regulation [START_REF] Corton | 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?[END_REF][START_REF] Henin | Stimulation of rat liver AMP-activated protein kinase by AMP analogues[END_REF].

-indirectly on AMPK through replenishing the pools of AMP. During the purine nucleotide pathway, AICAR is transformed into inosine 5'monophosphate (IMP), a precursor of AMP.

-independently of AMPK through ZMP which interacts with other AMP-regulated enzymes such as fructose-1,6-bisphosphatase [START_REF] Vincent | Inhibition by AICA Riboside of Gluconeogenesis in Isolated Rat Hepatocytes[END_REF] or glycogen phosphorylase [START_REF] Longnus | 5-Aminoimidazole-4-carboxamide 1-beta -D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms[END_REF] (Longnus et al., 2013) and inhibits or respectively Drugs such as reservatol, berberine and biguanids are able to block mitochondrial ATP synthesis and thus to increase AMP: ATP ratio which leads to AMPK activation. AICAR is a metabolic drug which is up-taken by the cells and transformed in ZMP which mimics AMP and which binds to ɣ-subunit and activates AMPK. A-769662 has also the ability to mimic AMP and to activate AMPK but through another subunit, the β-subunit. PT1 increases AMPK activity through its interaction with AMPK α-subunit and inhibition of autoinhibitory domain (Adapted from Fogarty and Hardie, 2009).

activates them, blocking gluconeogenesis. One well established AMPK-independent function of AICAR is its ability to induce astroglial differentiation of neural stem cells (Zang et al., 2008).

***

Altogether, these data demonstrated that AMPK is an LKB1 substrate as well as an energy sensor controlled by AMP/ATP ratio and other different regulators like AICAR.

2.2 mTOR downstream pathway and functions LKB1/AMPK have many downstream targets (Figure 23) being involved in several cellular processes like cell growth, autophagy, metabolism and polarity. Below I focused only on serine/threonine kinase mTOR, the most studied downstream target of this pathway.

mTOR is present in two complexes: mTORC1, which is a major growth regulator and mTORC2 which promotes cellular survival (Figure 24). AMPK blocks mTOR activation by two mechanisms:

1. it directly phosphorylates Tuberous Sclerosis Complex 2 (TSC2) at serine 1387 (in human) and therefore stimulates the GTPase activating protein (GAP) activity of TSC1/TSC2 complex in order to transform Rheb GTP in Rheb GDP . Rheb is a G protein which activates mTORC1 only when it is bound to GTP (Inoki et al., 2003;Shaw, 2009a).

2. it also directly phosphorylates Raptor, a subunit of mTORC1 complex, at two conserved sites: serines 722 and 792 and therefore induces binding of Raptor with the scaffolding 14-3-3 protein which leads to mTORC1 inhibition (Gwinn et al., 2008;Shaw, 2009a).

mTOR activation promotes protein biosynthesis, lipid anabolism and nucleotides production, all these processes being important for cell growth and proliferation [START_REF] Wullschleger | TOR signaling in growth and metabolism[END_REF]. The main mTOR downstream targets that promote expression of factors involved in cell growth and proliferation are:

-S6 protein kinase1 (S6K1) (Figure 24) which activates S6 ribosomal protein (S6RP)

and translational initiation factor eIF4B [START_REF] Guertin | Defining the role of mTOR in cancer[END_REF].

-eIF4E-binding protein (4E-BP1) (Figure 24) which inhibits the activation of translation initiation factor eIF4E [START_REF] Holz | mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events[END_REF].

In addition to the control of cell proliferation by TORC1 complex, LKB1/AMPK/mTOR pathway has other cellular functions such as:

-regulating actin cytoskeleton dynamic [START_REF] Jacinto | Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive[END_REF]. TORC2 activates Rho1

GTPase which activates Protein Kinase C (PKC) and in turn signals to the actin cytoskeleton via a Mitogen-Activated Protein (MAP) kinase pathway (Figure 24) [START_REF] Wullschleger | TOR signaling in growth and metabolism[END_REF].

-monitoring autophagy in mammalian cells [START_REF] Lum | Growth factor regulation of autophagy and cell survival in the absence of apoptosis[END_REF] (Figure 24). -controlling cell metabolism such as amino acid biosynthesis, glucose homeostasis and fatty acids metabolism [START_REF] Kim | Regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis[END_REF][START_REF] Um | Absence of S6K1 protects against age-and diet-induced obesity while enhancing insulin sensitivity[END_REF].

To conclude, LKB1/AMPK signaling regulates numerous downstream targets, one of them being mTOR. In particular through this regulation, this signaling pathway controls several cellular processes such as energy metabolism. LKB1/AMPK also regulates cell growth, migration, polarity and autophagy through other downstream substrates than mTOR not described in this manuscript.

III.A.3. Cellular regulations by LKB1/AMPK signaling

3.1 LKB1 is a major metabolic regulator LKB1 is a key regulator of energy homeostasis and it controls the lipid and glucose metabolism in several tissues such as liver, adipose tissue and skeletal muscles.

Regulation of lipid metabolism

LKB1 regulates lipids and cholesterol synthesis through AMPK activation which phosphorylates and inhibits Acetyl-CoA carboxylase (ACC), glycerol phosphate acyltransferase (GPAT) and 3-hydroxy-3-methyl glutamyl (HMG)-CoA reductase (Figure 23) [START_REF] Carlson | Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation[END_REF]Hardie and Alessi, 2013;[START_REF] Hardie | AMPK: a nutrient and energy sensor that maintains energy homeostasis[END_REF]. There are two ACC isoforms. ACC1 is expressed in lipogenic tissues like liver and adipose tissues and is involved in fatty acid synthesis [START_REF] Davies | Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freezeclamping. Effects of high fat diets[END_REF] ACC2 is expressed in oxidative tissues like heart and muscles and is involved in fatty acid oxidation (Merrill et al., 1997). Both isoforms are directly phosphorylated and inhibited by AMPK. GPAT is involved in triglyceride and phospholipid synthesis and HMG-CoA reductase (HMGR) is localized at the membrane of endoplasmic reticulum and regulates cholesterol synthesis. AMPK negatively regulates HMGR through its direct phosphorylation but also by regulating its gene expression via degradation of Forkhead transcription factor 1a (FoxO1a) [START_REF] Fisslthaler | Activation and signaling by the AMP-activated protein kinase in endothelial cells[END_REF].

Regulation of glucose metabolism

LKB1 regulates glucose uptake and gluconeogenesis by increasing Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) levels through AMPK activation [START_REF] Foretz | Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state[END_REF][START_REF] Shaw | The Kinase LKB1 Mediates Glucose Homeostasis in Liver and Therapeutic Effects of Metformin[END_REF]. AMPK directly interacts and phosphorylates PGC1α [START_REF] Jäger | AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha[END_REF]. This phosphorylation represents a priming signal for the subsequent PGC1α deacetylation by Sirtuin 1. Hence AMPK, in opposition with GCN5 presented in paragraph 1.3.2, regulates glucose metabolism through PGC1α activation [START_REF] Cantó | PGC-1alpha, SIRT1 and AMPK, an energy sensing network that controls energy expenditure[END_REF][START_REF] Jeninga | Reversible acetylation of PGC-1: Connecting energy sensors and effectors to guarantee metabolic flexibility[END_REF]. However, AMPK is not the only substrate of LKB1 which regulates gluconeogenesis. *** For example, Salt-inducible kinase (SIK2) is also able to phosphorylate CREB regulated transcription coactivator 2 (CRTC2). This phosphorylation stimulates CRTC2 cytosolic translocation and inhibits transcriptional activation of its hepatic gluconeogenic target genes [START_REF] Patel | The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver[END_REF].

Regulation of mitochondria dynamic

Another important mechanism regulated by LKB1/AMPK is mitochondrial biogenesis [START_REF] O'neill | AMP-activated protein kinase (AMPK) beta1beta2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise[END_REF][START_REF] Winder | Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle[END_REF]. In hematopoietic stem cells (HSC), Lkb1 inactivation decreases mitochondrial biogenesis and energy production by downregulating PGC1α levels (Figure 23) [START_REF] Gan | Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells[END_REF]Gurumurthy et al., 2010;[START_REF] Nakada | Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells[END_REF][START_REF] Saito | The role of the Lkb1/AMPK pathway in hematopoietic stem cells and Leukemia[END_REF]. PGC1α is a transcription coactivator which not only regulates glucose metabolism, as seen above, but it also enhances the expression of some mitochondrial genes [START_REF] Lin | Metabolic control through the PGC-1 family of transcription coactivators[END_REF]. Thus, LKB1/AMPK pathway regulates mitochondria biogenesis. This pathway controls also mitochondria degradation by autophagy, named mitophagy [START_REF] Egan | Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy[END_REF][START_REF] Hardie | AMPK: a nutrient and energy sensor that maintains energy homeostasis[END_REF][START_REF] Kim | Mitochondrial degradation by autophagy (mitophagy) in GFP-LC3 transgenic hepatocytes during nutrient deprivation[END_REF] as well as their mobility (Courchet et al., 2013) and fragmentation (Toyama et al., 2016).

Moreover, mitochondria defects are associated with increased oxidative damage which controls the production of reactive oxygen species (ROS) (see Chapter IV.3.2). In addition, several researchers have observed that LKB1 is also a regulator of ROS levels. It controls the activity and expression of antioxidant proteins such as superoxide dismutase 2 (SOD2) and catalase and thereby protects the genome integrity (Gurumurthy et al., 2010;Xu et al., 2015).

AMPK is also capable to increase antioxidants production through activation of Forkhead box O (FoxO) transcription factor [START_REF] Chiacchiera | The AMPK-FoxO3A axis as a target for cancer treatment[END_REF].

LKB1/AMPK pathway connects the energy metabolism to ROS production. Upon energy stress like glucose starvation, activated AMPK induces metabolic reprograming and subsequently controls redox balance and ROS production [START_REF] Zhao | ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway[END_REF]. Thus, LKB1 is a metabolic regulator which, by activating AMPK and other substrates, controls both glycolysis and oxidative phosphorylation (OXPHOS) as well as ROS formation.

It is also involved in protein metabolism mainly by controlling mTOR pathway, in lipid metabolism, autophagy and mitochondrial functions (biogenesis, degradation, dynamic and fission).

LKB1 establishes and maintains cell polarity

Another process controlled by LKB1 is cell polarity which represents the asymmetric distribution of molecules inside cells. Regulation of cell polarity is evolutionary conserved (Figure 25). The role of LKB1 in cell polarity was discovered for the first time in C. elegans. The Lkb1 ortholog Par4, controls the first asymmetric division of cells during early *** embryogenesis, which leads to the formation of the anterior-posterior axis during embryogenesis (Figure 25) [START_REF] Kemphues | Identification of genes required for cytoplasmic localization in early C. elegans embryos[END_REF][START_REF] Watts | The C. elegans par-4 gene encodes a putative serine-threonine kinase required for establishing embryonic asymmetry[END_REF]. In mammalian cells, LKB1 controls cell polarity of several cell types (Figure 25). For example, Lkb1 expression induces axon formation in hippocampal neurons [START_REF] Shelly | LKB1/STRAD promotes axon initiation during neuronal polarization[END_REF] and in neurons of developing cerebral cortex [START_REF] Barnes | LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons[END_REF]. Lkb1 also establishes and maintains polarity in various cells including pancreatic acinar cells (Hezel et al., 2008), intestinal epithelial cells (Baas et al., 2004a;[START_REF] Shorning | Lkb1 Deficiency Alters Goblet and Paneth Cell Differentiation in the Small Intestine[END_REF], Sertoli cells [START_REF] Tanwar | Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis[END_REF] and hepatocytes [START_REF] Fu | Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway[END_REF]. However, the molecular mechanisms by which LKB1 controls cell polarity are tissue specific (Hezel and Bardeesy, 2008).

Interestingly the capacity of LKB1 to regulate cell polarity in mammalian cells is due to its cytoplasmic functions (Baas et al., 2004a(Baas et al., , 2004b;;[START_REF] Sebbagh | Regulation of LKB1/STRAD localization and function by E-cadherin[END_REF]. Our team has shown that the C-terminal region of LKB1 is essential to control cell polarity of both intestinal epithelial cells and migrating astrocytes via the AMPK pathway [START_REF] Forcet | Functional analysis of Peutz-Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity[END_REF]. Our team also showed that Lkb1-AMPK pathway regulates cranial NCC polarized migration through the sequential activation of the Rho/Rock kinases, phosphorylation of Myosin Regulatory Light Chain (MRLC) and actin dynamic (cf Results Project1.1; (Creuzet et al., 2016)). These results are in line with the literature. It is known that AMPK activates Rock kinase which phosphorylates MRLC through MLC kinase (Figure 26) [START_REF] Miranda | AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells[END_REF]. Moreover, the expression of MRLC phosphomimetic mutant in Drosophila embryos rescued the polarity defects observed after inactivation of ampk or lkb1 [START_REF] Lee | Energy-dependent regulation of cell structure by AMP-activated protein kinase[END_REF]. Another AMPK downstream effector important for cell polarity is cytoplasmic linker protein-170 (CLIP-170) (Figure 26). CLIP-170 is a plus-end tracking protein (+TIP). AMPK phosphorylates directly CLIP-170 on serine 311. Hence, it promotes microtubules dynamic and polymerization which is crucial for cell polarity [START_REF] Nakano | LKB1 and AMP-activated protein kinase: regulators of cell polarity[END_REF].

AMPK is not the only kinase which mediates LKB1 functions in regulating cell polarity.

NUAK1 is also able to enhance MRLC phosphorylation by inhibiting MLC phosphatase [START_REF] Lee | Energy-dependent regulation of cell structure by AMP-activated protein kinase[END_REF]. There are also MAPK, BRSK and SAD-A and B kinases which are mainly associated with epithelial and neuronal polarity (Figures 25,26) [START_REF] Barnes | LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons[END_REF]Courchet et al., 2013;[START_REF] Hurov | The Par-1/MARK family of protein kinases: from polarity to metabolism[END_REF][START_REF] Jansen | LKB1 and AMPK family signaling: the intimate link between cell polarity and energy metabolism[END_REF][START_REF] Kojima | Suppression of tubulin polymerization by the LKB1-microtubule-associated protein/microtubule affinity-regulating kinase signaling[END_REF]. Thus, LKB1 controls both energy metabolism and polarity. However the link between these two processes is not very well characterized. Recently it has been shown that cells submitted to a contractile force stimulate the translocation of LKB1 to the plasma membrane and the LKB1-dependent recruitment of AMPK to E-cadherin cell-cell junctions. Here, AMPK regulates the actomyosin dynamic and therefore cell polarity. This function is dependent on glucose uptake and ATP production [START_REF] Bays | Linking E-cadherin mechanotransduction to cell metabolism through force mediated activation of AMPK[END_REF]. LKB1 phosphorylates kinases like AMPK, MARK and BRSK which directly activate proteins involved in microtubules dynamic such as cytoplasmic linker protein-170 (CLIP-170), Tubulin-associated unit (Tau), Microtubule-Associated Proteins (MAP) 2 and 4. AMPK is also able to control actin dynamic through indirect (dotted line) phosphorylation of Myosin Regulatory Light Chain (MRLC) [START_REF] Nakano | LKB1 and AMP-activated protein kinase: regulators of cell polarity[END_REF].

To conclude, LKB1 has different biological functions including cellular metabolism and polarity control. This metabolic regulator is involved in gluconeogenesis, fatty acid synthesis and oxidation as well as in mitochondrial biogenesis and energy metabolism, mostly by phosphorylating and activating AMPK. It also controls the polarity of cells and their polarized migration in physiological conditions such as neuronal migration and cranial NCC migration or in pathological conditions like cancer cell metastasis.

III.A.4. Role of LKB1 in development

Role of Lkb1 in embryogenesis

Lkb1 full inactivation (Lkb1 -/-) in mice is embryonic lethal at embryonic day 11 (E11) (Figure 27) [START_REF] Jishage | Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis[END_REF][START_REF] Ylikorkala | Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice[END_REF]. Lkb1 -/-embryos developed normally up to E8. At E8.5-E9.5 Lkb1-inactivated mice presented defects in angiogenesis due to elevated levels of VEGF (Vascular endothelial growth factor) and anomalies in head formation.

Embryos exhibited defects also in neural tube closure and an absence of the first branchial arch which is derived from neural crest cells and form head structures at late stages [START_REF] Ylikorkala | Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice[END_REF]. This is a phenotype compatible with a dysgenesis of cranial neural crest cells.

The extra embryonic tissues of Lkb1 -/-embryos, such as yolk sac and placenta, presented also anomalies [START_REF] Ylikorkala | Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice[END_REF]. Therefore, to test if embryonic defects were due to Lkb1 absence and not to extra embryonic tissue deformations, Lkb1 was specifically inactivated in epiblast by using the Mox2-Cre driver [START_REF] Londesborough | LKB1 in endothelial cells is required for angiogenesis and TGFbeta-mediated vascular smooth muscle cell recruitment[END_REF]. These mice mimic the embryonic lethal phenotype of full Lkb1 inactivation, thereby demonstrating the direct role of Lkb1 in embryonic tissue formation.

***

LKB1 regulates the differentiation of human embryonic stem cells (hESC) [START_REF] Lai | LKB1 controls the pluripotent state of human embryonic stem cells[END_REF].These pluripotent cells differentiate in all cell types of human body. LKB1 inactivation in hESC inhibited their differentiation (decreased differentiation markers like Nestin, Runx1, GAT, Sox17) and increased the pluripotency marker genes (for example Oct4 and Nanog). LKB1 controls p21/WAF1 transcription in hESC, as in somatic cells. Thus, in absence of LKB1 p21/WAF1 is downregulated and therefore there is an increased proliferation correlated with a decreased differentiation of these cells.

Other studies have analyzed LKB1 roles during the development and maintenance of specific tissues such as liver, nervous system and skeletal muscles, using different transgenic murine models [START_REF] Ollila | The tumor suppressor kinase LKB1: lessons from mouse models[END_REF]. In the next paragraph, I will present only the role of LKB1 in the nervous system since this is a part of my PhD program.

4.2 Role of Lkb1 in nervous system 4.2.1. Lkb1 is essential for neuronal development

Barnes and his colleagues deleted Lkb1 specifically in cerebral cortex of developing mice, by using Emx::Cre driver (Emx is a protein marker for pyramidal neurons of the cerebral cortex). They observed an absence of axon formation in neurons of studied mice and proposed that Lkb1 is activating BR serine/threonine kinase (BRSK) in neurons which phosphorylates Mitogen-activated proteins (MAP) and affects the cytoskeleton dynamics allowing axon specification [START_REF] Barnes | LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons[END_REF]. In the same year Shelly M. and her coworkers also observed that Lkb1 is essential for neuronal differentiation [START_REF] Shelly | LKB1/STRAD promotes axon initiation during neuronal polarization[END_REF]. Absence of Lkb1 in the spinal cord, hypothalamus and in the endocrine pancreas (Rip2-Cre driver) induced hind-limb paralysis in 7 weeks old mice. The neurons and axons were developed normally. Instead, with age, the lack of Lkb1 promotes demyelination and axon degeneration in spinal cord (Sun et al., 2011). Thus, Lkb1 is essential for both axon formation and maintenance.

Milbrand team also showed that Lkb1 is essential for axonal maintenance through Schwann cell communication. They deleted Lkb1 specifically in Schwann cells of adult mice by using PLP-Cre ERT driver (PLP is a myelin proteolipid protein) and observed a massive axonal loss. The most affected axons were the unmyelinated sensory ones. Thereby, the axonal degeneration was not due to demyelination but to metabolic changes in Schwann cells.

Lkb1-deficient Schwann cells exhibited mitochondrial dysfunction with increased mitochondrial reactive oxygen species (ROS) production and increased lactate levels (Beirowski et al., 2014). These metabolic changes affected neuronal survival.

All these data demonstrate that LKB1 is essential for neurons formation and function as well as for the metabolic dialogue between Schwann cells and neurons which is essential for neuronal survival.

Lkb1 is essential for glial cells development

Shen et al. also deleted Lkb1 specifically in Schwann cells but this time the inactivation was done in developing mice, by using 2'-3' cyclic nucleotide 3' phosphodiesterase CNP::Cre driver, CNP being a myelin-associated enzyme (Shen et al., 2014). With this mouse model the researchers showed that Lkb1 is essential for Schwann cell maturation. Lkb1 is asymmetrically localized in the cytoplasm of Schwann cells, at the interface between these cells and neurons.

Lkb1 phosphorylation on Serine 431 by Protein Kinase A (PKA) attracts Par3, a mediator of cell polarity, in the same compartment and activates the initiation of myelination.

In the same time, Dasgupta lab showed that Lkb1 controls Schwann cells differentiation by another mechanism, via its metabolic regulation capacity. Cellular differentiation is a process which demands a lot of energy and therefore is associated with a metabolic shift from glycolysis to oxidative phosphorylation. Lkb1 loss in Schwann cells decreased oxidative phosphorylation, including the synthesis of citrate which is a Krebs cycle substrate and consequently decreased myelin lipid levels (Pooya et al., 2014).

Altogether, these data highlighted that Lkb1 regulates Schwann cell mitochondrial metabolism and controls their differentiation. Still, the underlying molecular mechanisms are not very clear.

III.A.5. Association of LKB1 mutations with human pathologies

LKB1 was for the first time linked to human disease when germline mutations in one allele of LKB1 were found to be responsible for the Peutz-Jeghers syndrome (PJS; [START_REF] Hemminki | A serine/threonine kinase gene defective in Peutz-Jeghers syndrome[END_REF][START_REF] Jenne | Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase[END_REF][START_REF] Mehenni | Loss of LKB1 kinase activity in Peutz-Jeghers syndrome, and evidence for allelic and locus heterogeneity[END_REF].

PJS is a human autosomal dominant disease characterized by the presence of hamartomatous gastrointestinal polyps, melanin spots on the lips, buccal mucosa, fingers and toes (Figure 28A) and a high predisposition to develop malignant tumors in various organs (Figure 28B) [START_REF] Mcgarrity | Peutz-Jeghers syndrome[END_REF]. The incidence of PJS is estimated to be 1 in 120,000 births [START_REF] Lindor | The concise handbook of family cancer syndromes. Mayo Familial Cancer Program[END_REF].

It has been shown that approximately 80% of individuals with PJS have an LKB1 pathogenic variant, suggesting that LKB1 is the major gene responsible for this pathology [START_REF] Volikos | LKB1 exonic and whole gene deletions are a common cause of Peutz-Jeghers syndrome[END_REF]. Lkb1 monoallelic inactivation in mice (Lkb1+/-) was sufficient for the development of hamartomatus gastrointestinal polyps similar to PJS patient polyps (Bardeesy et al., 2002;[START_REF] Jishage | Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis[END_REF][START_REF] Miyoshi | Gastrointestinal hamartomatous polyposis in Lkb1 heterozygous knockout mice[END_REF][START_REF] Rossi | Induction of cyclooxygenase-2 in a mouse model of Peutz-Jeghers polyposis[END_REF]. These mice developed later gastrointestinal carcinoma due to loss of heterozygosity of Lkb1. Genetic analyses confirmed that loss of Lkb1 wildtype allele is responsible for predisposition of PJS patients to develop cancer [START_REF] Banno | Hereditary gynecological tumors associated with Peutz-Jeghers syndrome (Review)[END_REF][START_REF] Wang | Germline mutations of the LKB1 (STK11) gene in Peutz-Jeghers patients[END_REF]. Additional somatic mutations in other genes such as p53 accelerate the polyposis observed in PJS mouse.

LKB1 somatic mutations have also been described in patients with sporadic cancers and are associated with:

-30% of non-small cell lung cancers [START_REF] Sanchez-Cespedes | Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung[END_REF]. Moreover, it has been observed that LKB1 regulates lung cancer initiation, differentiation and metastasis [START_REF] Ji | LKB1 modulates lung cancer differentiation and metastasis[END_REF].

-20% of cervical cancers [START_REF] Wingo | Somatic LKB1 mutations promote cervical cancer progression[END_REF].

-27% of intraductal papillary mucinous neoplasms [START_REF] Sahin | Loss of Stk11/Lkb1 expression in pancreatic and biliary neoplasms[END_REF].

-10% of cutaneous melanomas [START_REF] Liu | LKB1/STK11 Inactivation Leads to Expansion of a Prometastatic Tumor Subpopulation in Melanoma[END_REF].

In conclusion, as seen in this chapter, the tumor suppressor LKB1 is a metabolic regulator involved in embryonic development and cell fate. It is a master kinase which has 14 substrates members of AMPK-family, being therefore able to regulate a multitude of cellular outcomes. However, mutations of LKB1 are associated so far only with tumor formation: germline mutations are responsible for the human Peutz-Jeghers syndrome whereas somatic mutations are associated with sporadic tumors. Owing to all the cellular processes regulated by LKB1 signaling, we could expect a broader implication of LKB1 in human diseases. To this regard, LKB1 has been proposed as a genetic risk factor for multiple sclerosis [START_REF] Boullerne | A single-nucleotide polymorphism in serine-threonine kinase 11, the gene encoding liver kinase B1, is a risk factor for multiple sclerosis[END_REF]. However so far there are not many studies supporting this hypothesis. [START_REF] Levine | The first 30 years of p53: growing ever more complex[END_REF], being mutated or repressed in more than half of cancers (Figure 29) [START_REF] Brady | p53 at a glance[END_REF]. Its tumor suppressor role was confirmed by the fact that p53 null mice display a totally penetrant cancer phenotype [START_REF] Kenzelmann Broz | In vivo analysis of p53 tumor suppressor function using genetically engineered mouse models[END_REF]. Inheritance of mutant p53 allele predisposes humans to the Li-Fraumeni cancer syndrome [START_REF] Olivier | TP53 mutations in human cancers: origins, consequences, and clinical use[END_REF]. p53 prevents cancer development by multiple manners, however its exact contributions to embryonic development are less clear. In this manuscript I will focus mainly on p53 functions during embryogenesis.

During my PhD, I initiated the characterization of the link between p53 and LKB1 signalings and its importance in pathologies due to neural crest cells (NCC) defects.

Therefore, the guideline of this chapter is the known connections between p53 & energy metabolism and p53 & NCC development. The bibliographic data that I will further present regarding p53 are not exhaustive. After a brief description of p53 structure and activation by cellular stresses, I will succinctly report an overview of some mechanistic regulations exerted by p53 in various cellular responses such as cell cycle arrest, metabolic reprogramming, autophagy and differentiation. I will then describe how p53 contributes to embryo development and neural crest cells in particular and how p53 deregulations are associated with neurocristopathies and ribosomopathies.

III.B.1. Structure and activation of the p53 protein

p53 is a transcription factor composed of 393 amino-acids organized in several domains (Figure 30) as indicated below from the N-terminal to the C-terminal part of the protein [START_REF] Hainaut | p53 and human cancer: the first ten thousand mutations[END_REF][START_REF] Joerger | The Tumor Suppressor p53: From Structures to Drug Discovery[END_REF]:

-two transactivation domains (TAD): TAD1 (residues 1-42) and TAD2 (43-73) -a prolin-rich region ( 61-94) -a DNA-binding domain (102-292) -a flexible linker region (300-318)

-a tetramerization domain (326-355) which contains a regulatory domain.

-a basic domain p53 is localized in both cytoplasm and nucleus, since it contains :

-two nuclear export signals (NES), one located within the C-terminal oligomerization domain and the other within the N-terminal TDA1 domain [START_REF] Stommel | A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking[END_REF]Zhang andXiong, 2001a, 2001b) (Figure 30).

-three nuclear localization signals (NLS), one located just before the oligomerization domain (316-325) and two minor NLS which reside in basic domain [START_REF] Shaulsky | Subcellular distribution of the p53 protein during the cell cycle of Balb/c 3T3 cells[END_REF]) (Figure 30).

p53 has a short half-life, around 30 minutes in most cells and tissues, therefore, its basal level is low and often undetectable [START_REF] Stommel | Accelerated MDM2 auto-degradation induced by DNAdamage kinases is required for p53 activation[END_REF]2005); Hainaut and Wiman, 2005). This low level of p53 is regulated by several E3 ubiquitin ligases (Figure 31) including Mdm2 [START_REF] Haupt | Mdm2 promotes the rapid degradation of p53[END_REF][START_REF] Kubbutat | Regulation of p53 stability by Mdm2[END_REF], COP1 [START_REF] Dornan | The ubiquitin ligase COP1 is a critical negative regulator of p53[END_REF], Pirh2 [START_REF] Leng | Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation[END_REF][START_REF] Sheng | Molecular basis of Pirh2-mediated p53 ubiquitylation[END_REF] and ARF-BP1. All these regulators control p53 ubiquitination and consequently p53 degradation via 26S proteasome. Interestingly, p53 activates the expression level of genes encoding the E3 ubiquitin ligases thereby establishing a negative feedback loop (Figure 31) [START_REF] Brooks | p53 ubiquitination: Mdm2 and beyond[END_REF][START_REF] Hollstein | Massively regulated genes: the example of TP53[END_REF]Hainaut and Wiman, 2005).

In the following paragraph, I focused on Mdm2 as it is the most studied p53 regulator and it is essential during embryogenesis.

Regulation of p53 by ubiquitination

Mdm2 in mammals (Hdm2 in human) is an E3-ubiquitin ligase that covalently binds one or several ubiquitin proteins on lysine residues. It regulates p53 via two key mechanisms:

(i) first it interacts with p53 TAD thereby blocking p53-dependent transcription [START_REF] Chen | Mapping of the p53 and mdm-2 interaction domains[END_REF][START_REF] Minsky | The RING Domain of Mdm2 Mediates Histone Ubiquitylation and Transcriptional Repression[END_REF][START_REF] Momand | The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation[END_REF][START_REF] Nag | The MDM2-p53 pathway revisited[END_REF].

(ii) second, it promotes p53 degradation by ubiquitination-mediated proteolysis [START_REF] Haupt | Mdm2 promotes the rapid degradation of p53[END_REF][START_REF] Kubbutat | Regulation of p53 stability by Mdm2[END_REF][START_REF] Wade | The p53 orchestra: Mdm2 and Mdmx set the tone[END_REF] (Figure 32). Mdm2 mono-or poly-ubiquitinates p53 and this is essential for p53 function: mono-ubiquitination leads to p53 nuclear export and mitochondrial translocation [START_REF] Marchenko | The role of ubiquitination in the direct mitochondrial death program of p53[END_REF][START_REF] Marchenko | Stress-mediated nuclear stabilization of p53 is regulated by ubiquitination and importin-alpha3 binding[END_REF] while polyubiquitination induces its degradation (Figure 32) [START_REF] Li | Mono-versus polyubiquitination: differential control of p53 fate by Mdm2[END_REF]. The balance between these two types of ubiquitination depends on Mdm2 levels: increased Mdm2 levels promote p53 polyubiquitination as low Mdm2 levels mediate mono-ubiquitination [START_REF] Li | Mono-versus polyubiquitination: differential control of p53 fate by Mdm2[END_REF]. However, other studies suggest that the interaction of Mdm2 with different proteins such as p300, Gankyrin, Kap1 and Sival regulates Mdm2 to perform poly-ubiquitination [START_REF] Du | Suppression of p53 activity by Siva1[END_REF][START_REF] Grossman | Polyubiquitination of p53 by a ubiquitin ligase activity of p300[END_REF][START_REF] Higashitsuji | The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53[END_REF][START_REF] Jain | Making sense of ubiquitin ligases that regulate p53[END_REF][START_REF] Sui | Yin Yang 1 is a negative regulator of p53[END_REF][START_REF] Wang | MDM2 interaction with nuclear corepressor KAP1 contributes to p53 inactivation[END_REF]. p53 half-life is controlled by several E3 ubiquitin ligases. There are different negative feedback loops, including Mdm2, Pirh2, COP1 which regulate p53 ubiquitination and are regulated by p53 at the transcriptional level [START_REF] Hollstein | Massively regulated genes: the example of TP53[END_REF] Proteasomes of both nucleus and cytoplasmic compartments are able to degrade polyubiquitinated p53 but with a preference for nuclear p53 turnover as p53, Mdm2 and Mdm4 (Mdmx) colocalize in nucleus. Mdm2 in presence or not of Mdm4 (Mdmx) poly-ubiquitinates p53 which is degraded by proteasome. Mdm2 also mono-ubiquitinates p53 which promotes its nuclear export and its translocation to mitochondria. Mdm4 (Mdmx) is also able to bind p53 and renders the p53 transcriptionally inactive [START_REF] Jain | Making sense of ubiquitin ligases that regulate p53[END_REF].

Mdm4, also known as Mdmx, is another regulator of p53 stability, but not directly since it is not an E3 ubiquitin ligase (Figure 32) [START_REF] Stad | Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms[END_REF]. Mdm4 forms a heteromeric complex with Mdm2, stabilizes Mdm2 and promotes Mdm2-mediated degradation of p53 [START_REF] Gu | Mutual dependence of MDM2 and MDMX in their functional inactivation of p53[END_REF]. Similarly to Mdm2, it also binds to p53 and inhibits its transcriptional activity.

Mdm4 can also be recruited to p53 target gene promoters where it binds to p53 TAD and thereby blocks the ability of p53 to interact with the transcriptional machinery [START_REF] Jain | Making sense of ubiquitin ligases that regulate p53[END_REF].

p53 stabilization and activation upon stress

Many cellular stresses are responsible for p53 stabilization and activation [START_REF] Bálint | Activation and activities of the p53 tumour suppressor protein[END_REF]. Various post translational modifications regulate p53 stability and function such as ubiquitination, phosphorylation, acetylation and methylation.

Ubiquitination, as previously described, is associated to p53 degradation and mitochondrial trafficking. In response to many stress signals, p53 is accumulated partly via Mdm2 and Mdm4 inhibition. Upon DNA damage, Mdm2 mediates its own ubiquitination as well as ubiquitination of its partner Mdm4 leading to their inhibition [START_REF] Kawai | DNA damageinduced MDMX degradation is mediated by MDM2[END_REF][START_REF] Pan | MDM2 promotes ubiquitination and degradation of MDMX[END_REF][START_REF] Stommel | Accelerated MDM2 auto-degradation induced by DNAdamage kinases is required for p53 activation[END_REF]. Other stress signals can induce different post translational modifications of both Mdm2 and p53 which perturb the Mdm2-p53 interaction and thereby stabilize p53. Ribosomal stress also inhibits Mdm2 and Mdm4 and promotes p53 activation. It has been shown that several mutated ribosomal proteins such as RPL5, RPL11

and RPL23 (for ribosome proteins functions see chapter II.3.2) interact with the acidic domain of Mdm2 and blocks the ubiquitination of p53 [START_REF] Gilkes | MDMX regulation of p53 response to ribosomal stress[END_REF].

Phosphorylation, methylation and acetylation of p53 are mostly associated to its accumulation and activation [START_REF] Bode | Post-translational modification of p53 in tumorigenesis[END_REF][START_REF] Chuikov | Regulation of p53 activity through lysine methylation[END_REF][START_REF] Gu | Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain[END_REF][START_REF] Ito | MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation[END_REF][START_REF] Lake | Protein methylation and DNA repair[END_REF]. For example, ionizing radiations activate DNAdependent protein kinase (DNA-PK) [START_REF] Morozov | DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps[END_REF] or ataxia telangiectasia mutated (ATM) [START_REF] Morgan | p53 and ATM: cell cycle, cell death, and cancer[END_REF] which phosphorylate p53 on serine 15. This serine is localized in the Mdm2-binding domain, therefore its phosphorylation disrupts Mdm2-p53 binding and stabilizes p53. UV exposure activates Chk1 and Chk2 which phosphorylate p53

on Serine 20 and therefore also perturbs MDM2-p53 binding [START_REF] Chehab | Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53[END_REF]. Genotoxic factors such as UV and ionizing radiations promote ROS formation thereby triggering oxidative DNA damage. ROS levels also regulate p53 stability by inducing its phosphorylation on serine 15 through activation of several kinases like p38αMAPK [START_REF] Bragado | Apoptosis by cisplatin requires p53 mediated p38alpha MAPK activation through ROS generation[END_REF], ATM [START_REF] Kurz | DNA damage-induced activation of ATM and ATM-dependent signaling pathways[END_REF] and ERK [START_REF] Persons | Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin[END_REF] and as described above this phosphorylation affects Mdm2-p53 binding thus inducing p53 accumulation and activation.

III.B.2. p53 biological functions

p53 is known to possess several biological functions, the most characterized one being its transcription factor capacity. Thus, p53 acts principally as an activator or repressor of genes expression, being therefore able to dictate several cellular outcomes such as cell cycle arrest, autophagy, energy metabolism and cellular differentiation [START_REF] Brown | Awakening guardian angels: drugging the p53 pathway[END_REF][START_REF] Loughery | Switching on p53: an essential role for protein phosphorylation?[END_REF]. In addition to its transcription-dependent functions, p53 has also cytoplasmic, independent transcription functions such as apoptosis activation and autophagy inhibition [START_REF] Green | Cytoplasmic functions of the tumour suppressor p53[END_REF]. Further I will briefly describe only some p53 biological functions which, as I will present later (Chapter III.B 3), are essential for embryonic development.

Cell-cycle arrest

p53, also known as the guardian of the genome, is capable of detecting anomalies in cell growth and in regulating genes expression mediating cell cycle arrest, in order to help the cell repair before division. p53 regulates many genes involved in this cellular process, but here I present only two cell cycle regulator-genes known to be involved also in neural crest development (see Chapter III.B.4).

The first p53 target gene identified was p21 which encodes for cyclin-dependent kinase inhibitor 1 responsible for G1 cell cycle arrest [START_REF] El-Deiry | Definition of a consensus binding site for p53[END_REF][START_REF] El-Deiry | p21(WAF1) Mediates Cell-Cycle Inhibition, Relevant to Cancer Suppression and Therapy[END_REF][START_REF] Harper | The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases[END_REF]. Several studies have shown that p53 capacity to upregulate p21 expression and thereby to block cell cycle is essential for its tumor suppressor activity [START_REF] Lozano | Mouse Models of p53 Functions[END_REF].

Interestingly, nuclear Lkb1 is also modulating p21 expression and consequently the cell cycle arrest via physical interaction with p53 [START_REF] Zeng | LKB1 is recruited to the p21/WAF1 promoter by p53 to mediate transcriptional activation[END_REF] and its downstream kinases either AMPK [START_REF] Bungard | Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation[END_REF] or NUAK2 [START_REF] Hou | A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation[END_REF] on the promoter regions of p21. However the direct phosphorylation of NUAK and AMPK by p53 remains controversial.

CyclinG1 (Ccng1) is another p53 target gene that causes cell-cycle arrest [START_REF] Okamoto | Cyclin G is a transcriptional target of the p53 tumor suppressor protein[END_REF][START_REF] Zauberman | A functional p53-responsive intronic promoter is contained within the human mdm2 gene[END_REF] and inhibits cell growth [START_REF] Zhao | Cyclin G1 Has Growth Inhibitory Activity Linked to the ARF-Mdm2-p53 and pRb Tumor Suppressor Pathways1 1 DEQ from the American Cancer Society (RSG-98-254-04-MGO) and NIH (RO1 CA90367)[END_REF]. Upon DNA damage or other cellular stresses, cyclinG1 promotes G2/M arrest [START_REF] Kimura | Cyclin G1 is involved in G2/M arrest in response to DNA damage and in growth control after damage recovery[END_REF].

Bungard et al. have shown that the nuclear complex Lkb1, AMPK, p53 is also essential for cyclinG transcription [START_REF] Bungard | Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation[END_REF].

Metabolism

As presented in the first chapter, cellular metabolism is a fundamental process involved in embryonic development. The most recent characterized ability of p53 is its capacity to mediate metabolic changes in cells. Depending on the type of stress, p53 modulates the metabolism via different mechanisms [START_REF] Berkers | Metabolic regulation by p53 family members[END_REF]Kruiswijk et al., 2015):

-it interacts with AMPK and mTOR -it regulates mitochondrial mass and activity -it controls lipid metabolism

As energy metabolism has been the guideline of my PhD project, I focused on the interaction of p53 with AMPK/mTOR pathway as well as its involvement in glycolysis and oxidative phosphorylation. Indeed, p53 controls glycolysis and oxidative phosphorylation by activating or inhibiting different regulators of these two processes.

v Crosstalk with AMPK/mTOR signaling pathway

Upon genotoxic stress, p53 inhibits mTORC1 in order to limit cell proliferation, growth and energy consumption. This inhibition relies on (Figure 33):

-transcriptional upregulation by p53 of Sestrins, a family of highly conserved stress-responsive proteins that protect cells from oxidative stress (Budanov andKarin, 2008a, 2008b). Sestrins activate AMPK [START_REF] Budanov | Stressin' Sestrins take an aging fight[END_REF][START_REF] Budanov | p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling[END_REF] to inhibit mTOR [START_REF] Feng | The coordinate regulation of the p53 and mTOR pathways in cells[END_REF].

-transcriptional upregulation of mTOR inhibitors such as AMPKβ, TSC2 among others [START_REF] Feng | The regulation of AMPK beta1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR pathways[END_REF]. Of note, LKB1 expression is also controlled by p53 [START_REF] Pappas | p53 Maintains Baseline Expression of Multiple Tumor Suppressor Genes[END_REF].

This capacity of p53 to regulate AMPK activity or expression and thereby to inhibit mTOR pathway was also observed in nongenotoxic conditions [START_REF] Drakos | Stabilization and activation of p53 downregulates mTOR signaling through AMPK in mantle cell lymphoma[END_REF].

v p53 regulates aerobic glycolysis p53 regulates glucose flux to pyruvate by either down-or up-regulating gene expression of glycolytic effectors that intervene at various steps of the glycolysis (Figure 34).

p53 downregulates the expression of : ( [START_REF] Cheung | Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death[END_REF][START_REF] Ruiz-Lozano | p53 is a transcriptional activator of the muscle-specific phosphoglycerate mutase gene and contributes in vivo to the control of its cardiac expression[END_REF][START_REF] Mathupala | Glucose catabolism in cancer cells. The type II hexokinase promoter contains functionally active response elements for the tumor suppressor p53[END_REF]. So far, this dual role of p53 in glycolysis is not well documented. Several studies suggest that the glycolytic regulation by p53 is tissue and context specific [START_REF] Maddocks | Metabolic regulation by p53[END_REF][START_REF] Vousden | p53 and metabolism[END_REF].

v p53 regulates mitochondrial and TCA activities p53 protects mitochondrial integrity, increases mitochondrial mass [START_REF] Kitamura | Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria[END_REF][START_REF] Lebedeva | Loss of p53 causes mitochondrial DNA depletion and altered mitochondrial reactive oxygen species homeostasis[END_REF][START_REF] Wang | p53 and mitochondrial function in neurons[END_REF] and enhances oxidative phosphorylation (OXPHOS).

Firstly, to increase OXPHOS, p53 regulates the activity of protein complexes of the mitochondrial electron transport chain (Figure 34) by directly upregulating the expression of apoptosis inducing factor (AIF) and synthesis of cytochrome C oxidase 2 (SCO2) which are important for the function of complexes I and IV respectively [START_REF] Matoba | p53 regulates mitochondrial respiration[END_REF][START_REF] Stambolsky | Regulation of AIF expression by p53[END_REF].

Secondly, p53 increases the TCA flux (Figure 34) by:

-positively regulating pyruvate dehydrogenase (PDH) levels through inhibition of PDH kinase 2 (PDK2) (Contractor T and Harris, 2012) or through activation of Parkinson Protein 2 (PARK2) [START_REF] Zhang | Parkin, a p53 target gene, mediates the role of p53 in glucose metabolism and the Warburg effect[END_REF].

positively regulating α-ketoglutarate (αKG) levels via the mitochondrial enzyme Glutaminase 2 (GLS2) which catalyzes the conversion of glutamine into glutamate that is catabolized into αKG and ammonia [START_REF] Hu | Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function[END_REF][START_REF] Suzuki | Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species[END_REF].

To conclude, p53 is involved in several aspects of energy metabolism, including downregulation of glycolysis and upregulation of mitochondrial respiration, as well as the control of AMPK/mTOR pathway essential for cell growth and the regulation of ATP pool inside the cells by activating catabolic reactions and inhibiting anabolic ones.

Autophagy

p53 regulates metabolism also by controlling autophagy, a cytoplasmic catabolic process (see Chapter IV.1.3). Depending on its subcellular localization, p53 has opposite effects on autophagy: cytoplasmic p53 turns autophagy off [START_REF] Morselli | Mutant p53 protein localized in the cytoplasm inhibits autophagy[END_REF]Tasdemir et al., 2008) whereas nuclear p53 turns autophagy on by positively regulating autophagy gene expressions such as Damage-Regulated Autophagy Modulator (DRAM) [START_REF] Balaburski | p53 and ARF: unexpected players in autophagy[END_REF][START_REF] Crighton | DRAM, a p53-induced modulator of autophagy, is critical for apoptosis[END_REF][START_REF] Levine | p53: The Janus of autophagy?[END_REF][START_REF] Tasdemir | Regulation of autophagy by cytoplasmic p53[END_REF].

As both nuclear p53 and inhibition of cytoplasmic p53 can induce autophagy, how does the cell "decide" between autophagy induction by cytoplasmic p53 inhibition or by nuclear p53?

The answer to this question could depend of the type of cellular stress. Autophagy is induced in response to various stress stimuli such as starvation, endoplasmic reticulum (ER) stress, DNA damage and oxidative stress (Figure 35) [START_REF] Kuma | The role of autophagy during the early neonatal starvation period[END_REF][START_REF] Shimizu | Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes[END_REF][START_REF] Yu | Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8[END_REF].

ER stress induces autophagy by promoting cytoplasmic translocation of p53 and its subsequent degradation by Mdm2 ubiquitination-mediated proteolysis [START_REF] Fabbro | Regulation of tumor suppressors by nuclear-cytoplasmic shuttling[END_REF][START_REF] Pluquet | Endoplasmic reticulum stress accelerates p53 degradation by the cooperative actions of Hdm2 and glycogen synthase kinase 3beta[END_REF][START_REF] Qu | Endoplasmic reticulum stress induces p53 cytoplasmic localization and prevents p53-dependent apoptosis by a pathway involving glycogen synthase kinase-3beta[END_REF][START_REF] Tasdemir | Regulation of autophagy by cytoplasmic p53[END_REF]. This cytoplasmic localization of p53 induced by ER stress is regulated by posttranslational modifications in the nuclear localization signal (NLS) such as phosphorylation of serines 315 and 376 [START_REF] Pluquet | Endoplasmic reticulum stress accelerates p53 degradation by the cooperative actions of Hdm2 and glycogen synthase kinase 3beta[END_REF][START_REF] Qu | Endoplasmic reticulum stress induces p53 cytoplasmic localization and prevents p53-dependent apoptosis by a pathway involving glycogen synthase kinase-3beta[END_REF].

On the other hand, other stimuli such as DNA damage and oxidative stress stabilize and activate p53 and enhance its nuclear functions, regulating positively the autophagy in a transcriptional dependent and independent manner (Figure 35) [START_REF] Feng | The coordinate regulation of the p53 and mTOR pathways in cells[END_REF][START_REF] Levine | p53: The Janus of autophagy?[END_REF][START_REF] Zeng | A novel role for DNA mismatch repair and the autophagic processing of chemotherapy drugs in human tumor cells[END_REF].

It is known that autophagy is essential for physiological processes such as preimplantation development, cell differentiation during adipogenesis, lymphogenesis and erythropoiesis [START_REF] Mizushima | Autophagy in mammalian development and differentiation[END_REF] and Schwann cell maturation because it properly reduces the cytoplasm of Schwann cells during myelination in the peripheral nervous system [START_REF] Jang | Autophagic myelin destruction by Schwann cells during Wallerian degeneration and segmental demyelination[END_REF]. Instead little is known about p53-dependent induction of autophagy during these processes. Thus, the capacity of p53 to modulate autophagy in a context-dependent manner might be essential to drive the rapid cellular changes necessary for proper differentiation and development.

To conclude, the link between p53 and autophagy is complex given that p53 is able to activate or suppress autophagy depending on the type of stress and its subcellular localization.

Cell differentiation

Another physiological process regulated by p53 is cell differentiation. Again, p53 has a dual role, inhibiting or promoting differentiation, depending on cell type and the specific differentiation program [START_REF] Molchadsky | p53 Plays a Role in Mesenchymal Differentiation Programs, in a Cell Fate Dependent Manner[END_REF].

For example, it has been shown that p53 accumulation and activation blocks muscle differentiation by directly binding to myogenin promoter and suppressing its transcription [START_REF] Yang | p53 suppresses muscle differentiation at the myogenin step in response to genotoxic stress[END_REF] and inhibits osteoblast differentiation by downregulating Runx2 and Osterix gene expression, two transcription factors essential for the mesenchymal stem cells commitment into osteoprogenitor lineage [START_REF] Lengner | Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling[END_REF][START_REF] Wang | p53 functions as a negative regulator of osteoblastogenesis, osteoblastdependent osteoclastogenesis, and bone remodeling[END_REF][START_REF] Zambetti | Skeletons in the p53 tumor suppressor closet: genetic evidence that p53 blocks bone differentiation and development[END_REF].

Later, Molchadsky et al. confirmed this inhibitory role of p53 but he also showed that p53 is able to positively regulate both myogenic and osteogenic differentiation of skeletal muscle cells [START_REF] Molchadsky | p53 Plays a Role in Mesenchymal Differentiation Programs, in a Cell Fate Dependent Manner[END_REF].

Thus, p53 is a metabolic regulator having a dual function regarding cell fate and it has been suggested that p53 acts as a "guardian of differentiation".

III.B.3 Role of p53 in development

Although most research on p53 focused on its tumor suppressor activity, several studies underlined the importance of p53 during development [START_REF] Almog | Involvement of p53 in cell differentiation and development[END_REF][START_REF] Brady | p53 at a glance[END_REF][START_REF] Hainaut | p53 and human cancer: the first ten thousand mutations[END_REF][START_REF] Lozano | Mouse Models of p53 Functions[END_REF].These studies suggest that p53 levels are important during embryogenesis since both loss or stabilization of p53 are responsible for developmental defects (Figure 36).

About 8-16% mouse embryos (mostly females) which are not expressing p53 exhibit defects in neural tube closure with an overgrowth of neural tissue in the midbrain region, an affection known as exencephaly [START_REF] Sah | A subset of p53-deficient embryos exhibit exencephaly[END_REF]. In parallel with exencephaly, affected mice also exhibit craniofacial malformations like ocular defects and anomalies in upper incisor tooth formation [START_REF] Armstrong | High-frequency developmental abnormalities in p53-deficient mice[END_REF]. However, the capacity of p53 to regulate mammalian embryogenesis remains controversial because the majority of p53-null mice embryos are developing normally and their only phenotype is a high risk to form tumors [START_REF] Donehower | Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours[END_REF][START_REF] Sah | A subset of p53-deficient embryos exhibit exencephaly[END_REF]. Absence of developmental defects can be due to the presence of two others p53 family members, p63 and p73 which have a similar structure to p53, are also expressed during early embryogenesis and are likely to have a compensatory effect for the loss of p53 [START_REF] Stiewe | The p53 family in differentiation and tumorigenesis[END_REF]. Even if they are expressed in early embryos, these two proteins are more involved in central nervous system development. p63 nullizygosity is perinatal lethal with defects in epithelium stratification, limb and craniofacial development and skeletal defects [START_REF] Mills | p63 is a p53 homologue required for limb and epidermal morphogenesis[END_REF][START_REF] Yang | p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development[END_REF]. p73 nullizygosity is lethal postnatally. The mice die several weeks after birth and until then they have a complex phenotype: reduced size, hippocampal dysgenesis, hydrocephalus, chronic infections, inflammation and pheromone sensing defects that impede breeding [START_REF] Yang | p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours[END_REF].

Recently, several labs have shown that combined loss of p53/p63, p53/p73 or p63/p73 have the same developmental defects than those described in single knockout embryos [START_REF] Lee | Evidence that DeltaNp73 promotes neuronal survival by p53-dependent and p53-independent mechanisms[END_REF][START_REF] Nemajerova | Targeted deletion of p73 in mice reveals its role in T cell development and lymphomagenesis[END_REF][START_REF] Nemajerova | TAp73 is a central transcriptional regulator of airway multiciliogenesis[END_REF][START_REF] Van Nostrand | The p53 family members have distinct roles during mammalian embryonic development[END_REF]. These results suggest that p53 family members have different roles during mammalian embryonic development.

In order to better analyze the possible compensatory pathways, Attardi lab generated p53, p63, p73 triple knockout mice (TKO). These TKO embryos are viable and showed no dramatic developmental defects compared with single knockout embryos [START_REF] Van Nostrand | The p53 family members have distinct roles during mammalian embryonic development[END_REF]. Instead, Wang et al., developed a TKO mouse model using CRISPR/Cas9 and observed that it promotes early defects in embryo development, starting at E7.5, due to an absence of mesendodermal differentiation from pluripotent embryonic stem cells. According to their model, these three protein members of p53 family have an important and redundant role during early embryo development [START_REF] Okuda | Discovery of a new role for the p53 family in the onset of mesendodermal differentiation of embryonic stem cells[END_REF][START_REF] Wang | The p53 Family Coordinates Wnt and Nodal Inputs in Mesendodermal Differentiation of Embryonic Stem Cells[END_REF]. L. Attardi suggested that the discrepancy between the two studies may relate to variations in genetic background of the mice or to a different time course of compensatory pathways upregulation due to the two different genetic technics used for p53, p63 and p73 inactivation.

As mentioned previously, not only p53 inactivation induces embryonic defects but also p53 stabilization and activation during development lead to embryonic lethality. When p53 is accumulated and activated in murine models, due to Mdm2 or Mdm4 inactivation, mice die at E3.5 or E7.5 respectively and this phenotype is fully rescued upon p53 loss [START_REF] Chavez-Reyes | Switching mechanisms of cell death in mdm2-and mdm4-null mice by deletion of p53 downstream targets[END_REF][START_REF] Jones | Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53[END_REF][START_REF] Migliorini | Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development[END_REF][START_REF] Montes De Oca Luna | Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53[END_REF][START_REF] Parant | Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53[END_REF]. In normal conditions p53 half-life is very short: 30 minutes in undifferentiated ESC and only 10 minutes in more differentiated cells [START_REF] Zhang | Parkin, a p53 target gene, mediates the role of p53 in glucose metabolism and the Warburg effect[END_REF].

Thus, p53 dosage and activity are particularly important for embryonic and tissue development.

Furthermore, several studies emphasized that p53 is essential during NCC ontogeny and defective p53 signaling is associated with neurocristopathies and ribosomopathies.

III.B.4. Role of p53 in neural crest cell formation

It is known that NCC proliferation is essential to fine-tune epithelium-mesenchymal transition (EMT)-driven delamination from neural tube. p53 regulates cranial NCC proliferation by controlling p21 and CyclinG1 expression (Rinon et al., 2011).

By using a chick embryo model, it has been shown that p53 is expressed in neural plate and later in cranial NCC progenitors and its expression decreases when cells undergo an EMT (Figure 37A). In line with this study, in Mdm4 knockout mice, p53 accumulation promoted cell cycle arrest which leads to embryonic lethality at E7.5 [START_REF] Chavez-Reyes | Switching mechanisms of cell death in mdm2-and mdm4-null mice by deletion of p53 downstream targets[END_REF].

Mdm2 is preferentially expressed in neural folds and at E8-9 it is expressed in migratory NCC in head region, in migratory NCC in the tail region and in the first three branchial arches.

At E9.5 Mdm2 becomes ubiquitously expressed [START_REF] Daujat | MDM2: life without p53[END_REF][START_REF] Jain | Making sense of ubiquitin ligases that regulate p53[END_REF].

Thus, at early stages Mdm2 expression may be essential for NCC migration. Mdm2 deletion, as Mdm4, may lead to an accumulation of p53 in NCC that could block their migration.

Hence, it has been shown that p53 accumulation and activation are responsible for disorders due to NCC defects like Treacher Collins syndrome and CHARGE syndrome (Table 8), among others neurocristopathies and ribosomopathies.

Co-expression of one p53 allele with mutations in both TAD1 and 2 (mutant p53 25, 26, 53, 54 ) along with a wild-type p53 allele in mice leads to a phenotype of CHARGE syndrome.

Mutation of residues 25 and 26 in TAD1 of p53 protein blocked the interaction of p53 with Mdm2 and thereby stabilized mutant p53. This mutant interacts, stabilizes and activates wild type p53 thereby triggering the CHARGE syndrome (Van Nostrand et al., 2014a).

Wild type p53 is accumulated in CHARGE syndrome also as a consequence of Chd7 mutations (Van Nostrand and Attardi, 2014). It has been suggested that Chd7 regulates p53 directly by binding to p53 promoter region and inhibiting its transcription. Chd7 also regulates p53 indirectly by inducing p53 post translational modifications or via ribosomal biogenesis defects which perturb Mdm2-p53 interaction and promote p53 stabilization (Van Nostrand et al., 2014). p53 inactivation in Chd7-deficient mice rescued their embryonic phenotype (Table 8) (Van Nostrand and Attardi, 2014).

By using a Treacher Collins syndrome (TCS) mice model (Tcof1 +/-) it has been shown that p53 is stabilized and activated in neuroepithelium and in premigratory cranial NCC and promotes craniofacial malformations (Jones et al., 2008). This accumulation of p53 increases CyclinG1 expression and promotes cell cycle arrest of cranial NCC progenitors (Jones et al., 2008). These observations support results from Rinon et al. presented above (Rinon et al., 2011).

Treacle, the protein encoded by Tcof1 is important for DNA damage repair. Treacle is ubiquitously expressed in all stages of embryo development but its expression is increased in neuroepithelium and pre-migratory cranial NCC at E8.5-E10.5. This period of time and cellular localization also correlates with an increase in ROS levels (Sakai and Trainor, 2016).

Thus, when Treacle is mutated or inactivated in TCS syndrome, the neuroepithelium and cranial NCC have high levels of ROS and DNA damage which activate p53 and promote apoptosis (Sakai et al., 2016). This apoptotic elimination of cranial NCC is the cause of TCS craniofacial malformations.

Limiting ROS formation and consequently oxidative DNA damage via treatment with antioxidants such as N-acetyl cysteine (NAC) decreases p53 activation and rescues craniofacial malformations (Sakai et al., 2016). Strikingly, p53 inactivation in Tcof1 +/-mice rescues the phenotype (Table 8) (Sakai and Trainor, 2016;[START_REF] Bursac | Activation of the tumor suppressor p53 upon impairment of ribosome biogenesis[END_REF]Van Nostrand and Attardi, 2014).

p53 mutants are also able to promote p53 accumulation and activation and lead to ribosomopathies. Homozygous expression in mice of p53 Δ31 mutant, which does not have a functionnal negative regulatory domain localized in the C-terminal region and thus leads to increased p53 activity, triggers a phenotype similar to the ribosomopathy Dyskeratosis congenita [START_REF] Simeonova | Mutant mice lacking the p53 C-terminal domain model telomere syndromes[END_REF].

Collectively, these data suggest an important role of p53 responses in the pathogenesis of ribosomo/neurocristopathies. Genetic engineered mouse models with alteration of genes causatives of neural crest cells (NCC) defects were bred with mouse models of p53 inactivation. p53 tm1TyJ allele: neomycin cassette replacing exons2-6 (Jacks et al., 1994). p53 tmBrd allele: pol II-neomycin cassette inserted in exon 5 with small deletions in exons 4 and 5 [START_REF] Donehower | Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours[END_REF]. NTD, neural tube closure defects; OFT, heart ventricular out flow tract.

IV. LYSOSOMES: a signaling hub to maintain cell metabolism and homeostasis

In the previous chapter, we have seen that both metabolic regulators p53 and LKB1 are able to control AMPK and mTOR pathways. In the past years, it has been shown that lysosomes are not only important for macromolecules degradation, recycling and material storage but are also essential for the regulation of AMPK and mTOR signaling pathways and therefore are involved in several cellular processes. In this chapter I will describe the processes underlying lysosomal biogenesis. I will then detail the cellular functions attributed to lysosomes and I will focus on their capacity to regulate signaling pathways. Further, I will present how the microenvironment can affect lysosomal homeostasis and what are the consequences of lysosomal dysfunctions.

Lysosomes: multifunctional organelles required for recycling and clearance

Historical context

Lysosomes were discovered in 1955 from rat liver fraction studies. At first, C. de Duve lab observed that an enzyme, acid phosphatase, is enclosed within a special type of cytoplasmic granules upon starvation stress. Next, they searched for liver enzymes that, like acid phosphatase, were concentrated in these granules and they found five other liver enzymes: glucuronidase, cathepsin D, ribonuclease, deoxyribonuclease and urate oxidase.

The presence, in the same membrane-limited pockets, of five acid hydrolases which have different substrates strongly suggested that these organelles are involved in intracellular digestion [START_REF] De Duve | The lysosome turns fifty[END_REF][START_REF] De Duve | Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue[END_REF].This discovery led de Duve to name these structures lysosomes, derived from the greek for "digestive body".

Nowadays, we know that lysosomes contain more than 50 acid hydrolases that can degrade proteins, lipids, polysaccharides, RNA and DNA.

Lysosome biogenesis

One important biological function for lysosomes is the degradation of material taken up from outside the cell by endocytosis. Endocytosis is not only crucial for lysosome function but also during lysosome formation. Material from outside the cell is taken up in clathrin-coated endocytic vesicles which fuse with early endosomes, the primary sorting station in the endocytic pathway. The early endosomes gradually mature into late endosomes, which are the precursors to lysosomes. During their formation, lysosomes will be progressively charged with hydrolases as these enzymes are transported into clathrin-coated vesicles from trans Golgi to the lysosome. One of the important modifications during endosome maturation is the pH acidification to 5.5 which is made possible due to the presence of vacuolar H + -ATPase at the membrane of the lysosome. Indeed, this protein will pump the protons inside the lysosome thus acidifying the lysosome lumen. Such an acidification is essential for hydrolases activity [START_REF] Cooper | The Cell[END_REF].

Classical lysosomal functions

These acidic vesicles are involved in several cellular processes such as:

• Macroautophagy, in this manuscript referred to as autophagy (digestion of misfolded or long-live proteins, organelles and other material inside the cell) (Figure 38) [START_REF] Hubert | LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes[END_REF]. Autophagy can be defined as a two-steps process with:

1. an induction step where an autophagosome is formed around the cytoplasmic components that have to be degraded (on-rate process).

2. a turnover step corresponding to the formation of the autolysosome after the fusion between the autophagosomes and lysosomes which are vesicles containing hydrolytic enzymes (off-rate process). During this second step, the cargo of autophagosome are degraded by lysosomal hydrolases [START_REF] Baehrecke | Autophagy: dual roles in life and death?[END_REF].

• Heterophagy (digestion of materials from outside the cell such as bacteria). Lysosomes are able to fuse with phagocytic vesicles, which ingest outside macromolecules or parasites, and form secondary lysosomes degrading macromolecules.

• Autolysis (complete break-down of died cells). Lysosomes promote autolysis by releasing digestive enzymes into the cytoplasm of cells.

• Exocytosis (release of enzymes and metabolites outside of the cell). Lysosomes promote exocytosis via the proteins present at their surface, necessary for lysosome movement and fusion with the plasma membrane.

All these lysosomal functions are important for recycling the products of biochemical reactions but lysosomes are also involved in others cellular functions such as [START_REF] Saftig | Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function[END_REF]: -plasma membrane repair, a calcium dependent process [START_REF] Idone | Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis[END_REF].

-cholesterol homeostasis via the lipidic transporter Niemann-Pick C1 protein (NPC1) localized at their membrane surface [START_REF] Chang | Cholesterol sensing, trafficking, and esterification[END_REF].

-Major Histocompatibility Complex (MHC) class IIdependent antigen presentation [START_REF] Van Niel | Dendritic cells regulate exposure of MHC class II at their plasma membrane by oligoubiquitination[END_REF][START_REF] Shin | Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination[END_REF].

-other physiological processes such as cell migration (e.g. neural crest cells via Ca 2+ storage), morphogenesis (e.g. limb development), synaptic plasticity and cell maturation (e.g.

Schwann cells

) that I will detail later, in paragraph IV.3.1.

Figure 38: Cellular mechanisms of autophagy

The first step of autophagy is the phagophore formation, an isolated membrane which maturates and forms autophagosome that contains the cargo for degradation. Autophagosome interacts with lysosome which contains the enzymes necessary for the degradation of autophagosome cargo. (Modified from Herzig and Shaw, 2017) Upon starvation, AMPK is recruited to the lysosomal membrane via Axin. Axin recrutes also LKB1 which phosphorylates AMPK on threonine 172. AMPK activation, as mTORC1, depends on v-ATPase and Ragulator activities. When AMPK is activated, mTORC1 is detached from lysosome and is inactivated (Zhang et al., 2014).

Lysosomes as a signaling hub to control nutrient homeostasis

Recently it was shown that lysosomes serve as a platform to activate signaling pathways such as mTOR and AMPK in an amino acid dependent manner, being therefore involved in the regulation of nutrient and energy homeostasis. Thus, in the following paragraphs, I will describe the mechanisms needed for mTOR and AMPK recruitment and activation at lysosomal surfaces and the crosstalk between p53 signaling and mTOR activation at this subcellular location.

mTOR pathway at the lysosomal surface

Recruitment of mTORC1 at the lysosome mTORC1 binds to lysosomal membrane via a small G protein named RagGTPase (Figure 39A). RagGTPase is a heterodimer (RagA or B with RagC or D) that interacts with Ragulator, a hetero-pentameric complex anchored within the lysosomal membrane (Figure 39A) [START_REF] Bar-Peled | An expanded Ragulator is a GEF for the Rag GTPases that signal amino acid levels to mTORC1[END_REF]. Thus, Ragulator recruits RagGTPase to lysosomal membrane but it can also activate RagGTPase by acting as a guanine nucleotide exchange factor for RagA or B (Figure 39A) (Kim et al., 2013b). Concomitantly, the active form of RagGTPase binds to p62/SQSTM1 which associates with Raptor, a subunit of mTORC1 complex, and therefore indirectly recruits mTORC1 to lysosomes [START_REF] Bitto | p62/SQSTM1 at the interface of aging, autophagy, and disease[END_REF][START_REF] Nicastro | The Architecture of the Rag GTPase Signaling Network[END_REF].

The guanine nucleotide exchange factor activity of Ragulator is controlled by vacuolar H + -ATPase (v-ATPase) which can be activated by amino acids present into the lumen of the lysosomes (Figure 39A) [START_REF] Bar-Peled | An expanded Ragulator is a GEF for the Rag GTPases that signal amino acid levels to mTORC1[END_REF]. Therefore, amino acids are also important regulators of mTORC1 localization to lysosomes. They can regulate the activity and conformation of RagGTPase and subsequently the translocation of mTORC1 to the lysosome surface [START_REF] Bar-Peled | An expanded Ragulator is a GEF for the Rag GTPases that signal amino acid levels to mTORC1[END_REF][START_REF] Demetriades | Regulation of TORC1 in Response to Amino Acid Starvation via Lysosomal Recruitment of TSC2[END_REF][START_REF] Sancak | Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids[END_REF]. α-Ketoglutarate (αKG) is an intermediate of the Krebs cycle and a by-product of many amino acid transaminations. This metabolite is also essential for mTORC1 lysosomal translocation because it stimulates RagGTPase activity by promoting GTP loading on RagA or B (Figure 39B). αKG production is regulated by leucine. This essential amino acid directly binds to GDH (glutamate deshydrogenase) which catalyzes the formation of αKG from glutamate (Figure 39A). However, leucine alone is not sufficient to activate mTORC1. It has been shown that intracellular glutamine is necessary for stimulation of mTORC1 by leucine (Figure 39B) [START_REF] Durán | Glutaminolysis activates Rag-mTORC1 signaling[END_REF]. Thus, mTORC1 localization to lysosomal membrane is controlled by RagGTPase and Raptor and regulated by amino acids such as leucine and glutamine. This specific localization of mTORC1 at the surface of lysosomes is essential for its activation.

Activation of mTORC1 at the lysosome

This specific lysosomal membrane localization brings mTORC1 near its regulator called Ras homologue enriched in brain (Rheb), a cell-membrane protein localized to lysosome and other endomembranes (Figure 39A andB). It is known that Rheb is crucial for mTORC1 activity but little is known about the underlying mechanism. What is known so far, based on in vivo and in vitro studies, is that the direct interaction between Rheb and mTORC1 is sufficient to promote mTOR kinase activity (Avruch et al., 2009a(Avruch et al., , 2009b;;[START_REF] Sato | Specific activation of mTORC1 by Rheb Gprotein in vitro involves enhanced recruitment of its substrate protein[END_REF].

TSC1 and TSC2 are negative regulators of Rheb. When TSC interacts with Rheb, it promotes the conversion of active Rheb GTP to inactive Rheb GDP , delocalizing it from lysosomes (Figure 39C). This delocalization consequently inactivates mTORC1.

It is also known that amino acids or growth factors maintain Rheb at the lysosome and therefore regulate mTORC1 activity [START_REF] Carroll | The lysosome: a crucial hub for AMPK and mTORC1 signalling[END_REF]. For example, arginine acts as a specific inhibitor of TSC2 recruitment to lysosomes (Figure 39C) [START_REF] Carroll | Amino acids and autophagy: cross-talk and cooperation to control cellular homeostasis[END_REF].

Another example is with MiCRoSpherule protein-1 (MCRS1) which avoids the Rheb-TSC2 interaction, in an amino acid dependent manner, maintaining Rheb at the lysosomal surface and connecting Rheb to mTORC1 [START_REF] Fawal | MCRS1 binds and couples Rheb to amino aciddependent mTORC1 activation[END_REF].

Altogether, these data point out a novel function of lysosomes in the regulation of mTOR activity and underline the importance of amino acids and growth factors for lysosomal localization and activation of the mTOR pathway.

Interestingly, lysosomes are also involved in recruitment and activation of AMPK, a physiological antagonist of mTOR.

Recruitment and activation of AMPK at the lysosomal surface

AMPK is localized in cytoplasm or nucleus, depending on the stimuli that the cells received such as energy stress, cell density etc. Recently, it has been shown that AMPK, under energy stress, is also localized and activated at lysosomes. One protein responsible for AMPK lysosomal localization and activation is Axin [START_REF] Zhang | Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-AXIN/LKB1 Axis[END_REF]. In 2013, Zhang Y.L et al.

have observed that Axin controls AMPK activity by regulating AMPK phosphorylation on threonine (Thr) 172 by LKB1 [START_REF] Chen | The negative influence of high-glucose ambience on neurogenesis in developing quail embryos[END_REF]. Indeed, Axin physically binds to both LKB1 and AMPK and stimulates the interaction between these two kinases (Figure 40). Axin/LKB1/AMPK complex formation is regulated by AMP and energy stress as follows:

Upon energy stress such as glucose deprivation, the N-terminal region of Axin interacts with LAMTOR1, a subunit of the Ragulator complex while the C-terminal region of Axin interacts with LKB1 and triggers its translocation to the lysosome (Figure 40) [START_REF] Zhang | The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism[END_REF][START_REF] Zhang | Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-AXIN/LKB1 Axis[END_REF]. Ragulator, and more precisely LAMTOR1, is localized on the late-endosome which will form the lysosome. Therefore, the formation of the complex between LAMPTOR1 and Axin/LKB1/AMPK occurs on late endosomes. When the maturation of the late endosome is damaged, it strongly impairs AMPK activation. Thus, lysosomes are important for AMPK activation upon energy stress. Moreover, upon glucose starvation, the v-ATPase changes its conformation, being capable of interacting with Axin but losing its capacity to activate GEF function of Ragulator (Figure 40). Thus, this new conformation promotes Axin/LKB1 translocation to the lysosome and AMPK activation and delocalizes mTORC1 from the lysosomal membrane (Figure 40) [START_REF] Zhang | The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism[END_REF]. This suggest that depending on stimuli, v-ATPase and Ragulator control the lysosomal translocation of either Axin/LKB1/AMPK or mTORC1 complexes.

Metformin (N,N-dimethylbiguanide), the most widely used treatment for type 2 diabetes, is using this Axin/LKB1/v-ATPase/Ragulator lysosomal pathway to increase AMPK activity and to inhibit mTOR [START_REF] Howell | Metformin Inhibits Hepatic mTORC1 Signaling via Dose-Dependent Mechanisms Involving AMPK and the TSC Complex[END_REF][START_REF] Zhang | Metformin Activates AMPK through the Lysosomal Pathway[END_REF].

To conclude, lysosomes are crucial for both mTOR and AMPK activation. mTOR is anchored at lysosomal membrane through RagGTPase, v-ATPase and Ragulator. Its presence at the lysosomal surface allows its interaction with the activator Rheb. Both localization and activation of mTOR are amino acid dependent. AMPK, under energetic stress, is also translocated at the lysosome surface via Axin interaction with Ragulator and v-ATPase. This interaction is also important for LKB1 localization to the lysosomal membrane where it phosphorylates AMPK at threonine172. However, this interaction delocalizes mTORC1 from the lysosome. Thus, the activation of AMPK and mTORC1 at the lysosomal surface is mutually exclusive.

Interestingly, the other tumor suppressor protein p53, previously described in this manuscript, also plays an important role during mTOR/AMPK activation at lysosome's surface.

p53 signaling and mTOR/AMPK activation at lysosomes surface

It has been shown by Xuetao Cao lab that p53 can also translocate to lysosomal membranes [START_REF] Li | Adaptor Protein LAPF Recruits Phosphorylated p53 to Lysosomes and Triggers Lysosomal Destabilization in Apoptosis[END_REF]. In general, p53 phosphorylation on serine15 is associated with its stabilization and with the activation of its nuclear function. However, in Nan Li et al. study, the phosphorylation of p53 on serine15 is essential for its lysosomal translocation.

Lysosome-localized adaptor protein (LAPF) is the protein which recruits phosphorylated p53 on Serine15 to the lysosome [START_REF] Li | Adaptor Protein LAPF Recruits Phosphorylated p53 to Lysosomes and Triggers Lysosomal Destabilization in Apoptosis[END_REF]. Upon apoptotic stimuli such as TNFα and ionizing irradiations, LAPF physically interacts with TAD domains of p53. This interaction is dependent on p53 phosphorylation on serine15. p53 phosphorylation on other serine positions such as 6, 9, 20, 33, 37 or 46 did not promote any interaction between p53 and LAPF and did not recruit p53 at the lysosomes [START_REF] Li | Adaptor Protein LAPF Recruits Phosphorylated p53 to Lysosomes and Triggers Lysosomal Destabilization in Apoptosis[END_REF].

At the lysosome, p53 promotes lysosomal membrane permeabilization which involves leakage of luminal lysosomal hydrolases into the cytoplasm and will induce apoptosis. For the moment, nothing is known about a possible regulation of mTORC1 by LAPF or by lysosomal phosphorylated p53 on Serine15 in vertebrates. However, it has been shown in the yeast Saccharomyces cerevisiae that the LAPF-like protein named Pib2, present at the lysosomes, promotes the lysosomal membrane permeabilization and subsequently cell death through mTORC1 regulation, upon ER stress [START_REF] Kim | A LAPF/phafin1-like protein regulates TORC1 and lysosomal membrane permeabilization in response to endoplasmic reticulum membrane stress[END_REF]. Thus, cytosolic p53 triggers apoptosis in a transcription-independent manner, through Thus, lysosomes are signaling hub that can integrate and relay external and nutritional information. In the next paragraph, I will describe the impact of lysosomes on physiopathological processes.

Lysosomal roles in normal and pathological processes

Lysosomal functions during development and adult maintenance

It is known that calcium (Ca 2+ ) is a cell migration-regulator because increased Ca 2+ levels modulate cytoskeleton dynamics and promote cell contraction and adhesion [START_REF] Praitis | The secretory pathway calcium ATPase PMR-1/SPCA1 has essential roles in cell migration during Caenorhabditis elegans embryonic development[END_REF][START_REF] Sumoza-Toledo | Dendritic cell maturation and chemotaxis is regulated by TRPM2-mediated lysosomal Ca2+ release[END_REF]. Lysosomes are acidic organelles capable of modulating intracellular Ca 2+ levels. They uptake Ca 2+ from cytosol via Ca 2+ /H + exchangers (CAX) channels [START_REF] Melchionda | Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo[END_REF] and are able to release it via other calcic channels such as TRMPL1 [START_REF] Lloyd-Evans | On the move, lysosomal CAX drives Ca2+ transport and motility[END_REF][START_REF] Patel | Evolution of acidic Ca 2+ stores and their resident Ca 2+ -permeable channels[END_REF].

It has been proposed that lysosomes regulate embryonic development [START_REF] Tsukamoto | Functional analysis of lysosomes during mouse preimplantation embryo development[END_REF]. In particular, it was shown that lysosomes are important for early developmental processes such as frog neural crest cell migration through calcic level regulation [START_REF] Lloyd-Evans | On the move, lysosomal CAX drives Ca2+ transport and motility[END_REF][START_REF] Melchionda | Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo[END_REF]. Morpholino-mediated knockdown of CAX expression, in frog, increased cytosolic Ca 2+ amount and blocked neural crest cell migration (Lloyd-Evans, 2016; [START_REF] Melchionda | Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo[END_REF].

The capacity of Ca 2+ to enhance cell attachment was also observed in cardiac NCC [START_REF] Heidenreich | Homocysteine enhances cardiac neural crest cell attachment in vitro by increasing intracellular calcium levels[END_REF]. Thus, lysosomes are essential regulators of neural crest cell migration through Ca 2+ level modulation.

Lysosomes control also more tardive developmental processes than NCC migration such as morphogenesis. It is known that the majority of organ-development is regulated by a well-defined zone of programmed cell death. Zuzarte-Luis et al., have shown that lysosomal cathepsins such as cathepsin D and B are crucial for mice and chick limb development because it regulates embryonic programmed cell death through Bone Morphogenetic Protein (BMP) pathway [START_REF] Zuzarte-Luis | Caspase redundancy and release of mitochondrial apoptotic factors characterize interdigital apoptosis[END_REF]. Thus, lysosomes have a sculpturing function during limb morphogenesis and are responsible for the difference in webbed digits between chick and duck.

Lysosomes are also able to sustain adult physiological processes such as plasma membrane repair, defense from parasites, energy pool maintenance, synaptic plasticity and cell maturation. Cells such as melanocytes, astrocytes or hematopoietic cells, harbor specialized secretory lysosomes. They contain, in addition to hydrolases, proteins necessary for secretion and, at their surfaces, proteins necessary for lysosome movement and fusion with the plasma membrane. These secretory lysosomes are called lysosome-related organelles and are important for plasma membrane repair and defense from parasites [START_REF] Andrews | Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells[END_REF]Divangahi et al., 2009aDivangahi et al., , 2009b;;[START_REF] Reddy | Plasma membrane repair is mediated by Ca(2+)regulated exocytosis of lysosomes[END_REF]. Moreover, it has been shown in astrocytes that extracellular glutamate induces partial exocytosis of lysosomes which contain abundant ATP. The release of ATP from astrocytes is required for Ca 2+ wave propagation among astrocytes and for feedback modulation of synaptic functions [START_REF] Li | Lysosomes Are the Major Vesicular Compartment Undergoing Ca2+-Regulated Exocytosis from Cortical Astrocytes[END_REF][START_REF] Zhang | Regulated ATP release from astrocytes through lysosome exocytosis[END_REF]. Synaptic plasticity is regulated through lysosomal function of degradation which decreases the number of dendritic spines, essential for long-term memory and cognition [START_REF] Goo | Activity-dependent trafficking of lysosomes in dendrites and dendritic spines[END_REF][START_REF] Kononenko | Lysosomes convene to keep the synapse clean[END_REF]. Lysosomes can also control Schwann cell maturation because, due to their degradation capacity, they properly reduce Schwann cell cytoplasm during myelination in the peripheral nervous system [START_REF] Jang | Autophagic myelin destruction by Schwann cells during Wallerian degeneration and segmental demyelination[END_REF]. Thus, lysosomes are essential for many physiological processes and therefore defects in lysosomal functions are generating a large panel of human pathologies.

As both LKB1 and p53 are regulators of reactive oxygen species production and as oxidative stress is responsible for some neurocristopathies (Chapter II.3), I have chosen to further detail the effect of oxidative stress on lysosomal homeostasis. I will then describe how defective lysosomes impact cell homeostasis and in particular how they trigger senescence, age-related diseases and other human pathologies called lysosomal storage disorders.

3.2 Oxidative stress and lysosome homeostasis 3.2.1. Reactive oxygen species production Reactive oxygen species (ROS) are chemical molecules containing oxygen such as hydrogen peroxide (H2O2), superoxide (O2 -) and hydroxyl radical ( . OH). They are produced by several organelles: mitochondria, peroxisomes, endoplasmic reticulum, plasma membrane through the oxidative metabolism (Muller F., 2000;Han D. et al., 2001).

In the cell, there are several non-enzymatic and enzymatic antioxidants like superoxide dismutase 1 and 2 (SOD1, SOD2) and catalase which are capable to detoxify ROS and to prevent oxidative stress (Figure 41) [START_REF] Zhao | ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway[END_REF].

In normal conditions ROS production by mitochondria through incomplete one-electron reduction of oxygen represent 1-2% of the total rate of oxygen consumption [START_REF] Zhao | ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway[END_REF].

However, if mitochondria are damaged higher levels of ROS are formed and antioxidants are not anymore able to prevent oxidative stress (Figure 41) [START_REF] Chance | Hydroperoxide metabolism in mammalian organs[END_REF][START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF][START_REF] Panieri | ROS homeostasis and metabolism: a dangerous liason in cancer cells[END_REF][START_REF] Zhao | ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway[END_REF].

Cells adjust differently depending on ROS levels. For example, moderate ROS levels are able to promote tumor growth and malignant progression by inactivating the protein tyrosine phosphatases and therefore regulating kinases pathways involved in tumor progression such as phosphoinositide 3-kinase (PI3K) and tyrosine kinase receptor (TKR) signaling [START_REF] Locasale | Metabolic flux and the regulation of mammalian cell growth[END_REF]. While, high ROS levels promote DNA damage and affects genome integrity, leading to cell senescence and death (Figure 41) [START_REF] Gorrini | Modulation of oxidative stress as an anticancer strategy[END_REF].

ROS & lysosomal membrane permeability

Mitochondrial dysfunction increases ROS production which affects the lysosomal membrane integrity via lipid peroxidation and causes lysosomal alkalinization [START_REF] Demers-Lamarche | Loss of Mitochondrial Function Impairs Lysosomes[END_REF][START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF][START_REF] Pivtoraiko | Oxidative Stress and Autophagy in the Regulation of Lysosome-Dependent Neuron Death[END_REF]. Under oxidative stress, H2O2 is able to translocate from cytoplasm into lysosome where it interacts with ferric ions and produces ferrous ions and hydroxyl radicals (Figure 42) (Fenton reaction). Hydroxyl radicals induce lysosomal membrane peroxidation which leads to membrane rupture (Pivtoraiko et al., 2008). Thus, oxidative stress causes dysfunction in lysosomes. The lysosomal leakage is directly proportional with the intralysosomal iron concentration [START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF] and with the increased lysosomal pH due to proton leakage [START_REF] Porter | Lysosomal basification and decreased autophagic flux in oxidatively stressed trabecular meshwork cells: implications for glaucoma pathogenesis[END_REF].

There is heterogeneity regarding lysosomal resistance to oxidative stress between cell types but also within cells. Scientists suggested that these differences between lysosomes depend on several parameters [START_REF] Chen | Phorbol ester induces elevated oxidative activity and alkalization in a subset of lysosomes[END_REF][START_REF] Nilsson | Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress[END_REF]:

-antioxidant properties of cells to eliminate H2O2 before it reaches lysosomes.

-iron concentration inside the lysosomes.

-lysosomal lumen pH.

Iron accumulation (mostly in mitochondria and lysosomes) which subsequently increases the sensitivity to oxidative stress [START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF] and lysosomal alkanilization [START_REF] Guha | Approaches for detecting lysosomal alkalinization and impaired degradation in fresh and cultured RPE cells: evidence for a role in retinal degenerations[END_REF] was found in many diseases associated with protein accumulation such as Alzheimer's disease and acute myeloid leukemia. Interestingly, Mitchell lab suggested that increasing cAMP concentration decreases the lysosomal pH and reduces the protein buildup in these pathologies [START_REF] Coffey | Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer's disease-linked presenilin 1 A246E mutation can be reversed with cAMP[END_REF].

To conclude, oxidative stress due most generally to mitochondrial malfunction causes lysosomal membrane permeabilization which perturbs the pH gradient and induces lysosomal enzyme delocalization inside the cytoplasm where they can affect other organelles such as mitochondrial permeability directly or indirectly via phospholipases.

Characteristics of defective lysosomes (lipofuscin granules)

Oxidative stress is responsible for lysosomal peroxidation which leads to accumulation of lipofuscin into the lysosomes (only 1% of lipofuscin is found free into the cytoplasm) [START_REF] Armstrong | Long-term effects of radiation exposure among adult survivors of childhood cancer: results from the childhood cancer survivor study[END_REF][START_REF] Carmona-Gutierrez | The crucial impact of lysosomes in aging and longevity[END_REF]Höhn et al., 2010a;[START_REF] Terman | Lipofuscin: mechanisms of formation and increase with age[END_REF]. Lipofuscin is an aging pigment [START_REF] Ames | Oxidants, antioxidants, and the degenerative diseases of aging[END_REF] discovered a long time ago, in 1842, by Hannover. Even if it was identified 175 years ago the exact cellular function of this material is still not well understood.

Lipofuscin is an electron dense, autofluorescent material detected by a range of excitation wavelengths including ultraviolet (330-380nm), blue (450-480nm) and green (510-560nm) light [START_REF] Tohma | Quantification of ceroid and lipofuscin in skeletal muscle[END_REF]. 30-70% of its composition is represented by oxidized proteins, 20-50% by lipids (Höhn et al., 2010b;[START_REF] Jung | Lipofuscin: formation, distribution, and metabolic consequences[END_REF][START_REF] Porter | Lysosomal basification and decreased autophagic flux in oxidatively stressed trabecular meshwork cells: implications for glaucoma pathogenesis[END_REF][START_REF] Terman | Lipofuscin: mechanisms of formation and increase with age[END_REF], low amounts of saccharides [START_REF] Benavides | Sequential histochemical studies of neuronal lipofuscin in human cerebral cortex from the first to the ninth decade of life[END_REF] and 2% by metals, including Fe, Cu, Zn, Al, Mn and Ca [START_REF] Jolly | Lipofuscin in bovine muscle and brain: a model for studying age pigment[END_REF]. Lysosomal proteolysis decreases with age and since proteins represent a high percentage in lipofuscin composition, decreased intralysosomal degradation can cause lipofuscin accumulation [START_REF] Skoczyńska | Melanin and lipofuscin as hallmarks of skin aging[END_REF].

Lipofuscin is accumulated with age in lysosomes of postmitotic cells such as neurons, glial cells, cardiac myocytes, muscle fibers, Sertoli cells and retinal pigment epithelial cells [START_REF] Terman | Lipofuscin: mechanisms of formation and increase with age[END_REF]. However, inhibition of lysosomal proteases and lipases [START_REF] Marzabadi | Mechanisms of lipofuscinogenesis: effect of the inhibition of lysosomal proteinases and lipases under varying concentrations of ambient oxygen in cultured rat neonatal myocardial cells[END_REF] or the presence of high amount of active iron into lysosomes (which increases oxidative stress and peroxidation processes as seen above) leads to increase rate of lipofuscin accumulation in an age independent manner [START_REF] Terman | Lipofuscin: mechanisms of formation and increase with age[END_REF]. Therefore, antioxidants such as Vitamin E, glutathion or ions chelators are protecting cells from lipofuscin accumulation while prooxidants have the opposite effect and significantly increase lipofuscin formation [START_REF] Terman | Lipofuscin: mechanisms of formation and increase with age[END_REF]. Lipofuscin is also able to stimulate its own production by increasing prooxidant molecules expression (Hohn et al., 2010) and by inhibiting oxidized protein degradation through proteasome inhibition (Hohn et al., 2011;[START_REF] Sitte | Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts[END_REF][START_REF] Powell | Aggregates of oxidized proteins (lipofuscin) induce apoptosis through proteasome inhibition and dysregulation of proapoptotic proteins[END_REF].

Lysosome functions in human pathologies

Age-related diseases

Lipofuscin granules are generally associated with aging and with age -related diseases such as neurodegenerative diseases like Alzheimer, Parkinson, and Huntington diseases [START_REF] Brunk | The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis[END_REF]. High levels of lipofuscin and lysosomal enzymes are observed in amyloid deposits in Alzheimer's patients [START_REF] Skoczyńska | Melanin and lipofuscin as hallmarks of skin aging[END_REF]. Lipofuscin accumulation was also observed in premature aging syndromes [START_REF] Brunk | The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis[END_REF].

Lipofuscin is frequently observed in association with senescence, an irreversible mechanism that arrests cell cycle [START_REF] Georgakopoulou | Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryopreserved and archival tissues[END_REF]. Senescent cells are associated with four biological processes: carcinogenesis [START_REF] Shay | Hallmarks of senescence in carcinogenesis and cancer therapy[END_REF], tumor suppression [START_REF] Collado | Senescence in tumours: evidence from mice and humans[END_REF], age-related disorders and tissue repair [START_REF] Rodier | Four faces of cellular senescence[END_REF].Considering the results I have obtained during my PhD linking LKB1 and p53 to lipofuscin granules formation (Results. Project 2.2), I will further focus on cellular senescence and aging.

Among the most compelling evidences that senescence promotes age-related disorders are the phenotypes of mice with hyperactivated p53. In particular, heterozygous mice harboring one p53 allele deleted in the first six exons of the p53 gene [START_REF] Tyner | p53 mutant mice that display early ageing-associated phenotypes[END_REF] or transgenic mice expressing the Δ40p53 or ΔNp53 isoforms [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF][START_REF] Rufini | Senescence and aging: the critical roles of p53[END_REF] exhibit an important resistance to develop spontaneous tumors. However these mice have a reduced lifespan characterized by accumulation of senescent cells in different tissues and a complex phenotype, including loss of fertility, with a "Sertoli cell only" phenotype in most of the seminiferous tubules, loss of subcutaneous fat, osteoporosis, sarcopenia and reduced hair growth [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF][START_REF] Tyner | p53 mutant mice that display early ageing-associated phenotypes[END_REF].

As presented in Chapter III.B.1.2, oxidative stress activates p53. Activated p53 induces a specific transcriptional response that regulates cellular senescence and aging [START_REF] Gambino | Oxidative stress activates a specific p53 transcriptional response that regulates cellular senescence and aging[END_REF]. p53-mediated senescent cells are associated with the formation and accumulation of lipofuscin and premature aging [START_REF] Georgakopoulou | Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryopreserved and archival tissues[END_REF].

In conclusion, lysosomes are important organelles which prevent premature aging by controlling signaling pathways, lipofusin formation and cell senescence.

Lysosomal storage disorders

Defects in lysosomal glycan, lipid or protein degradation or defects in lysosomal transporters or trafficking are responsible for lysosomal storage disorders. These human diseases constitute a group of metabolic developmental disorders characterized by accumulation of non-degraded material in the lysosomes [START_REF] Ballabio | Lysosomal disorders: from storage to cellular damage[END_REF][START_REF] Greiner-Tollersrud | Lysosomal Storage Disorders[END_REF][START_REF] Onyenwoke | Lysosomal Storage Diseases-Regulating Neurodegeneration[END_REF][START_REF] Schultz | Clarifying lysosomal storage diseases[END_REF].

There are more than 50 types of lysosomal storage disorders and many are associated to neurodegeneration. This neural death can be due to neuron mitochondrial defects or to glial cell dysfunctions which lead to demyelination [START_REF] Folkerth | Abnormalities of developing white matter in lysosomal storage diseases[END_REF][START_REF] Onyenwoke | Lysosomal Storage Diseases-Regulating Neurodegeneration[END_REF][START_REF] Rama Rao | Astrocytes and lysosomal storage diseases[END_REF][START_REF] Saher | Cholesterol in myelin biogenesis and hypomyelinating disorders[END_REF]. As discussed above, lysosomes also regulate synaptic plasticity and therefore long-term memory and cognition, cognitive defects representing the first sign of lysosomal storage disorders in patients [START_REF] Goo | Activity-dependent trafficking of lysosomes in dendrites and dendritic spines[END_REF].

To sum up, lysosomes are not only important for degradation processes but have many other cellular functions during embryonic development and adult physiological processes, such as cell migration, differentiation, cell protection and repair, nutrient and energy homeostasis. Therefore, lysosomal defects due to oxidative stress are responsible for various human pathologies as lysosomal storage disorders and neurodegenerative diseases. Momently, there is no cure for lysosomal storage disorders or lipofuscin accumulation. Treatments such as bone marrow transplantation and enzyme replacement therapy have partially decreased lysosomal storage disorders and Parkinson's disease symptoms and improved lysosomal enzyme activity [START_REF] Clarke | Enzyme replacement therapy of Fabry disease[END_REF][START_REF] Mcneill | Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells[END_REF]. Interestingly, treatments decreasing oxidative stress such as iron chelators, vitamin E and glutathione treatments, delay lipofuscin production and accumulation in murine models [START_REF] Brunk | The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis[END_REF][START_REF] Skoczyńska | Melanin and lipofuscin as hallmarks of skin aging[END_REF].

***

PhD context & objectives

As highlighted in the introduction, uncovering connections between metabolic responses and cell signaling pathways has recently encounter an impressive increase. One important signaling pathway regulating energy metabolism is controlled by the tumor suppressor LKB1. Before studies underlining LKB1 role in metabolic regulations through AMPK pathway, functions of Lkb1/AMPK pathway were predominantly explored in cell polarity in Caenorhabditis elegans (J.L. [START_REF] Watts | The C. elegans par-4 gene encodes a putative serine-threonine kinase required for establishing embryonic asymmetry[END_REF] and Drosophila melanogaster (Martina and St Johnston, 2003). These studies established that LKB1 signaling is a major regulator of cell polarity through in particular the establishment and maintenance of cell junctions. Data from the literature prompted us to better characterize if LKB1 coordinately regulates cell metabolism and polarity partly through the control of its downstream kinase AMPK and the negative regulation of the mTOR pathway.

In particular, it is now well established that cell polarity is essential for neural crest cells

Results

I. Project 1: LKB1 in cranial neural crest cells

Multipotent neural crest cells (NCC) originate from the dorsolateral folds of the neural tube and constitute a population of highly motile embryonic cells. These cells give rise to a broad range of derivatives including pigmented cells, most of the head skeleton, peripheral nerves and the enteric nervous system that allows the motricity of the digestive tract (Simões-Costa and [START_REF] Simões-Costa | Establishing neural crest identity: a gene regulatory recipe[END_REF].

The tumor suppressor gene LKB1 encodes a serine/threonine kinase that acts as a pivotal regulator of energy metabolism via the activation of AMPK, a sensor of the cellular energy status [START_REF] Force | Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition[END_REF]Hezel et al., 2008). LKB1 is a tumor suppressor gene and germline mutations of LKB1 are responsible for the human Peutz-Jeghers syndrome, an autosomal dominant disease characterized by the presence of mucosal gastrointestinal polyps and an increased risk to develop malignant tumors (see Introduction, Chapter III.A.5). Peutz-Jeghers syndrome also associates hyperpigmented macules of the skin, suggestive of defective melanocytes homeostasis and LKB1 role in the biology of melanocytes has been established [START_REF] Liu | LKB1/STK11 Inactivation Leads to Expansion of a Prometastatic Tumor Subpopulation in Melanoma[END_REF]. Homozygous disruption of Lkb1 in mice is embryonic lethal and causes defects in the neural tube closure, an absence of the first branchial arch (mostly composed of NCC) and facial reduction [START_REF] Ylikorkala | Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice[END_REF].

Altogether, these data were convincing arguments hinting at a role of the LKB1 protein kinase in the development of the neural crest lineage.

1. LKB1 signaling is essential for cranial neural crest cells development.

Article Nb1: Creuzet et al., Dev. Biol. 418:283 296, 2016 1

.1 Results summary

To address the contribution of Lkb1 during cranial NCC development, our team generated a mouse model with a temporal disruption of Lkb1 specifically in NCC just after their delamination from the neural tube, at embryonic day 8.5 (E8.5). Conditional inactivation of Lkb1 was achieved by breeding Lkb1 Flox mice (Bardeesy et al., 2002) with mice expressing the recombinase Cre under the human tissue plasminogen activator (Ht-PA) promoter using the Ht-PA::Cre transgene established by S. Dufour (Institut pour la Recherche Biomédicale, Créteil) (Pietri et al., 2003).

The lab also took advantage of the tractable system provided by the avian embryo. To inhibit chick Lkb1 expression specifically in cranial NCC, we used the RNA interference approach combined with a triple electrode system enabling a bilateral electroporation of the cephalic neural folds with double-stranded RNA molecules before the emigration of cranial NCC out of the neural tube (at 4-5 somites stages).

Combining studies on mouse and chick models of Lkb1 inactivation, our team has shown that Lkb1 is required for the development of cephalic NCC in vertebrates. Lkb1 regulates the delamination of pre-migratory NCC from the neural primordium as well as their polarization and survival. In the absence of this kinase, the embryos are affected by severe craniofacial and brain defects. These Lkb1-mediated effects on the development of cephalic NCC involve the sequential activation of the AMPK, the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Lkb1 mutant mice died around one day after birth due to cranial facial malformations that prevent the newborn pups to suckle milk from their mother.

The work was published in 2016 in Developmental Biology (Creuzet et al., 2016). Our work has been selected to illustrate the cover of the journal issue.

Results

Conclusions

In this study, we characterized the role of Lkb1 in cranial NCC delamination, polarized migration and survival. Lkb1 silencing by RNA interference in chick cranial NCC or genetic ablation of Lkb1 in mouse NCC just after their delamination led to craniofacial malformations. These defects included sever reduction of the telencephalic vesicle and of the development of naso-frontal and maxillo-mandibular structures.

Upon Lkb1 loss, the expression of Sox9, marker of pre-migratory NCC, and Sox10, marker of delaminating NCC, were decreased compared to control embryos. Furthermore, using the Golgi-nucleus axis as a maker of cell polarity, we demonstrated that Lkb1 loss resulted in a randomized positioning of chick migrating cranial NCC. We also explored viability of cranial NCC and found an expansion of cell death upon Lkb1 silencing. We have shown that Lkb1 controls cranial NCC formation through AMPK pathway by sequential activation of ROCK kinase and the actin molecular motor Myosin II.

Altogether, these data point out that Lkb1 pathway orchestrates several steps of cranial NCC development that are essential for cephalogenesis. Therefore, this study is of particular interest in order to better understand the molecular mechanisms responsible for craniofacial abnormalities which represent one-third of all congenital birth defects [START_REF] Dixon | Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities[END_REF].

Regulation of LKB1 by acetyl transferase GCN5.

Results not published which are part of an article in preparation (Ghawitian, Radu, et al., in preparation).

Scientific context

After underlining the role of Lkb1 signaling in cranial NCC fate and vertebrate head development, I also contributed to the investigation of the molecular mechanisms underlying LKB1 regulation by upstream factors. This project was initiated by Maya Ghawitian, a former PhD student in Marc Billaud lab.

As mentioned in the introduction of this manuscript, LKB1 undergoes post-translational modifications such as phosphorylation, prenylation, ubiquitination and acetylation (see Chapter III.A.1.3). Regarding acetylation, data from the literature described several acetylated residues of LKB1 [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation[END_REF]; Figure 19 B intro). In particular, lysine 48 (K48) deacetylation by the class III histone deacetylase Sirt1 regulates LKB1 subcellular localization and its catalytic activity [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation[END_REF]. Along this line, our team also identified by mass spectrometry that five lysine residues of LKB1 are acetylated (lysines 48, 64, 97, 108 and 178, see Introduction GCN5 is crucial during embryogenesis. GCN5 null mice are embryonic lethal early during development, just after the beginning of gastrulation [START_REF] Xu | Loss of Gcn5l2 leads to increased apoptosis and mesodermal defects during mouse development[END_REF]. Mice expressing GCN5 harboring mutations inactivating its catalytic domain (GCN5 HAT ) also lead to embryonic lethality but later on, at mid-gestation [START_REF] Bu | Loss of Gcn5 acetyltransferase activity leads to neural tube closure defects and exencephaly in mouse embryos[END_REF]. GCN5 HAT mutants exhibit neural tube closure defects limited to cranial region and reduced telencephalon formation. This suggests that GCN5 regulates early embryonic development via both acetyltransferase dependent and independent functions.

The malformations observed upon GCN5 loss or when GCN5 catalytic activity is abrogated are similar with those observed after cranial NCC agenesis [START_REF] Creuzet | Patterning the neural crest derivatives during development of the vertebrate head: insights from avian studies[END_REF][START_REF] Kurihara | Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1[END_REF] thereby suggesting that GCN5 is essential for cranial NCC development.

Interestingly, GCN5 mutant phenotype is evocative of the defects we described upon Lkb1 inactivation in cranial NCC (Creuzet et al., 2016).

To characterize the potential contribution of GCN5 upstream LKB1 signaling in NCC during vertebrate head formation, our team firstly explored the regulation of LKB1 by GCN5 in vitro. Secondly, we assessed how LKB1 acetylation and GCN5 contribute to neural crest formation in vivo.

Results

Nicolas Aznar and Maya Ghawitian, former PhD students in Marc Billaud lab, identified by mass spectrometry five acetylated lysine residues on LKB1, one of them being lysine 48 (K48). They validated by western blot on cell extracts that LKB1 can indeed be acetylated (Fig. 1A) and that GCN5 is the major acetyltransferase responsible for LKB1 acetylation (Fig. 1B). One of our collaborators, Vincent Mirouse (Genetic, Reproduction, Development (GReD), Clermont-Ferrand), has developed an antibody which recognizes specifically the acetylated form of LKB1 on K48. Validation of this home-made antibody was performed by comparing acetylation levels of LKB1 wildtype or a non-acetylatable mutant (K48R) (Fig. 1C). Next, our lab showed using overexpressed proteins that LKB1 is acetylated at K48 by GCN5 (Fig. 1D) and that both proteins are in the same complex (Fig. 1E).

During my PhD, I confirmed these results on endogenous immunoprecipitated LKB1 from HEK293T BOSC cells treated with Trichostatin A (TSA), a selective inhibitor of histone deacetylases class I and II, to stabilize acetylated proteins. Indeed, I identified by Western Blot that endogenous LKB1 is acetylated at K48 and that TSA treatment increased LKB1 acetylation at K48, suggesting that the signal observed is specific to an acetylated form of LKB1 (Fig. 2A). I have also shown that endogenous LKB1 and GCN5 interact together. The specificity of this interaction was confirmed by decreased GCN5 signal after immunoprecipitation of LKB1 in GCN5 silencing conditions (Fig. 2B). Moreover, the physical interaction between GCN5 and LKB1 was not impacted when the acetylation status of lysine 48 was modified. Indeed, LKB1 mutants such as LKB1 K48R and LKB1 K48Q (acetyl mimetic mutant) still interact with GCN5 as wild type LKB1 (Fig. 2C). Overall the interaction between LKB1 and GCN5 does not rely on acetyltransferase activity of GCN5 since GCN5 HAT mutant still interacts with LKB1 (Fig. 1D,E).

Altogether, these results emphasize that endogenous LKB1 is acetylated at K48 and that LKB1 is physically interacting with GCN5. Since acetylated K48 is nuclear and GCN5 is the acetyltransferase responsible for this acetylation I studied in which cellular compartment is taking place the physical interaction between Lkb1 and GCN5. It is known that GCN5, as LKB1, can be present in both nucleus and cytoplasm (Maralice Conacci-Sorrell.et al., 2010). As observed in figure 2G, I have shown that Lkb1 can interact with GCN5 in the cytoplasm. Instead, I did not detect any nuclear interaction. However, it is difficult to conclude that this interaction is only cytoplasmic because it is known that GCN5 in nucleus is part of large complexes, the best characterized one being the SAGA (Spt-Ada-GCN5 acetyltransferase) complex which has a size of 1.8-2 MDa (K.K. [START_REF] Garofalo | Oral l-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1[END_REF]Weake and Workman,2012). Therefore, it is possible that during LKB1 immunoprecipitation, nuclear GCN5 complexes were lost at centrifugation steps. Thus, other analyses have to be done in order to conclude in which compartment the two proteins interact.

As GCN5 is important for LKB1 translocation into the cytoplasm and as LKB1 kinase activity correlates with its cytoplasmic localization, I analyzed the role of GCN5 regarding LKB1 catalytic activity. AMPK is the most studied substrate of LKB1 (see Introduction Chapter III.A.2). Therefore, I analyzed AMPK phosphorylation at threonine 172 in presence or not of GCN5. As expected overexpressed LKB1 led to AMPK phosphorylation on threonine 172 (Fig. 2H). Preliminary results have shown that GCN5 transfection, in presence of overexpressed LKB1, led to increased AMPK phosphorylation compared to overexpression of LKB1 alone (Fig. 2H). This augmentation is not dependent of GCN5 acetyltransferase activity because GCN5 mutant in its catalytic subunit did not significantly decrease AMPK phosphorylation (Fig. 2H). These data suggest that GCN5 regulates LKB1 catalytic activity independently of its acetyltransferase activity. Further studies have to be done to confirm these results. Interestingly, the non-acetylatable form of LKB1 (LKB1 K48R) failed to limit cellular migration.

These preliminary results suggest that the acetylated form of LKB1 on K48 is essential to control cellular migration and that this LKB1 function is cytoplasmic. However, more experiments need to be done to confirm these findings.

In order to analyze the role of GCN5 during development and more precisely in cranial NCC, Marc Billaud team first studied GCN5 expression during embryogenesis by whole mount in situ hybridization in chick embryos. They cloned and sequenced chicken homologue of GCN5 cDNA and observed that 89% of the chicken GCN5 amino-acid sequence was conserved with the human long isoform of GCN5. Second they synthetized labeled probes that recognize GCN5 mRNA (antisens probes). After whole mount in situ hybridization, GCN5

mRNA were detected at 4 somites stage in the neural folds, just before neural tube closure. At 7 somites stage GCN5 mRNA accumulated in neural folds but also started to localize in migrating NCC. At 11 somites stages GCN5 transcripts continued to be observed in migrating cranial NCC.

Next, the team silenced GCN5 specifically in chick cranial NCC and observed that GCN5 absence leads to dramatic craniofacial malformations including reduction of the telencephalic vesicle and of the development of naso-frontal and maxilla-mandibular structures (Fig. 2J).

Collectively, our results suggested that Lkb1 is a downstream target of GCN5. We also demonstrated that GCN5 is essential for vertebrate head formation and that GCN5 loss mimics the phenotype associated with Lkb1 inactivation I previously described (Project 1.1, Creuzet et al., 2016). These results strongly suggest a crosstalk between Lkb1 and GCN5.

Conclusions

This study allowed us to identify the acetyltransferase GCN5 as a novel upstream regulator of LKB1. During this project, I showed that endogenous LKB1 and GCN5 are in the same complex, that GCN5 acetylates LKB1 on lysine 48 and that this acetylated form of LKB1 is nuclear.

I also observed that GCN5 controls LKB1 subcellular localization and consequently LKB1 catalytic activity independently of its acetyltransferase activity.

Interestingly, it has been shown that GCN5 has developmental functions that depend of its acetyltransferase activity but also that are independent of its catalytic activity. For example, homozygous point mutations in GCN5 catalytic domain (GCN5 Hat/Hat ) lead to embryonic lethality due to neural tube closure defects and exencephaly, demonstrating that the acetyl transferase activity is essential for neural tube closure and correct head patterning [START_REF] Bu | Loss of Gcn5 acetyltransferase activity leads to neural tube closure defects and exencephaly in mouse embryos[END_REF]. However, these mice survive longer than GCN5 knockout mice [START_REF] Xu | Loss of Gcn5l2 leads to increased apoptosis and mesodermal defects during mouse development[END_REF], suggesting that GCN5 exerts other developmental regulations independently of its acetyltransferase activity. The role of GCN5 in early embryonic development and neural tube closure was also described in humans. Human amniotic fluid and blood cells were isolated from pregnant women, in the second trimester, with normal fetuses or with fetuses displaying craniofacial abnormalities due to neural tube defects such as myelomeningocele and anencephaly [START_REF] Tsurubuchi | Amniotic fluid and serum biomarkers from women with neural tube defect-affected pregnancies: a case study for myelomeningocele and anencephaly: clinical article[END_REF]. In fetuses with myelomeningocele, GCN5 expression was decreased along with decreased histone H3 acetylation (in particular H3K9 and H3K18) [START_REF] Tsurubuchi | Amniotic fluid and serum biomarkers from women with neural tube defect-affected pregnancies: a case study for myelomeningocele and anencephaly: clinical article[END_REF]. This suggest that GCN5 and histone H3 acetylation are crucial for neural tube closure in human early development.

In our study, we have shown that GCN5 is expressed in cranial NCC and that GCN5

silencing in cranial NCC mimics the phenotype observed upon Lkb1 inactivation. We previously identified that Lkb1 regulates cranial NCC migration among several NCC behavior, through actin dynamic thereby controlling vertebrate head formation (Creuzet et al., 2016). In the present study, I have shown that the acetylation of LKB1 on K48 is important for the control of cell migration. This result suggests that GCN5 may have the capacity to control vertebrate head formation through LKB1 acetylation on K48. In order to test this hypothesis, further studies require to be performed. First, studying the impact of LKB1 acetylation in neural crest stem cells is primordial. To do so, Lkb1 could be silenced in the neural crest stem cell line morphology is differentially rescued depending on LKB1 form will be performed as previously (Creuzet et al., 2016).

Thus, several studies could be done in order to emphasize the role of LKB1 K48 acetylation by GCN5 in neural crest ontogeny and complete this study before submission.

Materials and methods

Cell line and cell culture

Bosc cells (human kidney epithelial cells) and Hela cells (human epithelial cervix adenocarcinoma cells) were obtained from American Tissue Culture Collection. Both were cultured in DMEM (Ref. 41966-029, ThermoFisher Scientific) with 10% of heat-inactivated fetal bovine serum, supplemented with 1% of penicillin/ streptomycin, at 37°C in 5% CO2.

During some experiments cells were treated for 4h before lysis with 1µM TSA Experiments were realized 48h post-transfection.

pSG5-LKB1 wild type vector was previously described (Nony et al., 2003). The human

Cell Fractionation

For cellular fractionation, Bosc cells were seeded in 2 dishes of 10 cm of diameter at 2x10 6 cells/dish. Cells were harvested and centrifuged for 4 minutes at 1200rpm. Cells pellet was incubated with 400µL lysis buffer containing 10mM Tris-HCl, ph 7.5 and 2 mM EDTA and 1% of protease inhibitors, for 1h in ice and vortexed every 15 minutes. Then, the lysate was passed 6 times in 26g syringes.

300ml of lysate was loaded on the top of a sucrose gradient formed by a 60% sucrose solution (500ml) and 10% sucrose solution (500ml). This solution contains 20mM Tris-HCl, ph 7.5, 0.5 mM EDTA, 100mM NaCl and the respective sucrose quantity.

Lysates were then centrifuged on a Sorval RC M120ex utracentrifuge using a RP55s-164 rotor at 16800rpm (35087g) during 30min at 4°C. Fractions were then collected and analyzed by Western Blot.

Immunofluorescence

Cells were plated at 10 5 cells/ well in a 6-well plate with 4 coverslips (12mm of diameter) per well. After 24h, cells were transfected with different vectors as previously presented. After 48h, cells were fixed in 4% paraformaldehyde, for 10 minutes. Then, cells were permeabilized with 0.5% Triton X-100, 0,3% BSA for 5minutes and blocked 1h in PBS-BSA3%. Cells were incubated for 1h45 with antibody anti Lkb1 N19 (Santa Cruz, sc-8185, lotJ1212) at 1/100 and anti GCN5L2 (C26A10) (Cell Signaling 3305S, lot4) at 1/100. Donkey anti-Rabbit Cya3

(1/1000) and anti goatAlexa488 (1/1000) secondary antibodies used were from Jackson

ImmunoResearch. Cells were mounted in Moviol/Dapi.

Cell Migration, wound healing assay

Hela cells were transfected with different vectors expressing WT-Flag-LKB1 or K48R-Flag-LKB1 or K48Q-Flag-LKB1 for 48h. After that cells were re-suspended and seeded at the 1*10 4 cells/well (total volume 50µl/well) in both ibidis wells ( ibidis Culture-Insert with 2 wells, 80201). The ibidi was used on a 12 well plate with 1 ibidi per well of 12 wells plate. The outer area was filled with 500µl of cell medium. The plate was incubated at 37°C and 5% CO2. After 24h, the ibidi was gently removed with sterile tools and the well was filled with 500µl medium (total 1ml/well). The images were taken at 0h, 24h, 48h and 72h after wounding.

II. Project 2: LKB1 metabolic and signaling regulations during NCC commitment.

As described in Project 1, we recently uncovered that Lkb1 governs several aspects of cranial NCC development that are crucial during vertebrate head formation (Creuzet et al., 2016). In addition to this first project, I also investigated the role of Lkb1 in truncal, vagal and sacral NCC which give rise to various derivatives including melanocytes, glial and neural cells in the peripheral nerves and in the enteric nervous system (Introduction Chapter II.2).

Based on a second mouse model of Lkb1 conditional inactivation in NCC, as well as an in vitro model of NCC differentiation, we identified that LKB1 signaling is essential during NCC lineage and exert crucial metabolic regulations.

1. The tumor suppressor LKB1 controls NCC fate through pyruvate-alanine transamination.

Article Nb2: Submitted, Radu, Torch et al.

Scientific context and results summary

To after their migration to target tissues (Delmas et al., 2003).

We reported that Lkb1 mutant mice die between 2 to 6 weeks after birth and exhibit coat depigmentation, intestinal pseudo-obstruction and a severe peripheral neuropathy. Using this mouse model, we established that LKB1 controls the development of Schwann cells in the peripheral nerves and the post-natal maintenance of ganglion cells of the enteric nervous system. Metabolomic profiling of peripheral nerves highlighted that Lkb1 loss triggers increased alanine and glutamate levels.

To better understand the metabolic defects in the absence of Lkb1, I used a neural crest stem cells line that can be committed, in vitro, into the early steps of glial lineage. Using this cell line, I confirmed that Lkb1 is key for glial differentiation. Mechanistically, Lkb1 loss led to increased alanine and glutamate levels as observed in vivo. Interestingly, inhibition of the alanine amino-transaminase responsible of pyruvate and glutamate conversion into alanine (transamination reaction) rescued glial differentiation of Lkb1-silenced NCC, in a mTORdependent manner. Furthermore, glutamate synthase inhibition, the enzyme which converts glutamate into glutamine, phenocopied Lkb1 loss as it impaired glial differentiation in a mTORdependent manner.

One hypothesis explaining impaired glial differentiation upon Lkb1 loss was that NCC lineage is blocked early in glial comitment due to a lack of energy. Interestingly, AICAR (5aminoimidazole-4-carboxyamide), a known agonist of AMPK but only in the presence of LKB1, has been described with AMPK-independent functions [START_REF] Guigas | AMPactivated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside[END_REF]. Furthermore, AICAR is an AMP precursor (see Introduction, Chapter III.A.2.1.2) which was shown to replenish IMP/AMP pools upon glutamate synthase inhibition (Tardito et al., 2015) and to induce astroglial differentiation (Zang et al., 2008). We demonstrated in vitro that AICAR rescued glial differentiation of Lkb1-deficient NCC and in vivo treatment of Lkb1-deficient mice with AICAR prevented the peripheral nerve and ENS phenotypes (Radu et al., submitted).

Altogether, these findings highlighted the novel and crucial role of Lkb1 during neural crest cell fate and uncovered a link between Lkb1-mediated pyruvate-alanine cycling and glial differentiation. These results provide new insights to understand the metabolic regulations exerted by Lkb1 during development and tumorigenesis.

Introduction

Results

Lkb1 deletion results in a complex neural crest phenotype

To explore Lkb1 functions in neural crest derivative cells in vivo, Lkb1 conditional mutant mice (Lkb1F/F) (Bardeesy et al. 2002) were interbred to the Tyr::Cre deleter (Delmas et al. 2003;[START_REF] Puig | The tyrosinase promoter is active in a subset of vagal neural crest cells during early development in mice[END_REF]) and the R26R reporter [START_REF] Soriano | Generalized lacZ expression with the ROSA26 Cre reporter strain[END_REF]) strains (Figure S1A). to thrive and died within a few weeks after birth (Figure 1C). cKO mice exhibited a reduced weight gain (Figure 1D) and a distended abdomen (Figure S1E). Necropsy revealed an intestinal pseudoobstruction with abnormal dilation of the small intestine and/or colon, atrophy of the cecum and at more advanced stage, a reduction of the stomach accompanied by hypertrophy of the cecum (Figure 1E). The mutant mice also displayed significantly reduced mobility due to progressive hindlimb paralysis (Figure S1F). When lifted by their tail, 21 days old (P21) cKO mice reflexively contracted their limbs, sometimes unilaterally, but more often bilaterally whereas control littermates extended their legs when challenged to the same test (Figure 1F).

Gait tests were also performed by footprint analysis at P21 (Figure S1G). The stride distance was significantly reduced in cKO mice, while the sway distance was unaffected, resulting in a shorter gait in the absence of Lkb1. At the macroscopic level, the sciatic nerves of cKO mice appeared thinner and more translucent than in control littermates and lacked the characteristic banded pattern of myelin (Figure 1G).

Thus, conditional deletion of Lkb1 in truncal and vagal NCC results in a complex phenotype consisting of coat depigmentation, intestinal pseudo-obstruction and a peripheral neuropathy.

Lkb1 loss causes ENS degeneration

To explore the mechanisms leading to the intestinal pseudo-obstruction in cKO mice, we first analyzed by immunohistochemistry the expression pattern of To determine whether the ENS was affected in cKO mice, we used several complementary approaches. expression as a reporter of Cre expression on wholemount intestine of P21 animals (Figure S2E). In these conditions, the enteric network was stained in wild-type animals whereas the ENS of cKO animals showed a drastic decrease of labeling.

The results were corroborated by neural (Tuj1) and glial (GFAP) staining on intestine and colon sections of P21 mice, which revealed that Lkb1-deficiency resulted in a strong reduction of both neural and glial cells labeled by Tuj1 and GFAP respectively along the intestine and colon (Figure 1K). The individual analysis of cKO mice showed that all mutants had at least one area of the digestive tract with hypoganglionosis. The most frequently hypoganglionic area was the distal intestine while the proximal colon did not appear to be affected (Figure S2F). To quantify these anomalies, we counted the number of enteric neurons per length of tunica muscularis (Figure 1L). In cKO animals, the number of Tuj1-positive neurons was 2-fold lower in the distal intestine and 1.6-fold lower in the distal colon as compared to WT controls. Thus, the lack of Lkb1 leads to an ENS hypoganglionosis.

We next sought to evaluate if the defects observed at P21 were also present at earlier stages. As shown in By immunohistochemistry we observed that phospho-AMPK staining was specifically decreased in the remaining enteric ganglia of cKO mice compared to control littermates, whereas no changes were seen in smooth muscle cell layers or enterocytes (Figure S2I). Consistently, phosphorylation of the S6 ribosomal protein (S6RP), a substrate of the p70S6 kinase acting downstream of mTORC1, was enhanced in the enteric ganglia of intestine and colon regions of cKO mice (Figure 1M).

Collectively, our results provide evidence that Lkb1 function is key for the postnatal maintenance of the ENS.

The peripheral neuropathy arises from a blockade of Schwann cell differentiation

To explore the role of Lkb1 in peripheral nerves, we 2Cd,e respectively). Large clusters of unsorted axons were also identified (Figures 2Cf andS3H). Nonmyelinating SC of cKO mice did not enwrap axons individually as in wild-type animals (Figures 2Cc,f) but wrapped axons in bulk. Several axons achieved an appropriate 1:1 relationship with SC but others showed aberrant sorting or remained unsorted (Figure 2Cg,h). To quantify axon myelination defects in cKO animals, we measured the g-ratio: the ratio of the axonal diameter to the myelinated fiber diameter.

We found an increased g-ratio in mutant mice (Figure Next, we examined the distribution of E-cadherin, a cell adhesion protein concentrated in subdomains corresponding to Schmidt-Lanterman incisures and paranodal loops adjacent to nodes of Ranvier in myelinating Schwann cells [START_REF] Tricaud | Adherens junctions in myelinating Schwann cells stabilize Schmidt-Lanterman incisures via recruitment of p120 catenin to Ecadherin[END_REF]. In dissociated nerve fibers of cKO animals, E-cadherin staining was diffuse and the typical localization of Ecadherin was lost suggesting that SC polarity is disorganized in absence of Lkb1 (Figure 2E), an observation consistent with a recent report (Shen et al., 2014).

While studying transverse and longitudinal sections of cKO sciatic nerves, we observed an increased number of Schwann nuclei at P6 and at P21 (Figure S3K). To assess the nature of these supernumerary cells, we labeled immature/promyelinating SC with Sox2 immunostaining [START_REF] Bremer | Sox10 is required for Schwanncell homeostasis and myelin maintenance in the adult peripheral nerve[END_REF]) and observed that cKO mice exhibited a marked increase in Sox2 staining at P6 compared to WT, and this was also evident to a lower extent at P21 (Figure 2F). We confirmed these data using an antibody that recognizes Krox20, a transcription factor that is a pivotal regulator of SC myelination, to visualize myelinating SC differentiation (Figure S3L). At P6 no Krox20 nuclei were detected in cKO animals, thus suggesting a delay in myelinating SC differentiation in the absence of Lkb1.

Altogether, these results suggest that in the absence of Lkb1, SC failed to differentiate, thus resulting in the accumulation of SC precursors localized in the nerve.

Lkb1 controls glial fate specification

To examine the consequences of Lkb1 inactivation on NCC fate, at the early steps of glial commitment of the Schwann cell lineage, we utilized the JoMa1.3 mouse neural crest stem cell line, which can either be maintained in a progenitor state or differentiated into glial cells among other NCC derivatives (Figure 3A) (Maurer et al., 2007). Cells are kept in the progenitor state through the inducible expression of the c-myc ER oncogene which exerts its transcription factor activity upon treatment of cells with 4-OHT (Figure S4A). In

Lkb1 ablation impairs mitochondrial activity

Owing to the crucial role of Lkb1 and AMPK in controlling mitochondrial activity (Beirowski et 3F). Cellular distribution of mitochondria in isolated sciatic nerve fibers is altered and appeared more dispersed in myelinated fibers of cKO mice compared to WT littermates (Figure 3G).

Electron microscopy showed dilated mitochondrial cristae in the absence of Lkb1 (Figure 3H). Next, we assessed the respiratory capacities of Lkb1-deficient glial JoMa1.3 cells with the Seahorse metabolic analyzer and found a significant diminution of oxygen consumption during basal, maximal and spare respiration. In addition, ATP production was significantly reduced (Figures 3I andS4J). Taken together, these results confirm that Lkb1 regulates the distribution and the activity of mitochondria during glial differentiation (Beirowski et al. 2014;Pooya et al. 2014).

Several reports have shown that Lkb1 and AMPK inactivation trigger a metabolic shift towards aerobic glycolysis [START_REF] Faubert | Loss of the tumor suppressor LKB1 promotes metabolic reprogramming of cancer cells via HIF-1α[END_REF][START_REF] Kishton | AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival[END_REF]Kottakis et al. 2016;Shackelford 2013). Thus, our data demonstrated that Lkb1 has critical functions in the control of energy metabolism during glial differentiation, acting in particular to maintain mitochondria structure and activity.

Rewiring of metabolism in Lkb1-deficient nerves

The mitochondrial effect of Lkb1 ablation in our systems prompted us to characterize the metabolome of sciatic nerves. For that purpose, we performed high-resolution magic angle spinning (HRMAS) proton NMR spectroscopy on sciatic nerves, a technique that does not require prior solubilization of the samples. With the original application of this technique to structurally preserved nerves, we were able to record variations in both polar metabolite and lipid levels in Lkb1 cKO sciatic nerves compared to WT (Figure 4A). The broad resonances (L) arise from lipids and macromolecules, mainly methyl (L1) and methylene groups (L2 to L6) along fatty acids chains while sharp peaks arise from small molecules, i.e. metabolites (Figures 4A andB). We could detect and assign 17 metabolites (Figure 4C), and the main spectral contributions were due to total creatine (8), phospholipid intermediates [START_REF] Bhatt | Signals and Switches in Mammalian Neural Crest Cell Differentiation[END_REF]10,11,12), taurine ( 14) and myo-inositol (15). Peaks corresponding to aliphatic chains of lipids and macromolecules were all down-regulated (Figure 4D). The multivariate statistical model showed a very good separation between the two mouse genotypes (Figure 4E), mainly due to downregulation of all acyl chain groups (L4 to L6). These analyses also showed that the most discriminant changes in metabolite levels between WT and cKO groups concerned b-D-glucose (17), glutamate (6) and alanine (2) (Figure 4C).

Quantification of these metabolites confirmed the statistically significant decrease of b-D-glucose and increase of glutamate and alanine levels in sciatic nerves of cKO animals (Figure 4F). Altogether, these results indicate that Lkb1 loss rewires sciatic nerves metabolism (Figure 4G).

Pyruvate-alanine transamination dictates glial differentiation.

To further characterize how Lkb1 loss triggers acidification rate (ECAR) measurement, were slightly reduced while the glycolytic reserve was diminished in the absence of Lkb1 (Figure S5E). However, upon ALAT inhibition with Chloro-alanine treatment, the extracellular pH of cells with or without Lkb1 was similar, thus demonstrating that glycolysis was not affected (Figure 5C). These data strongly support the hypothesis that the pyruvate is preferentially transformed into alanine upon Lkb1 knockdown independently of glycolytic rate.

Metabolomic profiling of sciatic nerves also identified an increase of glutamate level. Glutamate is required for the transamination of pyruvate (see Figures 4G and5B). We thus determined glutamate levels in JoMa1.3 cells in conditions of glial differentiation.

Lkb1 knockdown during glial differentiation led to glutamate accumulation but only when ALAT is inhibited, most probably because glutamate is readily transformed into alanine by ALAT (Figure 5D).

Analyses of the expression of a set of genes coding for metabolic enzymes during glial differentiation revealed that progenitor cells exhibited an increased expression of the genes coding for the Lactate Dehydrogenase A (LDHA) and the Pyruvate Kinase M2 (PKM2) whereas glial cells had enriched expression of the Lactate Dehydrogenase B (Figure S5F). LDHA and PKM2 are often associated with cell proliferation [START_REF] Wang | p53 and mitochondrial function in neurons[END_REF] and their enriched expression in progenitor cells therefore fits with the neural crest stem phenotype of JoMa1.3 cells.

However, Lkb1 knockdown did not drastically change the expression levels of these metabolic genes (Figure S5G). With a low threshold (over -0.5 or 0.5),

we nevertheless observed that GPT2 expression, which encodes the mitochondrial form of ALAT, was enriched in glial cells upon Lkb1 knockdown, thereby suggesting that alanine accumulation observed in the absence of Lkb1 could result, at least partly, from an elevated expression of GPT2. We also assessed by RT-qPCR the expression of GLUL, which encodes Glutamine Synthetase (GS), and observed that GLUL expression was high in progenitor cells (Figure S5F).

Of note, JoMa1.3 progenitor cells are maintained in a proliferative status by inducible c-Myc ER expression and c-Myc is known to trans-activate the expression of GLUL (Tardito et al. 2015). Thus, progenitor cells artificially express high levels of GS compared to glial cells as confirmed by western blotting (Figure S5H).

Western blot analyses confirmed that the level of GS protein is not affected by the absence of Lkb1 in glial cells (Figure S5I), thereby indicating that GS regulation did not occur neither at transcriptional nor at translational levels.

To examine the impact of alanine and glutamate levels during differentiation of NCC-derived glial cells, we used the ALAT inhibitor Chloro-alanine, since we found that this compound normalized alanine and glutamate concentrations. In these conditions, Chloro-alanine treatment of Lkb1-knockdown cells led to a drastic reduction of actin stress fibers formation (Figure 5E). Furthermore, incubation with the ALAT inhibitor rescued expression of the glial marker S100 (Figure 5F).

We next evaluated the mitochondrial respiration under Chloro-alanine treatment and observed that the impaired oxygen consumption due to Lkb1 silencing was not prevented (Figures 5G).

Conversely, we observed a significant decrease of mTOR activation (Figure 5H), suggesting that this kinase acts downstream of the signaling pathway involving Lkb1 and pyruvate-alanine cycling.

Normalizing NEAA levels or mTOR activity rescues glial differentiation

As glutamate conversion into glutamine is known to be essential during glial differentiation (Saitoh and Araki 2010), we reasoned that GS inhibition should phenocopy Lkb1 silencing in JoMa1.3 differentiation in glial cells. These cells were treated with Methionine Sulfoximine (MSO; Figure 6A), a specific nonreversible inhibitor of GS [START_REF] Ghoddoussi | Methionine sulfoximine, an inhibitor of glutamine synthetase, lowers brain glutamine and glutamate in a mouse model of ALS[END_REF].

MSO treatment triggered glutamate (Figure 6B) as well as alanine (Figure 6C) accumulation in a dose dependent manner. Moreover, incubation of JoMa1.3 cells cultivated in glial differentiation conditions with MSO induced stress fibers formation in a dose dependent manner (Figure 6D) and impaired S100 expression in NCC-derived glial cells (Figure 6E).

We also observed that GS inhibition by MSO led to mTOR activation (Figure 6F). Interestingly, although MSO treatment promoted a reorganization of the mitochondrial network that was similar to the one observed upon Lkb1 knockdown (Figure S5J), the mitochondrial respiration was not decreased but rather increased (Figure S5K). Furthermore, glycolysis was not affected by MSO treatment (Figure S5L). These results therefore establish that GS inhibition blocked glial differentiation and phenocopied Lkb1 silencing.

Interestingly, it has been reported that treatment of cells with AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), the purine precursor for inosine monophosphate (IMP), favored astroglial differentiation (Zang et al. 2008) while AICAR was depleted in glioblastoma cells following GS inhibition (Tardito et al. 2015). AICAR is phosphorylated by adenosine kinase inside the cells and is converted to purine nucleotides, among them ZMP and AMP, two allosteric activators of AMPK [START_REF] Rattan | 5-Aminoimidazole-4-carboxamide-1-beta-Dribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase[END_REF].

Although AICAR has been described as an AMPK agonist in the presence of Lkb1, there is increasing evidence that this molecule exerts AMPKindependent metabolic effects [START_REF] Liu | Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK[END_REF][START_REF] Vincent | Differential effects of AMPK agonists on cell growth and metabolism[END_REF]. Given that GS inhibition phenocopies Lkb1 inactivation, it prompted us to test whether AICAR rescued glial differentiation upon Lkb1 knockdown. AICAR treatment decreased actin stress fibers formation (Figure S6A) and rescued S100 expression (Figure 6G). Similarly, this compound reduced stress fiber formation (Figure S6B) and rescued S100 expression of MSO-treated cells (Figure 6H). Interestingly, glutamate levels were decreased after AICAR addition to Lkb1-silenced cells (Figure S6C) or MSO-treated cells (Figure S6D)

whereas AICAR had no effect on alanine levels (Figure S6E). AICAR treatment affected neither mitochondrial respiration (Figure S6F) nor glycolysis (Figure S6G) but significantly diminished mTOR activation without restoring AMPK phosphorylation (Figure 6I). These findings indicate that AICAR compensated for Lkb1 loss by acting downstream of GS and upstream of mTOR.

Our data hinted at a mechanistic link between the sensing of alanine-glutamate levels and mTOR activity. To address this question, we treated Lkb1 knockdown JoMa1.3 cells with the mTOR inhibitor Torin1. We first showed that Torin1 normalized mTOR activation in JoMa1.3 cells silenced for Lkb1

(Figure S6H). Then, immunofluorescence staining with the glial marker S100 revealed that Torin1 treatment rescued glial differentiation of Lkb1 knockdown cells (Figure 6J). Altogether, these data provide evidence that Lkb1 controls glial cell differentiation through the regulation of alanine and glutamate levels upstream of the mTOR pathway.

AICAR treatment prevents peripheral nerves and ENS defects.

We next explored the in vivo effects of AICAR on Lkb1 cKO mice. Lkb1-deficient mice were treated by intraperitoneal injections of AICAR (0.5 mg/g of body weight) every two days from P10 to P21. We first observed that weight loss of cKO animals was reduced upon AICAR treatment (Figure 7A).

Furthermore, at P21, mutant mice treated with AICAR displayed increased mobility and ameliorated leg reflex extension when lifted by their tail compared to mutant mice treated with vehicle (Figure 7B). In addition, P21 mutant mice treated with AICAR showed less intestinal dilation (Figure 7C). Inhibition of the mTOR signaling was restored upon AICAR treatment both in sciatic nerves (Figure 7D) and in the enteric nervous system (Figure 7E). We did not detect any undesirable side effects induced by AICAR on both wild-type and cKO animals. Strikingly, neuronal and glial enteric cells were maintained in mutant mice with AICAR (Figure 7F). These results

show that AICAR treatment prevented, at least partly, expression of the neurocristopathy phenotype of cKO mice. Collectively, this study and our precedent report (Creuzet et al., 2016) identify the LKB1 pathway as a central orchestrator of neural crest development and highlight the pivotal role of energy metabolism in the formation of NCC derivatives. These results should also extend our understanding of the metabolic etiology of human neurocristopathies.

Discussion

Methods

Animals and genotyping

Floxed Lkb1 mice were obtained from RA. DePinho (Boston, USA) and R26R mice were provided by P.

Soriano (New York, USA). Characterizations of Lkb1

Floxed (Bardeesy et al. 2002), Tyr::Cre (Delmas et al. 2003;[START_REF] Puig | The tyrosinase promoter is active in a subset of vagal neural crest cells during early development in mice[END_REF]) and R26R [START_REF] Soriano | Generalized lacZ expression with the ROSA26 Cre reporter strain[END_REF] mice have been reported previously. Since Tyr::Cre transgene is located on the chromosome X, the study was restricted to male animals. Heterozygous (Lkb1 +/F ) and homozygous (Lkb1 F/F ) males were crossed with Lkb1 +/F Tyr::Cre /° females to generate Lkb1 homozygous conditional knockout in neural crest-derived tissues (referred to as cKO in the text) and wild-type littermate animals (WT: Lkb1 +/F , Lkb1 F/F and Lkb1 +/+ ;Tyr::Cre male mice). Embryos and mice were genotyped by multiplex PCR using DNA extracted from tails with primers indicated in Table 3 and PCR conditions in original references cited previously. For timed pregnancies, mice were bred and the time of plug identification was counted as day 0.5. Mice were maintained under standard housing conditions at the animal facility PHTA of Grenoble, and maintained on a mixed genetic background.

Tyr::Cre/°;Z/EG/° mice were described previously [START_REF] Colombo | Genomic localization of the Z/EG transgene in the mouse genome[END_REF][START_REF] Novak | Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cremediated excision[END_REF]. All animal

Electron microscopy

For electron microscopy, mice were anesthetized with Ketamine/Xylazine, perfused with 2% The same protocol was applied for electron microscopy on JoMa1.3 cells.

Teased fibers preparation and staining

Sciatic nerves were isolated from wild-type or cKO mice, teased on glass-slides, fixed for 30 minutes in 4% paraformaldehyde and subsequently washed in PBS [START_REF] Tricaud | Adherens junctions in myelinating Schwann cells stabilize Schmidt-Lanterman incisures via recruitment of p120 catenin to Ecadherin[END_REF]. After teasing, the nerves

Seahorse analyses

The extracellular acidification rate (ECAR) and O2 consumption rate (OCR) were analyzed by using the Residual water signal was presaturated during the 1.5 s relaxation delay time.

Spectra pre-processing

All pre-processing steps were performed using the TOPSPIN 3.1 Bruker software (Bruker Biospin, Karlsruhe, Germany). Spectra were aligned to TSP signal at zero ppm and phase corrected. A five-order polynomial was systematically applied for baseline correction.

All spectra from 4.678 to 0.60 ppm were divided into intervals or buckets equal to 10 -3 ppm using the AMIX software (Bruker). Each bucket was normalized to total spectrum amplitude. Since this procedure can mask minor peak variations when very intense peaks are present in the spectra, e.g. lipid peaks (fig 4A, L2 peak), we also performed bucketing on a reduced spectral region starting from 1.825 ppm (fig 4A into brackets). Resonance assignment was performed as previously described (for metabolites: [START_REF] Govindaraju | Proton NMR chemical shifts and coupling constants for brain metabolites[END_REF][START_REF] Fauvelle | Prediction of neuroprotective treatment efficiency using a HRMAS NMR-based statistical model of refractory status epilepticus on mouse: a metabolomic approach supported by histology[END_REF]for lipids: Behar et al. 1994;[START_REF] Zietkowski | Detection of cancer in cervical tissue biopsies using mobile lipid resonances measured with diffusionweighted (1)H magnetic resonance spectroscopy[END_REF].

Multivariate statistical Analysis

Buckets were then imported into SIMCA V13 software (UmetricsAB, Umea, Sweden) for multivariate statistical analysis. Buckets were mean centered, scaled to unit variance (i. Tyr::Cre/°;Z/EG/° E14.5 embryo were fixed in PFA 4% for 1h30 at 4°C washed in PBS, incubated in 30% sucrose for 4 hours, embedded in 30% sucrose-50%

OCT and sectioned at 7 mm.

Sections for histology were stained with Harris

Hematoxylin and Eosin (Sigma-Aldrich) according to the supplier's protocol. For immunofluorescence, sections and cells were treated with standard protocols using primary antibodies as described in Table 3. Secondary

Whole-mount Tuj1 immunostaining of enteric nervous system

The enteric neural network was visualized according to the protocol previously described [START_REF] Barlow | Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET[END_REF]Pingault et al. 2010). Briefly, embryonic (E15.5)

and postnatal intestines (P0) were fixed for 2 hours in 4% paraformaldehyde at 4°C. After washing twice in PBT (phosphate buffer saline-0.1% Triton X-100), tissues were incubated overnight with the neural marker Tuj1 (mouse, Covance, 1:1000), diluted in PBT containing 1% BSA and 0.15% Glycine (PBG).

After extensive washings, tissues were then incubated overnight with anti-mouse Cya3conjugated secondary antibody (1/500; Jackson ImmunoResearch). After washing three times in PBG, tissues were counterstained with the nuclei dye Hoechst 33342, mounted in Mowiol and analyzed with a Zeiss AxioImager M2 microscope, equipped with a Hamamatsu Orca R2 camera.

Whole-mount Acetylcholinesterase (AChE) activity staining

Acetylcholinesterase activity was visualized histochemically according to previously described protocols [START_REF] Chalazonitis | Homeodomain interacting protein kinase 2 regulates postnatal development of enteric dopaminergic neurons and glia via BMP signaling[END_REF][START_REF] Enomoto | GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys[END_REF]. Briefly, after gut dissection of P21 animals and removal of mesenteric attachments, tissues were post fixed with 4% PFA for 1-2 hours at 4°C, and transferred to saturated sodium sulfate overnight at 2 for antibodies description).

Tails were collected at the time of dissection for genotyping of the embryos.

For ßgalactosidase immunodetection we used a chicken polyclonal antibody against ßgalactosidase (Abcam) followed by a goat anti chicken IgY secondary antibody conjugated to Cy TM 2 (Abcam).

Neuronal cells were identified using the monoclonal antibody against neuronal class III tubulin (Tuj1, Covance) and glial cells using the rabbit polyclonal anti GFAP (Thermofisher).

Foot printing measurement

Walking pattern was documented in both WT and LKB1 cKO mice at P21 by dipping foot pads of the hind feet into black Chinese ink and allowing the animal to walk on paper strips. Parameters of footprints were evaluated by measuring left and right stride length and sway length (distance between the two feet).

Western blot analysis.

Western blot analysis was performed as described previously (Nony et al. 2003). Briefly, JoMa1.3 cells were lysed in classical lysis buffer (Hepes 50 mM, NaCl 150 mM, EDTA 5 mM, 1% NP40) and 20 mg of protein extracts were separated in SDS-10% polyacrylamide gels and transferred to PVDF Immobilon-P membranes (Millipore). After blocking, membranes were incubated overnight with different antibodies as described above and in Table 4 in blocking solution at 4°C and with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (Biorad) for 1 h at room temperature. Immunoreactive proteins were detected with Amersham ECL chemiluminescence reagents (GE Healthcare) according to the manufacturer's instructions. Quantifications were performed using ImageJ software. Each band intensity for phosphoand total-proteins were first normalized to actin and then phospho-proteins were normalized to total proteins. Values for siLkb1 condition were indexed to siCont.

MitoTracker staining

After the first cycle of siRNA transfection against Lkb1, 2x10 4 JoMa1.3 cells/ml were seeded on

RNA extraction and quantitative Real-Time PCR (RT-qPCR)

RNAs were extracted from about 10 7 JoMa1.3 cells per condition using the Nucleospin miRNA Kit (Macherey-Nagel) according to the manufacturer's protocol and was quantified using a Nanodrop 1000.

A total of 1mg was reverse transcribed using the i-Script gDNA Clear cDNA Synthesis (Biorad). For primer pairs giving no Ct before 30 cycles (SOX10, p75 NTR , GFAP and S100B), a pre-amplification step was performed using the SsoAdvanced Preamp Supermix (Biorad) according to the protocol. Relative quantitative PCRs were performed using Sso Advanced Universal SYBR Green Supermix (Biorad) with 2ml of cDNAs diluted 1/5 (in triplicates) and run on the CFX96 Touch Real-Time PCR Detection System (Biorad). Negative controls were performed in the same conditions without the transcriptase reverse. The housekeeping genes AUP1 and RELA were used as controls. For each gene, expression levels were normalized to AUP1 housekeeping genes using the Delta Ct method. For each point, results

were expressed as a ratio (progenitor/glial or siLkb1/siCont.) of the means of DeltaDeltaCt values +/-standard deviation using data from 4 or 5 independent RT-qPCR experiments.

Conclusions

During this second project which represents my main PhD project, I identified that Lkb1 is crucial for the specification and maintenance of NCC-derivatives in the peripheral nervous system. Our work unveils a critical function of Lkb1 in Schwann cell lineage as well as in the post-natal maintenance of myenteric and submucosal plexi of the enteric nervous system.

Several articles have already underlined the role of Lkb1 in Schwann cell maturation and differentiation (Beirowski et al., 2014;Pooya et al., 2014;Shen et al., 2014). In these studies Lkb1 was inactivated specifically in Schwann cells by using:

-myelin protein zero (P0)::Cre driver expressed at E14.5 in immature Schwann cells (Beirowski et al., 2014;Feltri et al., 1999) (Figure 1).

or by using 2'3'-cyclic nucleotide 3'-phosphodiesterase (Cnp)::Cre driver. Cnp is expressed at E12.5 in neural crest cells and in central nervous system, in oligodendrocytes [START_REF] Chandross | Identification and Characterization of Early Glial Progenitors Using a Transgenic Selection Strategy[END_REF][START_REF] Genoud | Notch1 control of oligodendrocyte differentiation in the spinal cord[END_REF]Shen et al., 2014).

In a later stage, Cnp is expressed in myelin-forming Schwann cells (Figure 1) (Pooya S et al., 2014). Cnp::Cre expression is higher in the later stage and Lkb1 role, in the literature, was associated only to this myelinating stage (Pooya S et al., 2014;Shen et al., 2014).

However, both existing models are not appropriated to study Lkb1 functions in NCC lineage because P0 is expressed after NCC specification and Cnp is expressed also in central nervous system. Thereby, in our study, by using Tyrosinase (Tyr)::Cre driver we inactivated Lkb1 specifically in NCC before their commitment to melanocytic, enteric or Schwann lineages (Figure 1), making it a good model to study NCC lineage.

Furthermore, by using this model and a NCC line able to be differentiated in vitro in glial cells, I uncovered a link between pyruvate-alanine cycling, the mTOR pathway and glial differentiation. Lkb1 controls glial commitment by regulating the non-essential amino acids maturation. This phenotype is restored by Rapamycin, an mTORC1 inhibitor (Beirowski et al., 2017). The value-added contribution of our study to the existing literature is the original mechanism by which Lkb1 regulates mTOR activation through alanine-pyruvate transamination. Moreover, in our study we highlighted that mTOR is not only important for Schwann cell maturation, but also earlier during NCC specification.

Several researchers have shown that AICAR reverts metabolic syndromes in murine models like insulin-resistant diabetic (ob/ob) mice [START_REF] Song | 5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice[END_REF], obese (fa/fa) Zucker rats [START_REF] Bergeron | Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis[END_REF][START_REF] Buhl | Chronic treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside increases insulinstimulated glucose uptake and GLUT4 translocation in rat skeletal muscles in a fiber type-specific manner[END_REF] and insulin-resistant high-fat fed rats [START_REF] Iglesias | AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats[END_REF]. In our study we have shown that AICAR rescued glial differentiation of Lkb1-deficient NCC and treatment of Lkb1-deficient mice with AICAR prevented the peripheral nerve and enteric nervous system phenotypes.

Even if AICAR has promising effects in murine models, it is not used in treatment of human pathologies like type 2 diabetes or other metabolic diseases because of its short half-life and poor bioavailability which compels to use high doses causing side effects of liver hypertrophy and lactic acidosis [START_REF] Coughlan | AMPK activation: a therapeutic target for type 2 diabetes?[END_REF][START_REF] Winder | Can Patients with Type 2 Diabetes Be Treated with AMPK-Activators?[END_REF]. Therefore it will be interesting to explore if other treatments like inhibitors of transaminases or diet alimentation are also able to prevent Lkb1-dependent NCC defects.

The phenotypes of the two mouse models with conditional inactivation of Lkb1 specifically in NCC, presented in Projects 1 and 2 ("early" and "late" models respectively) recapitulate many clinical features of human NCC disorders called neurocristopathies (see Introduction, Chapter II.3). This suggests that defects of Lkb1 signaling could be involved in a wider range of human pathologies than only the Peutz-Jeghers syndrome as described so far. depending on the stress level, on prooxidant or antioxidant actors. Along this line, ROS limitation, p53 genetic loss or p53 inhibition with the chemical inhibitor pifithrin-α prevented neural crest defects in several genetic mouse models (Hayano et al., 2015;Jones et al., 2008;[START_REF] Mcgowan | Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects[END_REF]Noack Watt et al., 2016;Pani et al., 2002a;Sakai et al., 2016;[START_REF] Van Nostrand | The p53 family members have distinct roles during mammalian embryonic development[END_REF][START_REF] Watkins-Chow | Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes[END_REF]; Introduction, Chapter III.B.4). Altogether, these data support the important contribution of oxidative stress and p53 signaling to neural crest formation and open new therapeutic strategies to counteract neural crest defects (Danilova and Gazda, 2015a;Van Nostrand and Attardi, 2014). and Kalcheim, 1999). In human, defects in NCC formation lead to a wide group of disorders called neurocristopathies or ribosomopathies when genetic mutations affect ribosomal functions.

Cranial NCC naturally produce high levels of endogenous oxidative stress (Sakai et al., 2016).

Furthermore, NCC are very sensitive to exogenous oxidative stress. Alcohol exposure in fetal alcohol disorders or high glucose levels in case of maternal diabetes are examples of causative agents leading to accumulation of reactive oxygen species (ROS) in NCC (Chen et al., 2013a[START_REF] Chen | The negative influence of high-glucose ambience on neurogenesis in developing quail embryos[END_REF]Morgan et al., 2008;Sakai and Trainor, 2016;Wei and Loeken, 2014).

Additionally, aberrant signaling of the tumor suppressor and transcription factor p53 was observed in several genetic models targeting NCC formation (Danilova et al., 2010;Jones et al., 2008;Noack Watt et al., 2016;Pani et al., 2002b) as well as in patients with neural crest defects (Van Nostrand and Attardi, 2014). Half-life stabilization of p53

protein by blocking its degradation by Mdm2 triggers typical neural crest defects (Rinon et al., 2011;Van Nostrand et al., 2014b). Genetic loss or chemical inhibition of p53 prevented neural crest defects in several genetic models (Hayano et al., 2015;Jones et al., 2008;Noack Watt et al., 2016;Pani et al., 2002b;Van Nostrand et al., 2014b). It was shown recently that p53 activation is the result of increased oxidative stress and subsequent DNA damages (Sakai et al., 2016). Altogether, these data support the essential contribution of oxidative stress and aberrant p53 signaling to defects in crest formation (Danilova and Gazda, 2015b;Van Nostrand and Attardi, 2014).

We recently discovered the tumor suppressor and serine/threonine kinase LKB1 (encoded by the STK11 gene) as a major actor of NCC formation. We described that LKB1 is required for vertebrate head formation through the control of delamination, migration, survival and differentiation of cephalic NCC (Creuzet et al., 2016). We also highlighted that LKB1 is essential for the maintenance of the enteric nervous system as well as for glial commitment of the Schwann cell lineage (Radu et al., submitted). Our mechanistic analyses uncovered that LKB1 is required for glial commitment through the control of pyruvate-alanine cycling upstream of mTOR limitation. Interestingly, AICAR, an AMP precursor, prevented neural crest defects due to Lkb1 loss in vivo (Radu et al., submitted).

LKB1 acts through the activation of 14 AMPK-related kinases that regulate cell polarization and energy metabolism during cell proliferation and differentiation among several cellular processes (Alessi et al., 2006b;Hezel and Bardeesy, 2008;Shackelford and Shaw, 2009;Shorning and Clarke, 2011). Regulation of energy metabolism by LKB1 occurs through the activation of the AMP-activated protein kinase (AMPK), a sensor that adapts energy supply to the nutrient demands of cells facing metabolic stress (Shackelford, 2013). LKB1 has been also shown to limit oxidative stress, thereby protecting the genome from oxidative damage (Xu et al., 2015). Furthermore, LKB1 preserves genome integrity and therefore protects cells from genotoxic stress through activation of the DNA repair BRCA1 pathway (Gupta et al., 2015).

To better understand if oxidative stress and p53 signaling contribute to LKB1-dependent control of NCC fate, we measured oxidative stress levels upon Lkb1 knockdown during glial commitment of NCC.

We report here, using a neural crest stem cell line,

Results

Lkb1 loss triggers oxidative DNA damages Among metabolites previously identified by 1 H HRMAS RMN metabolomics profiling of sciatic nerves from wildtype or conditional NCC Lkb1 knockout animals (Tyr::Cre;Lkb1 F/F mice; Radu et al., submitted), we observed that several antioxidant metabolites were upregulated upon Lkb1 loss (Fig. 1A) as quantified for total creatine (Fig. 1B). This observation is suggestive of increased oxidative stress upon Lkb1 inactivation as supported by data from the literature (Gupta et al., 2015;Xu et al., 2015). We therefore measured intracellular ROS levels in vitro using DCFDA, a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity, in the JoMa1.3 mouse neural crest stem cell line that can either be maintained in a progenitor state or differentiated into glial cells among other NCC derivatives (Maurer et al., 2007). DCFDA fluorescence intensity was increased upon Lkb1 knockdown in JoMa1.3 cells committed to glial lineage compare to control cells (Fig. 1C). This fluorescence indeed corresponds to increased ROS levels as it was completely abrogated by pre-treating the cells with the antioxidant N-Acetyl-Cystein (NAC) (Fig. 1D). Next, we assessed if Lkb1 knockdown was also associated with oxidative DNA damage as a direct consequence of elevated ROS.

Immunostaining of cells of -H2AX, a DNA damage marker, showed a clear increase in labelling (Fig. 1E). Pre-treatment of cells with the antioxidant NAC partially prevented the accumulation of DNA damage (Fig. 1F). Altogether, these data demonstrate that Phosphorylation of p53 at serine 15 has been associated with transcriptional activity of p53 (Khanna et al., 1998). We therefore evaluated p53 transcriptional activity upon Lkb1 knockdown in Together, these data thereby highlighted aberrant p53 signaling upon Lkb1 loss as a consequence of increased oxidative stress.

Lkb1 sustains lysosomal proteolytic activity p53 signaling is intimately related to autophagy flux (Kruiswijk et al., 2015). We therefore explored if hyper-phosphorylated p53 resulted in defective autophagy. Lkb1 silencing in JoMa1.3 cells upon led to massive LC3-II accumulation and p62 degradation as assessed by western blot analyses (Fig. 3A).

Increased autophagy was also detected in vivo by immunofluorescence staining of LC3 on sciatic nerves sections of Lkb1 cKO animals compared to WT mice (Fig. 3B).

While performing immunofluorescence analysis of JoMa1.3 cells, we noticed upon Lkb1 loss the presence of vesicles with a strong autofluorescence.

Cells analyses without any labelling, confirmed the presence of autofluorescent vesicles with green brighter than blue or red fluorescence (Fig. 3C).

Using confocal spectral microscopy, we better characterized the spectral properties of these autofluorescent inclusions and showed that control In vivo, we did not detect strong accumulation of such autofluorescent inclusions neither in dissociated nerves fibers nor in nerve sections upon Lkb1 loss.

However, while analyzing electron microscopy data performed on sciatic nerve sections of Lkb1 cKO animals, we observed that myelinating Schwann cells upon Lkb1 loss harbored aberrant lysosomal structures corresponding to polymorphic late endosomes (Fig. 3F).

Off note, we observed that although phosphorylated p53 was localized in the nucleus of some cells, in others it localized at specific perinuclear structures that correspond to autofluorescent inclusions (Fig. 3G).

AICAR rescues glial differentiation by preventing p53 activation and lysosomal defects

In our previous study, we demonstrated that AICAR, an AMP precursor, was able to rescue glial differentiation in vitro upon Lkb1 silencing and prevented in vivo the neural crest defects due to Lkb1 inactivation (Radu et al., submitted). Surprisingly, when analyzing immunofluorescence staining of JoMa1.3 cells with the glial marker S100, we noticed a disappearance of the perinuclear inclusions upon AICAR treatment (Fig. 4A). Western blot analyses upon AICAR treatment showed that AICAR limited p53 phosphorylation at Serine 15 (Fig. 4B).

Immunofluorescence staining in JoMa1.3 cells confirmed that AICAR prevented phospho-p53 accumulation (Fig. 4C). Together, these data suggest that AICAR treatment prevented p53 activation and rescued lysosomal homeostasis. LKB1/AMPK and mTOR signaling during metabolic modulations during differentiation (Radu et al., submitted). Both mTOR and AMPK control lysosomal homeostasis. mTOR activity triggers lysosomal tubulation, a dynamic process that requires lysosome moving along microtubules (Hipolito et al., 2018).

Discussion

Indeed, mTOR stimulates the level of Arl8b GTPase on lysosomes which boosts the motor protein kinesin-1 activity and therefore lysosome extension.

AMPK inhibits both mTORC1/2 and the transcription factor TFEB, a master regulator of a transcriptional program of cellular adaptation to stress (Hipolito et al., 2018;Martina and Puertollano, 2017).

Collectively, our data uncovering the crucial involvement of LKB1/AMPK in NCC formation also highlighted how this signaling impacts cytoskeleton dynamic. Indeed, LKB1 contributes to cranial NCC formation through a sequential activation of the AMPK/Rho/ROCK1 pathway to control phospho-MRLC and actin dynamic (Creuzet et al., 2016). We also described that metabolic deregulations of amino acid levels and impaired glial differentiation are associated with massive actin and tubulin cytoskeleton reorganization upon Lkb1 loss (Radu et al., submitted). Moreover, LKB1 through its downstream kinase NUAK1, immobilizes mitochondria along microtubules, an essential process during terminal axon branching in neurons (Courchet et al., 2013).

Our observations, collectively with massive data from the literature, suggest that impaired LKB1/AMPK activity triggers a metabolic energy imbalance with a drop cellular ATP pool. Along this line, we identified that the AMP precursor AICAR is able to rescue glial differentiation (Radu et al., submitted). In this study, we also observed that AICAR limits oxidative DNA Neurocristopathies represent a large group of human diseases due to defects in neural crest formation.

These pathologies associate several clinical phenotypes such as craniofacial abnormalities, hearing loss, megacolon (as a result of enteric ganglia agenesis), cardiac or pigmentation defects (Mayor and Theveneau, 2013) (Mayor and Theveneau, 2013). Similar abnormal development of the neural crest also occurs in diseases with deficient ribosome biogenesis called ribosomopathies, besides defects in blood cells formation leading in particular to anemia (Danilova and Gazda, 2015b).

Many genetic factors are associated with the etiology of these pathologies. However, these diseases often present a phenotypic variability that is independent of the genotype and which could be the results of environmental factors leading to oxidative stress.

Multiple reconstructive surgeries improve the condition of patients with craniofacial malformations whereas removal of the aganglionic segment is the current treatment for megacolon. Nonetheless, surgery is rarely fully corrective and efforts have been made recently to explore preventive therapeutic approaches. Limiting oxidative stress in particular gave very promising results (Sakai et al., 2016).

Therefore, future exploration of LKB1-controlled oxidative DNA damages during neural crest formation will be an essential part of a more general approach to identify molecular events limiting neural crest defects upon oxidative stress and which will be of crucial importance in developing potential preventing therapies. For immunofluorescence, sections and cells cultivated on glass coverslips were treated with standard protocols using primary antibodies as described in Table 3. Secondary antibodies used Western blot analysis.

Methods

Animals and genotyping

Western blot analysis was performed as described previously (Radu et al., submitted). Antibodies and blotting conditions used for this study are listed in Table 1. Quantifications were performed using ImageJ software. Each band intensity for phosphoand total-proteins were first normalized to actin and then phospho-proteins were normalized to total proteins. Values for siLkb1 condition were indexed to siCont.

RNA extraction and quantitative Real-Time PCR (RT-qPCR)

RNAs from glial committed cells transfected with nontargeting or Lkb1-targeting siRNA were extracted, reverse transcribed and relative quantitative PCRs were performed as previously described (Radu et al., submitted). Negative controls were performed in the same conditions without the transcriptase reverse.

The housekeeping genes AUP1 and RELA were used as controls. For each gene, expression levels were normalized to AUP1 housekeeping genes using the Delta Ct method. For each point, results were expressed as a ratio (progenitor/glial or siLkb1/siCont.) of the means of DeltaDeltaCt values +/-standard deviation using data from 4 or 5 independent RT-qPCR experiments.

Electron microscopy

Electron microscopy analyses were performed on sciatic nerve sections of Tyr::Cre;Lkb1;R26R mice and JoMa1.3 cells using the same protocol as described previously (Radu et al., submitted).

Graphical representations and Statistical analyses

All cell culture experiments were realized at least by 5A). For in vivo analyzes, Anthony Lucas has undertaken crossing of "early" NCC-inactivated Lkb1 mice (Ht-PA::Cre, Lkb1 Fl/Fl ) with heterozygous or homozygous p53 loss mice (Figure 5B). Phenotypic characterization of these mice will be performed on tissues previously described to exhibit defects upon Lkb1 loss:

formation of the head, enteric nervous system, sciatic nerve and melanocytes. Epithelia are polarized cells that form barriers crucial for tissue homeostasis [START_REF] Marchiando | Epithelial barriers in homeostasis and disease[END_REF]. The primary characteristic of epithelial barrier formation is the intercellular tight junction present along plasma membrane [START_REF] Marchiando | Epithelial barriers in homeostasis and disease[END_REF]. This is particularly true in testes, where Sertoli cells, which are epithelial somatic cells of the seminiferous tubules, are forming the blood-testis barrier through tight, adherent and gap junctions [START_REF] Mruk | The Mammalian Blood-Testis Barrier: Its Biology and Regulation[END_REF]. This structure supports the maturation of germ cells and prevents their recognition by immune cells [START_REF] Jiang | Blood-testis barrier and spermatogenesis: lessons from genetically-modified mice[END_REF]. Perturbation of these junctions represents both the effect and the cause of human diseases. For example, patients with Peutz-Jeghers syndrome have modifications of tight junctions in the blood-testis barrier [START_REF] Bertoldo | AMPK: a master energy regulator for gonadal function[END_REF]. Disruption of blood-testis barrier cause immunological or other damages to meiotic and postmeiotic germ cells and consequently promotes alteration of sperm production and male infertility [START_REF] Jiang | Blood-testis barrier and spermatogenesis: lessons from genetically-modified mice[END_REF].

Interestingly, Sertoli cells provide the adequate ionic and metabolic environment (export of alanine and lactate) essential for germ cells maturation. Thus, the metabolic communication between Sertoli cells-germ cells relies on a very similar process that the one for glial-neuronal cells described in Introduction (Chapter I.3.2).

Like Sertoli cells, intestinal epithelial cells are forming a barrier, but only through tight junctions, which is crucial for tissue homeostasis. It controls microbial interactions and contributes to nutrient and water transport [START_REF] Peterson | Intestinal epithelial cells: regulators of barrier function and immune homeostasis[END_REF]. Intestinal epithelial cells metabolism is also important for a cellular dialogue with their neighboring cells (see Introduction, Chapter I.3.2).

LKB1 is required for both Sertoli cells and intestinal epithelial cells polarity to control the establishment of blood-testis barrier [START_REF] Tanwar | Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis[END_REF] and intestinal barrier respectively [START_REF] Partanen | Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4[END_REF]. Several studies identified a link between energy metabolism and polarity [START_REF] Ghosh | The stress polarity pathway: AMPK 'GIV'-es protection against metabolic insults[END_REF]see Introduction Chapter III.A.3.2). For instance, AMPK stabilizes tight junctions and regulates cell polarity and intestinal epithelial barrier (Aznar et al., 2016) and malfunctions of this barrier have been associated with metabolic disorders [START_REF] Bauer | Targeting the gastrointestinal tract to treat type 2 diabetes[END_REF][START_REF] Mccreight | Metformin and the gastrointestinal tract[END_REF][START_REF] Wu | New insights into the anti-diabetic actions of metformin: from the liver to the gut[END_REF]. Therefore, targeting AMPK-dependent stress polarity pathway has been proposed as a therapeutic strategy to control the gut barrier and consequently to correct metabolic disorders [START_REF] Ghosh | The stress polarity pathway: AMPK 'GIV'-es protection against metabolic insults[END_REF].

However, the exact molecular mechanisms by which LKB1 regulates energy metabolism in Sertoli and intestinal epithelial cells have been less explored.

Thus, we first analyzed LKB1 functions in Sertoli polarity and cell metabolism as well as its role in cellular communication between Sertoli cells and the neighboring cells. Mullerien Hormone (AMH) Receptor [START_REF] Tanwar | Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis[END_REF]. This inactivation leads to Sertoli cell polarity loss, perturbation of blood-testis barrier as well as suppression of AMPK and activation of mTOR signaling. However metabolic defects were not explored in this study.

To explore LKB1 metabolic regulations in Sertoli cells, our team generated a mouse model of conditional ablation of Lkb1 specifically in these cells. To do so, Lkb1 Flox mice were crossed with mice expressing the recombinase Cre under the promoter of AMH, generated by F. Guillou and P. Crepieux, INRA, Rennes [START_REF] Lécureuil | Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice[END_REF]. Using the AMH::Cre model, the team confirmed the crucial role of Lkb1 in fertility since mutant male mice were sterile with no mature germ cells neither in testis nor epididymis. Seminiferous tubules were totally disorganized in the absence of Lkb1. Sertoli cell polarity was impaired and the hematotesticular barrier was absent. The team also observed increased proliferation of myoid and Leydig cells.

During my PhD I contributed to the phenotypic characterization of these mutant mice.

I have shown that Lkb1 loss triggers a mislocalization of immature germ cells.

Further, the team explored whole testis metabolism and observed an aberrant upregulation of the nutrient-sensing mTOR pathway as well as lipids and carbohydrate accumulation within and around seminiferous tubules in the absence of Lkb1. Furthermore, HRMAS RMN metabolomics analyses of testes revealed profound metabolic deregulations upon Lkb1 loss with increased levels of several metabolites such as alanine and glutamine.

To spermatocytes II that have 50% of the number of chromosomes. Spermatocytes II enter also in meiosis and produce haploid round spermatids which maturate into spermatozoa [START_REF] Laiho | Transcriptome profiling of the murine testis during the first wave of spermatogenesis[END_REF]. The first wave of maturation of spermatogonia into round spermatids starts a few days after birth, takes approximately 35 days and is synchronized [START_REF] Oakberg | Gamma-ray sensitivity of spermatogonia of the mouse[END_REF][START_REF] Tanaka | Gene expression in spermiogenesis[END_REF]. Spermatocytes appears at 9 days after birth (P9) and spermatid appear at P20 [START_REF] Bellvé | Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization[END_REF]. The seminiferous tubules are surrounded by myoid cells while Leydig cells are localized in interstitial spaces adjacent to seminiferous tubules (Fig. 1A).

The team has recently demonstrated that Lkb1 in Sertoli cells is crucial for fertility since male mice which are not expressing Lkb1 specifically in Sertoli cells were sterile. Macrosopic observations of testis revealed that cKO mice exhibited increased testis weight at 3 weeks after birth (P21) called macro-orchidy followed by a progressive regression of the testicular volume and a marked micro-orchidy at 3 months after birth (P90). In order to better characterize the macro-orchidy, I quantified the surface of seminiferous tubules of wild type and Lkb1 conditional knockout mice at P21, on testis cross-sections. Thus, I have shown that the increased testicular weight is associated to an increase of the seminiferous tubules area (Fig. 1B).

The team underlined that Lkb1 in Sertoli cells limits neighboring cells proliferation and preserves tissue homeostasis of the testis. Indeed, in the absence of Lkb1 in Sertoli cell, they observed an aberrant hyper-proliferation of myoid and Leydig cells which correlates with the macro-orchy. The team also observed an absence of spermatozoid in seminiferous tubules or epididymis of mutant mice at 90 days after birth (P90). However, the role of Lkb1 in Sertoli cell on germ cell proliferation and differentiation remained to be clarified.

To address this question I first analyzed the number of germ cells in seminiferous tubules in the presence or absence of Lkb1 and uncovered that Lkb1 loss did not affected the total number of germ cells per mm² (Fig. 1C). However, considering that the surface of seminiferous tubules is increased, the total number of germ cells also increased. This result suggests that Lkb1 expressed in Sertoli cells regulates germ cell proliferation.

Second, I performed immunolabelling of VASA, a cytoplasmic protein specifically expressed in germ cells from spermatogonia to round spermatids stages, with its highest expression being at the early spermatocyte stage. In spermatogonia, the accumulation of VASA protein is much lower and depends on the stage of differentiation. It has been suggested that weak VASA staining corresponds to type A spermatogonia (undifferentiated state with self-renewal capacity) also called germ stem cells, while intermediate staining corresponds to type B spermatogonia (differentiated into spermatocyte) [START_REF] Castrillon | The human VASA gene is specifically expressed in the germ cell lineage[END_REF][START_REF] Toyooka | Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development[END_REF].

As the first wave of germ cell maturation in the testis is synchronized, the most mature germ cells at P21 are spermatocytes [START_REF] Bellvé | Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization[END_REF][START_REF] Laiho | Transcriptome profiling of the murine testis during the first wave of spermatogenesis[END_REF]. As observed in Fig. 1D, I have shown that in the absence of Lkb1 there is the same number of cells with low VASA staining (at the basal membrane) compared to control and therefore, same number of spermatogonia. In our experiments, VASA immunostaining did not allow us to distinguish between spermatogonia type A (Ad and Ap) or Type B (Figure 1.A right panel).

Further, in order to better characterize the germ cell differentiation, I performed immunostaining for synaptonemal complex protein 3 (SCP3), a specific marker of meiotic germ cells (spermatocyes I and II). At P21 there was no significant difference between the numbers of SCP3 positive cells in wild type or Lkb1 conditional knock out murine testes (Fig. 1E).

Instead, SCP3 positive cells localization in seminiferous tubules was aberrant upon Lkb1 loss.

In normal conditions, SCP3 positive cells are more at the basal membrane than in absence of Lkb1 (Figu.1E). Mislocalization of SCP3 positive cells upon Lkb1 loss was even more pronounced at P90 (Fig. 1F). In normal conditions, spermatocytes are organized in one cell layer near the basal part of the tubules whereas spermatozoa (which have a smaller nucleus) are localized in the tubule's lumen. Instead, in Lkb1 conditional knockout murine testes, the germ cell distribution is disorganized, with many spermatocytes in the tubule's lumen and no spermatozoa (Fig. 1F).

Interestingly, the number of spermatocytes was not affected at P21 upon Lkb1 loss.

However, Lkb1 absence triggered their mislocalization in the seminiferous tubule. This suggest that impaired maturation of germ cells into spermatozoa may be a consequence of spermatocytes mislocalization which probably is due to the disruption of blood-testis barrier integrity. Thus, these results suggest that Lkb1 expressed in Sertoli cells regulates germ cell proliferation, localization and consequently is involved in their differentiation.

Lkb1 inactivation compromises Sertoli cells metabolism.

Previous data of the team have established that the absence of Lkb1 in Sertoli cells rewires testis metabolism. First, the team showed on transverse sections of testes from wild type and Lkb1 conditional knockout mice that Lkb1 controls AMPK/mTOR pathway known to be essential for metabolic regulation. Second, they observed that Lkb1 loss affects lipid and glucid metabolism. Finally, they characterized the metabolome of testes. For that purpose, they performed high-resolution magic angle spinning (HRMAS) proton NMR spectroscopy on testes at P21 and P90 (with F. Fauvelle, GIN, Grenoble, France). Most discriminant changes in metabolite levels between WT and Lkb1 conditional knockout testes concerned alanine, glutamine, lactate, succinate and glucose, all being increased.

In order to better understand the mechanisms underlying Lkb1-dependent metabolic regulation, I used a murine Sertoli cell line. By using this cell line, I confirmed that absence of Lkb1 in Sertoli cells led to decreased AMPK activity associated with up-regulation of the mTOR pathway (Fig. 2 A). I also assessed the energy metabolism of Lkb1-deficient Sertoli cells with the Seahorse metabolic analyzer. I uncovered that the basal glycolysis, glycolytic capacity and the glycolytic reserve, based on extracellular acidification rate measurement, were not affected by Lkb1 knockdown (Fig. 2B). However, oxygen consumption during basal and maximal respiration was strongly increased (Fig. 2C). In addition, ATP production was significantly increased (Fig. 2C).

To evaluate how polarity and metabolism impact Sertoli functionality, I also analyzed Lkb1 role during phagocytosis, a well characterized Sertoli cell function. Preliminary results have shown that absence of Lkb1 triggered a decrease in phagocytosis (Fig. 2D). Further, our team will confirm this result and will analyze other Sertoli cell functions such as cell polarity through the establishment of cell-cell junctions and the role of metabolism in the control of these functions.

Taken together, these data demonstrate that Lkb1 has critical functions in the control of energy metabolism of Sertoli cells and tissue homeostasis and a publication is in preparation. [START_REF] Filipponi | Repression of kit expression by Plzf in germ cells[END_REF][START_REF] Phillips | Spermatogonial stem cell regulation and spermatogenesis[END_REF], need to be done in order to better characterize the role of Lkb1 in germ stem cell functions such as self-renewal capacity and differentiation.

Interestingly, similarly to our previous study on neural crest derivatives of the peripheral nervous system, Lkb1 loss in testis triggers an increase in non-essential amino acids such as alanine and glutamine. Altogether, these projects regarding the role of Lkb1 in neural crest cells and Sertoli cells allowed us to uncover that Lkb1 signaling exerts a crucial control of energy metabolism via non-essential amino acids levels during cell differentiation and maintenance and that this process is mainly conserved across tissues.

Materials and methods

Animals

Floxed Lkb1 mice were obtain from RA. DePinho (Boston, USA). For the generation of Sertoli cells specific Lkb1 null mice, conditional Lkb1 floxed mice were bred with AMH-Cre mice (Lecureuil et al., 2002). Homozygous floxed Lkb1 animals expressing Cre recombinase were considered as Sertoli specific knockout mice. Mice were maintained under standard housing conditions and maintained on a mixed genetic background.

Cell line and cell culture

The mouse TM4 Sertoli cells was obtained from American Tissue Culture Collection. For Immunofluorescence, P21 paraffin testes sections were treated with standard protocols using 10mM EDTA antigen retrival buffer (pH9) and SCP3 antibody (Abcam, ab15092) at 1/500 overnight. P90 frozen testes sections were incubated with PBS-BSA 0.3%-Triton100X 0.5% for 10 min and after were incubated for 1h with PBS-BSA 3%-GPI 10% and with the same SCP3 antibody at the same concentration as above, overnight. Secondary antibody used was from Jackson ImmunoResearch, a Donkey anti-Rabbit Cya3 (1/1000).

Nuclei were stained with Hoechst 33342 (10 µg/ml, Invitrogen).

Western blot analysis

Western blot analysis was performed as described previously (Radu et al., submitted). Briefly, 20g of protein extracts were separated in SDS-10% polyacrylamide gels and transferred to PVDF Immobilon-P membranes (Millipore). After blocking, membranes were incubated overnight with different antibodies as described Table1 in blocking solution at 4°C and with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (Biorad) for 1 h at room temperature. ImageJ software was used to quantify the intensity of phosho-AMPK, phosho-S6Rp, AMPK, S6Rp and β-actin bands. Each intensity measurement for phospho-AMPK and phospho-S6 was divided by the corresponding total AMPK and S6. The resulted values, were then divided by the corresponding standardized β-actin measurement, and the numbers are normalized to the control.

Table1. Primary antibodies used for western blot analysis

Seahorse analysis

The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analyzed by using the Seahorse XF96-Analyzer (Seahorse Bioscience). 1.5×10 4 TM4 cells were seeded in 96-well Seahorse plates in normal cell culture medium 16 h before assay.

Seahorse analyzes were done as previously described (Project 2.1; Radu et al, submitted).

Briefly, the media was replaced with Seahorse assay media specific for OCR or ECAR measurements and equilibrated in non-CO2

incubator for 1 hr. Basal OCR and ECAR were measured for 3 min every 10 min for 3 points, followed by sequential injection of different drogues (Table2). OCR and ECAR were normalyzed by cell density. For both respiration and glycolysis, quantifications were performed by calculating for each category the mean and standard deviation from the values obtained from four independent experiments.

Phagocytosis

To assay the phagocytosis activity, TM4 cells were plated at 2*10 4 cells/well, in a 6 well plate and 24h after, the cells were transfected with siRNA targeting Lkb1 (siLKB1) or Nontargeting control siRNA (siControl) for 72h. After the 72h, the cells were incubated with F8816 Several years ago, MEX3, an RNA-binding protein, has been characterized as a novel downstream target of Lkb1 important for Caenorhabditis elegans embryogenesis [START_REF] Draper | MEX-3 is a KH domain protein that regulates blastomere identity in early C. elegans embryos[END_REF][START_REF] Huang | MEX-3 interacting proteins link cell polarity to asymmetric gene expression in Caenorhabditis elegans[END_REF]. The nematode LKB1 homologue, Par4, negatively regulates MEX3 activity in the posterior part of the early embryo and thus controls the formation of the anterior-posterior embryo axis [START_REF] Huang | The RNA binding protein MEX-3 retains asymmetric activity in the early Caenorhabditis elegans embryo in the absence of asymmetric protein localization[END_REF]. M. Billaud lab then identified and characterized the 4 mammal MEX3 proteins called MEX3A to MEX3D [START_REF] Buchet-Poyau | Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies[END_REF]. However, MEX3 functions and contribution to LKB1 signaling in mammals are still largely unknown.

Our team and others identified that members of MEX3 family are involved in fertility by controlling Sertoli cell polarity [START_REF] Le Borgne | The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway[END_REF] or in energy metabolism balance (Jiao et al., 2012a(Jiao et al., , 2012b)). To further explore the coordinated regulation between cell polarity and metabolism, we developed a collaboration with Almeida R. lab. They have shown that MEX3A is involved in intestinal differentiation, polarization and stemness associated with gastrointestinal carcinogenesis [START_REF] Pereira | CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness[END_REF]. However, the exact mechanism by which MEX3A controls these biological functions is not known. Therefore, the aim of R. Almeida lab was to better define MEX3A involvement in regulation of cellular differentiation, metabolism and polarity in gastrointestinal tract during homeostasis and carcinogenesis. Furthermore, R.

Almeida lab showed a novel putative role for mammalian MEX3A on the regulation of the Lkb1-AMPK-mTOR signaling pathway.

Thus, in the continuity of my previous studies and in collaboration with R. Almeida lab, I characterized the metabolic status in another polarized cell type that is the colorectal adenocarcinoma (Caco2) cell line. At pre-confluency these cells are in an immature state while at confluency they exhibit spontaneous differentiated characteristics and they form a polarized epithelial monolayer characterized by brushborder microvilli and tight junctions. Thus, these cells, as Sertoli cells, are essential for the establishment of protective barriers through tight junctions formation. Pereira and its colleagues have shown that MEX3A overexpression in Caco2 cells alters the maintenance of these junctions [START_REF] Pereira | CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness[END_REF].

Results

During this collaborative metabolic study, I used Caco2 MOCK and MEX3A stably transfected cell lines. I have shown that upon MEX3A overexpression in pre-confluent cells, the oxygen consumption during basal and maximal respiration and thereby the ATP production are decreased (Fig. 1A) while the glycolytic capacity is increased (Fig. 1B). These results were further confirmed by Almeida R. team since they observed by RT-qPCR that several metabolic genes which regulate OXPHOS are down-regulated upon MEX3A overexpression (data not shown).

As presented above, Caco2 cells differentiate with time of culture. Thus, I also analyzed the energy metabolism of Caco2 cells in post-confluent conditions defined as cells cultured 12 days after reaching confluence. As presented in Fig. 1C (top), post-confluent MOCK cells undergo higher OXPHOS than pre-confluent cells. Instead, glycolysis is not increased although we observed some increase of glycolytic capacity and reserve in post confluence MOCK cells.

(Fig. 1C, bottom).

In post-confluent condition, upon MEX3A overexpression, both mitochondrial respiration (Fig. 1D) and glycolysis (Fig. 1E) are impaired and limitation of cell respiration associated with MEX3A overexpression is stronger in post-confluence than in pre-confluence (Fig. 1F).

Conclusions

It is known from the literature that pluripotent cells or stem cells exhibit a stronger dependence on glycolytic metabolism rather than OXPHOS while differentiated cells switch their energy metabolism to OXPHOS, producing more energy (see Introduction, Chapter I.2).

In this line, I have shown in control Caco2 cells that post-confluence conditions, in which the cells are less proliferative and more differentiated, trigger an increase of cell respiration in comparison with pre-confluent cells.

I also observed that respiration is impaired upon MEX3A overexpression in preconfluent conditions and that the glycolytic reserve is strongly increased. Therefore, these results support R. Almeida lab hypotheses that MEX3A regulates energy metabolism to control cellular differentiation.

In post-confluency, MEX3A overexpression promotes a decrease of both OXPHOS and glycolysis. Thus, suggesting they might use other metabolic pathways to fuel cellular processes. Maybe the pentose phosphate pathway is activated or glutamine or fatty acid catabolism are used to produce ATP. Therefore, further studies need to be done in order to better characterize the energy metabolism of MEX3A overexpressed cells in post-confluency.

Hence, this study brings new insights regarding the connection between energetic stress and cell polarity. Moreover, both metabolic results presented in this study and MEX3A effects observed by Almeida R team on cellular differentiation, strongly suggest that MEX3A controls intestinal cell fate by regulating the energy metabolism and this control may involve the LKB1-AMPK-mTOR pathway. Further explorations are currently ongoing by Almeida R lab and a paper is in preparation. All the experiments were performed three times, with three replicates in each experiment.

Materials and methods

Cell line and cell culture

The CaCo2 (heterogeneous human epithelial colorectal adenocarcinoma) cells transfected with MEX3A or MOCK were sent by Almeida R. lab. For these transfections they used pCMV-MEX3A expression vector together with a pCMV-Tag3B empty vector (Agilent Technologies) (Peirera et al., 2013).

Cells were cultured in 37C with DMEM (Ref. 41966-029, ThermoFisher Scientific) supplemented with 10% FBS and 1% penicillin-streptomycin. These are stable clones and G418 has been included in the medium at a concentration of 300ug/m. The medium was replaced every two days.

Seahorse analysis

Cells were plated at 1x10^5 cells per well of a 6-well plate. The metabolic measurements by Seahorse were done with cells in pre-confluency conditions, 3 days after plating (pre-confluency) or 12 days after reaching confluence (post-confluency). To do so, 3×10 4 cells (pre-confluency) or 2.5×10 5 cells (post-confluency) were seeded in 96-well Seahorse plate coated with fibronectin, in normal cell culture medium 30 minutes before assay.

The plate was centrifuged at 200g for 1min (no centrifugal braking). After 30 minutes, the medium was replaced with Seahorse assay medium specific for OCR or ECAR measurements and equilibrated in non-CO2 incubator for 1 hr. Seahorse analyzer uses a cartridge with 96 optical fluorescent O2 and pH sensors to measure OCR (pmol/min) and ECAR (mPh/min).

Basal OCR and ECAR were measured for 3 min every 10 min for 3 points, followed by sequential injection of different drogues presented in the previous project (see Tabel2 of Project3 1.2). Each treatment was measured for 3 min every 10 min for 3 points. OCR and ECAR were normalized by cell density. For both respiration and glycolysis, quantifications were performed by calculating for each category the mean and standard deviation from the values obtained from 3 independent experiments.

Conclusions

During my PhD I highlighted the role of LKB1 as an energy metabolic regulator in several polarized cells such as neural crest cells (NCC) and Sertoli cells. I also uncovered that MEX-3A, an LKB1 potential downstream target, is a metabolic regulator of colorectal adenocarcinoma cells, another polarized cell type. (Figure 1).

The first project revealed that Lkb1 regulates vertebrate head formation by controlling cranial NCC delamination, migration, polarity and survival through actin dynamic. Lkb1 in cranial NCC activates AMPK pathway which positively regulates ROCK kinase and consequently the actin molecular motor Myosin II. In the second part of this first project we uncovered the acetyl transferase GCN5 as a novel modulator of Lkb1 signaling during embryonic development and NCC formation. We showed that GCN5 controls LKB1 acetylation on K48 and suggested that this post translational modification may represent a crucial regulator of vertebrate head development.

The second project, emphasized the role of Lkb1 in truncal, vagal and sacral NCC differentiation and maintenance (Figure 1). We demonstrated that Lkb1 is essential for intestinal motricity, peripheral nerve formation and coat pigmentation through the control of NCC lineage. In particular, Lkb1 regulates neural crest specification into Schwann cells as well as the maintenance of neural crest derivatives involved in enteric nervous system such as satellite glial cells and enteric neurons. Our results pointed out a novel function of Lkb1 as a metabolic regulator of non-essential amino acids, like alanine and glutamate, during NCC specification. Our data brought complementary insights about how Lkb1 metabolic function impacts lysosomal activity, NCC fate and aging. We also uncovered a specific crosstalk between Lkb1 and p53 crucial for NCC ontogeny. Further, it will be of particular interest to analyze the contribution of p53 to NCC defects and to explore the link between GCN5 and p53.

The third project of my PhD was focused on a better characterization of Lkb1 signaling capacities to regulate energy metabolism in other polarized cells such as Sertoli cells and intestinal cells. We highlighted that Lkb1 expressed in Sertoli cells controls male fertility by regulating Sertoli cell polarity, hematotesticular blood barrier, myoid and Leydig cell proliferation, germ cell localization and maturation. Furthermore, our data described how LKB1 influences the energy metabolism of Sertoli cells potentially by limiting amino-acid levels such as alanine and glutamine (Figure 1). Finally, I showed that Mex-3A, a potential downstream target of Lkb1, controls the energy metabolism of intestinal cells and consequently their differentiation. (Result. Project 3.2) (Figure 1).

Altogether, these data obtained during my PhD projects delineated that LKB1 signaling is a key regulator of both cell polarity and energy metabolism during cell fate and could exert a coordinated connection between these two cellular processes.

General discussions & Prospects

I. LKB1 contributions to neural crest cells and underlying mechanisms.

LKB1 in neural crest cells lineage

As presented in the introduction chapters, neural crest cells (NCC) are embryonic multipotent cells that delaminate from neural tube and undergo polarized migration following stereotypical routes. NCC localization along the anterior-posterior axis of neural tube defines several NCC subpopulations that differentiate into many cell types (Introduction Chapter II.2).

Our findings showed that Lkb1 is essential for NCC lineage and governs several aspects of cranial, truncal, vagal and/or sacral NCC formation.

In particular, we identified that Lkb1 regulates cranial NCC delamination and migration through AMPK/ROCK pathway and actin dynamics. Cranial NCC have the particularity to delaminate from the neural tube all at the same time [START_REF] Theveneau | Collective cell migration of the cephalic neural crest: The art of integrating information[END_REF]. Thus they start their migration as multilayered cell group, in opposition to other NCC like trunk NCC that delaminate progressively from neural tube and migrate as a single chainlike group [START_REF] Theveneau | Collective cell migration of the cephalic neural crest: The art of integrating information[END_REF]. Therefore, it will be interesting to analyze if Lkb1 controls also the polarized migration of other NCC subpopulations like trunk NCC and if this regulation requires the AMPK/ROCK pathway.

Next, in order to better characterize the role of Lkb1 during NCC lineage, Anthony

Lucas, a 1 st year PhD student working with Chantal Thibert, is currently completing the phenotypic characterization of the mice with "early" Lkb1 inactivation in NCC used so far only to study the head formation. To begin, phenotypic characterization will be performed on enteric nervous system. Preliminary data of the team have shown that Lkb1 loss in this model led to an aganglionosis in the distal colon, a phenotype reminiscent to human Hirschsprung disease.

Moreover, Lkb1 loss triggered a hypoganglionosis in the intestine. Indeed, the number of enteric neurons and satellite glial cells were reduced in absence of Lkb1. However, NCC migration alongside the gut during embryonic development of the enteric nervous system was not affected. Further, A. Lucas will confirm these results and, by using primary cultures of enteric neurospheres, ex vivo, will analyze the proliferation, survival, differentiation and metabolism of enteric NCC and their derivatives. Next, he will characterize the NCC specification into Schwann cells and the NCC-derived melanoblasts in the skin. Finally, he will evaluate the impact of Lkb1 loss on the formation of other organs containing NCC-derivatives such as heart and thyroid. Providing an overall phenotyping of NCSC-derived tissues upon

Lkb1-deficiency will reinforce our understanding of the importance of LKB1 signaling in NCSC and its potential contribution to human neural crest disorders.

Furthermore, in the continuity of our two first projects, the team will analyze by the technics described in the result chapters, if LKB1 acetylation at lysine 48 by GCN5 contributes to NCC development (see Results, Project I.2.3) and if p53 signaling also participates to the neurocristopathy phenotype due to Lkb1 inactivation.

p53 accumulation blocks cranial NCC proliferation and impairs NCC delamination from the neural tube as well as their migration (Rinon et al., 2011;Van Nostrand et al., 2014b).

Thus, p53 levels during early development are essential for vertebrate face formation. Our first project provided evidence suggesting that GCN5, the upstream regulator of LKB1, may also be a regulator of cranial NCC migration and head development. From the literature, we know that deletion of GCN5 in mice led to early embryonic lethality and inactivation of both GCN5 and p53 partially rescued the phenotype: embryos lived longer although they still die at mid gestation [START_REF] Bu | Loss of Gcn5 acetyltransferase activity leads to neural tube closure defects and exencephaly in mouse embryos[END_REF]. This strongly suggest that p53 is accumulated also in absence of In conclusion, these studies will (i) reveal if the role of LKB1 in cell migration is conserved in other NCC subpopulations than the cephalic ones, (ii) underline the role of Lkb1

during NCC lineage and (iii) uncover if there is a crosstalk between GCN5 and p53 important for NCC development.

LKB1 & Neurocristopathies

LKB1, a modulator gene in human neurocristopathies?

The complex phenotype developed by the genetically engineered mice that we generated to inactivate Lkb1 in NCC recapitulates the symptoms of human neurocristopathies and suggests that LKB1 could contribute to the etiology of these disorders.

As a brief reminder, neurocristopathies are human diseases due to NCC defects at various developmental stages such as their formation, delamination, migration, differentiation and survival (Introduction, Chapter I.3.1). Neurocristopathies include a broad group of diseases and syndromes like colon aganglionosis, craniofacial malformations (cleft lip/palate, sensorineural deafness, smelling defects), heart malformations, skin/hair pigmentation defects and for some patients, demyelination. Although neurocristopathies have a high medical and societal impact, the main treatment is surgery but is often variable and rarely entirely corrective.

In some cases, the surgery is not even possible. Moreover, our understanding regarding the molecular and genetic networks responsible for these pathologies is still very preliminary.

In order to test if LKB1 is a modulator gene in these pathologies, in collaboration with scientists working in this field, we could sequence the genes coding proteins of the LKB1 holoenzyme (LKB1, MO25, STRADα and β) in patients with neurocristopathies to search for mutations.

Analyses of LKB1 holoenzyme in neurocristopathies are strongly relevant given that biallelic deletions of exons 9 to 13 of the STRADα gene are responsible for the human syndrome Polyhydramnios Megalencephaly Symptomatic Epilepsy (PMSE). This syndrome is a developmental disorder characterized by craniofacial abnormalities, infantile-onset epilepsy and neurocognitive delay [START_REF] Puffenberger | Polyhydramnios, megalencephaly and symptomatic epilepsy caused by a homozygous 7-kilobase deletion in LYK5[END_REF]. The PMSE-causing mutation in humans represents a loss-of-function mutation that destabilizes STRADα and prevents it from activating LKB1 [START_REF] Orlova | STRADα deficiency results in aberrant mTORC1 signaling during corticogenesis in humans and mice[END_REF][START_REF] Zeqiraj | ATP and MO25α Regulate the Conformational State of the STRADα Pseudokinase and Activation of the LKB1 Tumour Suppressor[END_REF]. Therefore, PMSE patients have an increased mTOR pathway. Interestingly treatment of 5 PMSE patients with Sirolimus, the mTORC1 inhibitor rapamycin, limited seizures frequency [START_REF] Parker | Rapamycin Prevents Seizures After Depletion of STRADA in a Rare Neurodevelopmental Disorder[END_REF].

The most interesting neurocristopathy to seek for mutations of genes of LKB1 signaling is the Peripheral demyelinating neuropathy, Central dysmyelination, Waardenburg syndrome, and Hirschsprung disease (PCWH), a subgroup of Waardenburg-Shah syndrome (WS4).

Indeed, the complex phenotypes developed by our mouse models (coat depigmentation, peripheral neuropathy and demyelination, intestinal pseudo-obstruction and distal colon aganglionosis) are very evocative of the symptom of PCWH patients. All PCWH patients harbor Sox10 mutations which are associated with about half of WS4 cases and 15% of Waardenburg Syndrome II cases (Pingault et al., 2010). Sox10 mutations often occur at the heterozygous state and can be germline or de novo mutations. High phenotypic variability with an incomplete penetrance of each symptoms has been observed in the few familial cases described yet. So far, the causes of this phenotypic discrepancy, especially between WS2, WS4 and PCWH patients is not clear although the nature of the mutations (truncating or missense mutations) has been proposed to explain phenotype variability [START_REF] Chaoui | Identification and functional analysis of SOX10 missense mutations in different subtypes of Waardenburg syndrome[END_REF][START_REF] Chaoui | Subnuclear re-localization of SOX10 and p54NRB correlates with a unique neurological phenotype associated with SOX10 missense mutations[END_REF]. Furthermore, modulator genes could contribute to NCC defects along with Sox10 mutations. Such genetic interactions have been already described between Sox10 and the transcription factor Zeb2, the receptor/ligand EDNRB/EDN3 or Integrin b1 [START_REF] Stanchina | Genetic interaction between Sox10 and Zfhx1b during enteric nervous system development[END_REF][START_REF] Watanabe | Sox10 and Itgb1 interaction in enteric neural crest cell migration[END_REF][START_REF] Watanabe | Differentiation of Mouse Enteric Nervous System Progenitor Cells Is Controlled by Endothelin 3 and Requires Regulation of Ednrb by SOX10 and ZEB2[END_REF]. It will thus be of interest to study the involvement of Lkb1 signaling in PCWH. However, WS4 and PCWH are very rare genetic disorders and less than 30 cases of PCWH have been reported in the literature. The identification and validation of mutations would then be difficult and hardly statistical due to low number of patients.

Is LKB1 involved in NCC defects associated with lysosomal storage disorders?

Lysosomes have recently been proposed as signaling hubs: they integrate and relay external and nutritional information thus controlling proteolysis and cell senescence to prevent premature aging [START_REF] Carmona-Gutierrez | The crucial impact of lysosomes in aging and longevity[END_REF]. As specified in results, project II.2, the team will further explore how the signaling pathways Lkb1 and p53 controls lysosomal proteolysis to limit lipofuscin formation, senescence and premature aging in NCC.

Lysosomes control intracellular Ca 2+ levels and defects in Ca 2+ uptake are responsible for several lysosomal storage disorders [START_REF] Lloyd-Evans | On the move, lysosomal CAX drives Ca2+ transport and motility[END_REF][START_REF] Lloyd-Evans | Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium[END_REF][START_REF] Styrt | An abnormal calcium uptake pump in Chediak-Higashi neutrophil lysosomes[END_REF] [START_REF] Hanson | Association of dermal melanocytosis with lysosomal storage disease: clinical features and hypotheses regarding pathogenesis[END_REF]. This pathology is part of the criteria for a subgroup of neurocristopathies named Phakomatosis pigmentovascularis [START_REF] Hanson | Association of dermal melanocytosis with lysosomal storage disease: clinical features and hypotheses regarding pathogenesis[END_REF]. Moreover, it has been reported in 2015 the case of a 3 years old child which developed symptoms of both Hirschsprung's disease, a neurocristopathy described in chapter II.3.1, and cystinosis, a lysosomal storage disorder characterized by body accumulation of the amino acid cysteine [START_REF] Mittal | Hirschsprung's disease with infantile nephropathic cystinosis[END_REF]. Finally, as presented in the introduction (Chapter II.3), many ribosomopathies with NCC defects are associated with acute myeloid leukemia (AML). Part of AML patients have accumulation of misfolded proteins [START_REF] Schardt | Activation of the unfolded protein response is associated with favorable prognosis in acute myeloid leukemia[END_REF] and lysosomal defects [START_REF] Sukhai | Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors[END_REF] probably caused by increased iron levels which subsequently enhance the sensitivity to oxidative stress and lysosomal alkalinization. Interestingly, treatments decreasing oxidative stress such as iron chelators, vitamin E and glutathione treatments, delay lipofuscin production and accumulation in murine models [START_REF] Brunk | The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis[END_REF][START_REF] Skoczyńska | Melanin and lipofuscin as hallmarks of skin aging[END_REF]. These treatments are also efficient for neurocristopathies like the Treacher Collins Syndrome [START_REF] Trainor | Treacher Collins syndrome: etiology, pathogenesis and prevention[END_REF]Sakai and Trainor, 2016;Sakai et al., 2016). However, for the moment, very little is known about the molecular links between neurocristopathies and lysosomal defects.

Apolipoprotein D (ApoD), which is a glial derived lipid binding protein produced by astrocytes and Schwann cells [START_REF] García-Mateo | Schwann cell-derived Apolipoprotein D controls the dynamics of post-injury myelin recognition and degradation[END_REF], is also accumulated in neurodegenerative diseases, aging brain and peripheral nervous system injury [START_REF] Dassati | Apolipoprotein D takes center stage in the stress response of the aging and degenerative brain[END_REF][START_REF] García-Mateo | Schwann cell-derived Apolipoprotein D controls the dynamics of post-injury myelin recognition and degradation[END_REF][START_REF] Martínez | Oxidative stress induces apolipoprotein D overexpression in hippocampus during aging and Alzheimer's disease[END_REF][START_REF] Sanchez | Aging without Apolipoprotein D: Molecular and cellular modifications in the hippocampus and cortex[END_REF]. ApoD has been proposed as a marker of lysosomes that are the most vulnerable to oxidative stress [START_REF] Ganfornina | ApoD, a glia-derived apolipoprotein, is required for peripheral nerve functional integrity and a timely response to injury[END_REF][START_REF] Pascua-Maestro | Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress[END_REF]. Therefore, we could study if there is an accumulation of ApoD in our mouse models and if this accumulation depends on alanine

Regulation of Schwann Cell Precursors by LKB1?

Schwann Cell Precursors (SCP) derive from cranial, truncal and vagal NCC. They conserve the multipotency and high plasticity of NCC, being therefore able to generate several cell types during both embryogenesis and adulthood such as Schwann cells, neurons and melanocytes (Introduction, Chapter II.2.3). SCP multipotency has been discovered many years ago [START_REF] Sherman | Basic fibroblast growth factor (bFGF) acts intracellularly to cause the transdifferentiation of avian neural crest-derived Schwann cell precursors into melanocytes[END_REF] by in vitro studies. However, only very recently it has been clearly

shown by in vivo analyses that SCP also differentiate into melanocytes, enteric neurons or bone marrow mesenchymal stem cells (see Introduction, Chapter II.2.3). Our results strongly suggest that Lkb1 signaling is essential for SCP maintenance.

While studying Cre expression in the "late" model of Lkb1 inactivation (using the Tyr::Cre driver) we identified that some cells in the bone marrow also expressed the recombinase Cre (see Results, Project II.1 submitted article Sup.Fig. 3 F). These cells most probably are SCPderivatives and Lkb1 may thus be also inactivated in SCP-derivated mesenchymal stem cells.

It could be interesting to further validate this hypothesis and evaluate if this is also the case in the "early" model of Lkb1 inactivation in NCC. Of note, AMPK controls the proliferation of mesenchymal stem cells [START_REF] De Meester | Role of AMP-activated protein kinase in regulating hypoxic survival and proliferation of mesenchymal stem cells[END_REF] and contributes to tight junctions formation in these cells [START_REF] Rowart | Mesenchymal Stromal Cells Accelerate Epithelial Tight Junction Assembly via the AMP-Activated Protein Kinase Pathway, Independently of Liver Kinase B1[END_REF]. Studying Lkb1 inactivation in mesenchymal stem cells and the consequences for the homeostasis of the hematopoietic niche as well as the establishment and maintenance of hematopoietic stem cells and derivatives would then be of great interest.

Bone marrow and blood composition analyses could be easily performed on the 2 mice models generated by the lab.

A biobank of mesenchymal stem cells has been collected at the hospital of Grenoble by S.

Park (IAB, EFS, UGA, CHU, Grenoble) from patients with myelodysplasia, some of them due to the 5q deletion responsible of a ribosomopathy (see chapter II.3). Control samples were collected from sternal puncture during cardiac surgeries. Assessing the status of LKB1/AMPK/mTOR signaling in this collection of cells could be a first step in the exploration of LKB1 defective signaling in NCC and SCP in human disorders. As the genetic screening for proteins of the LKB1 holoenzyme is difficult in neurocristopathies due to the low number of patients, we could undergo genetic analyses in mesenchymal stem cells of patients with myelodysplasia.

SCP have an important potential for clinical use and for developing new therapeutic approaches, due to their multipotency and the fact that they persist in adult nerves. However, we still do not know so far how energy metabolism regulates SCP fate. Therefore, this study about the role of LKB1 in mesenchymal stem cells could also represent an important step for a better characterization of the underlying molecular mechanisms.

II. LKB1 contributions to intercellular communication and tissue homeostasis

Our work highlighted that LKB1 is essential to maintain whole tissue homeostasis.

Indeed, Lkb1 conditional inactivation:

-in Schwann cells promoted neurodegeneration (Results, Project II.1).

-in Sertoli cells perturbed germ cell maturation and Leydig and myoid cells proliferation (Results.Project III.1).

-in enteric nervous system affected differentiation and/or maintenance of intestinal epithelial cells and potentially smooth muscle cells (data not shown).

The enteric glial cells are important for gastrointestinal motricity and to control the epithelial barrier integrity [START_REF] Neunlist | Enteric glial cells: recent developments and future directions[END_REF]. They help maintaining the intestinal barrier by forming a cellular and molecular bridge between enteric nerves, immune cells as well as enteroendocrine cells within the intestinal epithelium [START_REF] Sharkey | Emerging roles for enteric glia in gastrointestinal disorders[END_REF].

Preliminary observations during our studies, showed that Lkb1 inactivation in enteric neurons and satellite glial cells promoted a lack of Paneth cells in the intestinal epithelium as well as defects in stromal tissue such as loss of adherence between cells (data not shown). In order to better characterize this role of Lkb1 the team initiated a collaboration with R. Almeida lab. Immunostaining for various markers of intestinal or smooth muscle differentiation will confirm if Lkb1 absence in enteric nervous system affects cell differentiation and survival.

Hence, this project will contribute to understand the interaction between enteric nervous system and adjacent cells and analyze a novel function of Lkb1 as regulator of this cell-cell communication.

Cell to cell communication is an ancient process through which one cell is capable of modulating the behavior of another via the secretion of signal molecules.

In our studies, one explanation could be that Lkb1 loss leads to deregulated intercellular communication due to defective metabolites. This metabolic communication may be controlled by Lkb1-dependent regulation of transaminases, also known as aminotransferases.

III. Metabolic regulations exerted by LKB1

As a brief reminder aminotransferases are enzymes able to transfer nitrogenous groups between an amino acid and an α-keto acid. They belong to class EC. 2.6.1 and so far, there are 101 transaminases described on ExPASy database (Bioinformatics Resource Portable).

Our studies revealed that Lkb1 controls NCC specification into glial cells by repressing a non-essential amino acid biosynthetic program coupled to pyruvate-alanine transamination (Results, Project II.1). Our data thus led us to hypothesize that Lkb1 limits alanineaminotransferase (ALAT) activity whereas it favors glutamate synthetase (GS) activity. ALAT catalyzes alanine production by transferring an amino group from L-glutamate to pyruvate in order to form L-alanine and α-ketoglutarate (this reaction is reversible).

Lkb1 expressed in Sertoli cells controls germ cell maturation also by regulating nonessential amino acid levels (Results, Project III.1).

One of the most affected amino acid in both studies (NCC and Sertoli cells) was alanine which was up-regulated. Thus, this role of Lkb1 as a new metabolic regulator of pyruvatealanine cycling seems conserved across cell types sharing similar metabolic functions.

Feedback loops between aminotransferases and the mTOR pathway.

Coloff and its coworkers have shown that aminotransferases are important regulators of the proliferation -quiesence balance (see Introduction,. They observed that mTOR pathway regulates positively mRNA expression of aminotransferases such as PhosphoSerine AminoTransferase1 (PSAT1), Asparatate AminoTransferase1 (AAT1 or GOT1) and Alanine AminoTransferase2 (ALAT2 or GPT2) (Table1). However, mTOR inhibition with TORIN1, a drug which inhibits both mTORC1 and mTORC2 complexes, reduced only PSAT1 and ALAT2 protein expression and decreased only alanine levels (Coloff et al., 2016).

In my studies, I have shown that both ALAT and mTOR inhibition are able to restore glial differentiation in absence of Lkb1 (see Results, Project II.1). However, it is the alanine production which regulates mTOR pathway because ALAT inhibition, and therefore, alanine down-regulation, partially normalized (decreased) the mTOR pathway. This result correlates with the literature since it is known that mTOR is activated at lysosomal surface in an amino acid dependent manner (see Introduction, Chapter IV.2.1). So far, most research has been focused on leucine and glutamine/glutamate amino acids whereas the control of mTOR activity by alanine has received less attention, although alanine was shown a long time ago to be involved in proteolysis (lysosomal activity) regulation [START_REF] Caro | A combination of intracellular leucine with either glutamate or aspartate inhibits autophagic proteolysis in isolated rat hepatocytes[END_REF][START_REF] Leverve | Control of proteolysis in perifused rat hepatocytes[END_REF].

Our study showed for the first time that alanine also regulates mTOR and that Lkb1 controls this mTOR amino acid-dependent regulation. Thus, our results in association with Coloff et al. results suggest that there is a feedback loop: aminotransferases as well as amino acid levels regulate mTOR activity and activated mTOR controls aminotransferases and amino acids expression. To analyze if there is a feedback loop in our system, we could treat Lkb1 knockdown cells with mTOR inhibitor (TORIN1) and measure amino acid levels (alanine, glutamate) as well as ALAT and GS activity (in our system ALAT mRNA or protein expression is not affected).

How LKB1 signaling regulates aminotransferases activity?

Until last year, nothing was known about LKB1 and aminotransferases. Recently, the molecular links between LKB1 and these enzymes started to be a topic of high interest and several studies underlined this link (Table 1):

-N. Bardeesy lab has shown that LKB1 downregulates PSAT1 expression and activity leading to serine metabolism inhibition (Table1).

Serine metabolism fuel the methionine salvage pathway that produces the methyl donor S-adenosyl methionine (SAM) (Figure 1). Thus, LKB1 deficiency increased serine metabolism and consequently DNA methylation which regulates gene expression. This role of LKB1 was detected in pancreatic cancer cells and is essential for its tumor suppressor activity (Kottakis et al., 2016).

-Another aminotransferase linked to LKB1 signaling is Glutamine:Fructose-6phosphate AmidoTransferase 1 (GFAT1). It has been shown that AMPK, the LKB1 downstream substrate, inhibits GFAT1 activity by its phosphorylation on serine 243 (Table1) [START_REF] Scott | The sweet side of AMPK signaling: regulation of GFAT1[END_REF][START_REF] Zibrova | GFAT1 phosphorylation by AMPK promotes VEGFinduced angiogenesis[END_REF]. GFAT1 is a negative regulator of angiogenesis and it has been shown to be linked to diabetic state [START_REF] Cooksey | Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione[END_REF][START_REF] Hebert | Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance[END_REF].

GFAT1 activation promotes an increase in O-linked-β-N-glucosamine acetylation (O-Glc NAcetylation) of proteins which is a hallmark of vascular complications of type 2 diabetes (Ma ( Kottakis et al., 2017) and [START_REF] Ma | Protein O-GlcNAcylation in diabetes and diabetic complications[END_REF][START_REF] Srinivasan | Glutamine fructose-6-phosphate amidotransferase (GFAT) gene expression and activity in patients with type 2 diabetes: inter-relationships with hyperglycaemia and oxidative stress[END_REF]. Therefore, the AMPK-GFAT1 crosstalk regulates vascular dysfunction associated with metabolic disorders [START_REF] Zibrova | GFAT1 phosphorylation by AMPK promotes VEGFinduced angiogenesis[END_REF] - Our data suggest that post-translational modifications could regulate the enzymatic activities. As LKB1 is a master kinase acting through downstream kinases such as AMPK, the lab will thus explore if the phosphorylation status of ALAT enzyme is modified upon Lkb1 loss.

In particular, ALAT1 phosphorylation has been observed in large scale mammal Table1: Aminotransferases regulated by LKB1/AMPK/mTOR phosphoproteome analyses (Zhou et al., 2013;Bian et al., 2014). The sequence surrounding the phosphorylated site threonine 22 in ALAT partially matches the AMPK phosphorylation motif recently described [START_REF] Schaffer | Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction[END_REF]. We will thus further investigate in NCC-derived glial cells if ALAT activity is controlled by its phosphorylation status through AMPK kinases.

We will assess the phosphorylation by blotting immunoprecipitated ALAT with a mix of monoclonal antibodies that specifically recognizes phosphorylated serine/threonine in AMPK motifs (LXRXX(pS/pT); Cell Signaling Technology). If we identify that the potential sites in ALAT are indeed phosphorylated by AMPK, we will generate non-phosphorylatable mutants and assess the impact on their enzymatic activities.

Collectively, these data will complete the very recent literature regarding the link between LKB1 signaling pathway and aminotransferases and will provide evidence that LKB1 is a key regulator of cell behavior through the control of ALAT and alanine biosynthesis.

IV. Wider implication for LKB1 in human metabolic diseases and cancers?

So far, mutations of LKB1 are associated only with tumor formation: germline mutations are responsible for the human Peutz-Jeghers syndrome whereas somatic mutations are associated with sporadic tumors (Introduction,Chapter III.A.5). Owing to all the cellular processes regulated by LKB1 signaling, we could expect a broader implication of LKB1 in human diseases.

Our results suggested that aberrant LKB1 metabolic signaling underlines the etiology of various human pathologies such as neurocristopathies potentially associated with lysosomal storage disorders; metabolic syndromes, peripheral neuropathies as well as cancers.

Is LKB1 involved in neurocristopathies associated with metabolic syndromes?

Nowadays, little is known about the involvement of metabolism defects to neurocristopathies occurrence. There is so far only one article suggesting that some metabolic syndromes such as Pyruvate Deshydrogenase deficiency and Oxidative phosphorylation deficiency can be associated with neurocristopathies since these metabolic diseases are associated with similar facial abnormalities (Table 2) (Berio A., 2011).

The link between oxidative phosphorylation and transaminases activity is still not very clear. During our study, we surprisingly observed that the aminotransferase ALAT activity and the production of non-essential amino acids such as alanine, are crucial for NCC differentiation independently of oxidative phosphorylation (Results, Project II.1). Indeed, glial differentiation was restored whereas oxidative phosphorylation was still strongly impaired. These data underline the importance of transaminases and amino acid levels for cell fate.

In the last 10 years, several epidemiologic studies have shown that elevated levels of ALAT and aspartate aminotransferase (AST) in blood, known as biomarkers for liver diseases [START_REF] Kew | Serum aminotransferase concentration as evidence of hepatocellular damage[END_REF], can also be serum biomarkers for metabolic syndromes like type 2 diabetes even in the absence of hepatocellular damages [START_REF] Bonora | Increased risk of cardiovascular disease and chronic kidney disease in NAFLD[END_REF][START_REF] Goessling | Hepatic stellate cells and cirrhosis: fishing for cures[END_REF][START_REF] Goessling | Aminotransferase Levels and 20-year Risk of Metabolic Syndrome, Diabetes, and Cardiovascular Disease[END_REF][START_REF] Sookoian | Alanine and aspartate aminotransferase and glutamine-cycling pathway: Their roles in pathogenesis of metabolic syndrome[END_REF]. Thus, we could measure if there is an increase in serum alanine levels and ALAT activity in our mouse models reminiscent of neurocristopathies. However, we have to be precautious because it may be difficult to observe any increase of ALAT in blood upon specific Lkb1 inactivation only in some cell types (Schwann cells, enteric neurons, satellite glial cells, melanocytes, cranial NCC, etc.) which are not known to have a high ALAT activity.

We could also analyze ALAT activity in plasma of patients with neurocristopathies and study if this enzyme could be a biomarker of neurocristopathies associated with metabolic syndromes. These studies would complete the analyses proposed in paragraph I.1 regarding the screening of genes coding for proteins of the LKB1 holoenzyme in patients with neurocristopathies.

Does LKB1 contribute to regulate peripheral neuropathies through the control of alanine levels?

As already described, Lkb1 inactivation in neural crest cells promoted a peripheral neuropathy among other phenotypes (Results, Project 2.1).

Increased alanine levels have been associated with peripheral neuropathies such as hereditary sensory and autonomic neuropathy type 1 (HSAN1) (OMIM 162400). This disorder is characterized by sensory loss and lower limbs paralysis due to defective neurons with smaller mean axon diameter of myelinated and nonmyelinated fibers and abnormal Schwann cells [START_REF] Garofalo | Oral l-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1[END_REF]. However, this pathology is not caused by ALAT defects but rather by serine palmitoyltransferase (SPT) missense mutations [START_REF] Garofalo | Oral l-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1[END_REF][START_REF] Othman | Plasma deoxysphingolipids: a novel class of biomarkers for the metabolic syndrome?[END_REF]. Serine palmitoyltransferase catalyzes the formation of sphinganine, a precursor of sphingolipids (cell membrane lipids), from L-serine and palmitoyl-CoA (Figure 2A). This enzyme, in specific conditions, is able to use also other substrates such as L-alanine (Figure 3A). In presence of L-alanine it forms deoxysphinganine which is not degraded by the canonical sphingolipid degradation pathway and is neurotoxic (Figure 2B). Garofalo and his colleagues have shown that 10% of L-alanine enriched diet increases deoxysphinganine levels in plasma, sciatic nerves and liver and promoted a severe peripheral neuropathy in mice [START_REF] Garofalo | Oral l-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1[END_REF]. The levels of deoxysphinganine were measured by chromatographymass spectrometry (LC-MS), as described by Eckardstein team [START_REF] Penno | Hereditary Sensory Neuropathy Type 1 Is Caused by the Accumulation of Two Neurotoxic Sphingolipids![END_REF]. L-serine enriched diet rescued the phenotype [START_REF] Garofalo | Oral l-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1[END_REF]. This suggests that there is a balance between alanine and serine, their levels being associated to neurotoxic product formation and peripheral neuropathy development. Therefore, alanine accumulation observed in sciatic nerves of Lkb1 conditional knockout mice (Results, Project II.1) may correlate with an increase in deoxysphingolipids which aggravates the peripheral neuropathy. In order to test this hypothesis, we could analyze the levels of deoxysphingolipids first in vitro, in NCC-derived glial cells knockdown for Lkb1 and second in plasma and sciatic nerves of Lkb1 conditional knockout mice, by lipid extraction, sequential acid and basic hydrolysis and chromatographymass spectrometry (method described in [START_REF] Penno | Hereditary Sensory Neuropathy Type 1 Is Caused by the Accumulation of Two Neurotoxic Sphingolipids![END_REF]. If an increase in desoxysphingolipids is observed, the lab could further test if L-serine enriched diet rescues the peripheral neuropathy.

Clinically, HSAN1 is very similar to diabetic peripheral neuropathy. Recently, elevated plasma levels of deoxysphingolipids have been identified as a novel biomarker for diabetes and correlate with distal sensorimotor polyneuropathy [START_REF] Dohrn | ECEL1 mutation causes fetal arthrogryposis multiplex congenita[END_REF][START_REF] Hammad | Increased Plasma Levels of Select Deoxy-ceramide and Ceramide Species are Associated with Increased Odds of Diabetic Neuropathy in Type 1 Diabetes: A Pilot Study[END_REF], [START_REF] Mwinyi | Plasma 1-deoxysphingolipids are early predictors of incident type 2 diabetes mellitus[END_REF]Othman et al., 2015a). Decreasing plasma deoxysphingolipids, by oral L-serine supplementation, improves neuropathy in diabetic rats [START_REF] Othman | Lowering Plasma 1-Deoxysphingolipids Improves Neuropathy in Diabetic Rats[END_REF].

Altogether, these data suggest that increased alanine levels are responsible for deoxysphingolipids formation and accumulation which are neurotoxic and affect Schwann cells and lead to peripheral neuropathy. The project proposed here will bring evidence about the molecular mechanisms by which Lkb1-dependent metabolic control of non-essential amino acids impacts deoxisphingolipids formation and peripheral neuropathy regulation.

How metabolic regulations exerted by LKB1 contribute to its tumor suppressor activity?

As already described, during my PhD I studied the metabolic regulation exerted by Lkb1 (i) Pyruvate has different fates: it can be transformed into lactate by lactate dehydrogenase (LDH), into alanine by ALAT or it can enter into the mitochondria to fuel TCA cycle. Our results unveil a key role played by Lkb1 in limiting pyruvate-alanine cycling in both NCC and Sertoli cells. However, there are some tissue specificity since we observed that Lkb1 loss in Sertoli cells, by contrast to NCC, promotes lactate accumulation and oxidative phosphorylation augmentation. These increases of both alanine and lactate are also observed in cancer cells. In tumors, the concentration of lactate is 80 times higher than in normal situations, reaching 40 mM. Many clinical studies highlighted that lactate accumulation is a marker of poor prognosis in cancers [START_REF] Romero-Garcia | Lactate Contribution to the Tumor Microenvironment: Mechanisms, Effects on Immune Cells and Therapeutic Relevance[END_REF]. Recently it has been shown that cancer cells prefer to use lactate instead of glucose for oxidative phosphorylation [START_REF] Harjes | Metabolism: More lactate, please[END_REF][START_REF] Faubert | Lactate Metabolism in Human Lung Tumors[END_REF][START_REF] Hensley | Metabolic heterogeneity in human lung tumors[END_REF][START_REF] Hui | Glucose feeds the TCA cycle via circulating lactate[END_REF]. Thus, it will be interesting to study if LKB1 controls lactate turnover in cancer cells. In support to this idea AMPK has been shown recently to control LDH activity and metabolic homeostasis in muscle stem self-renewal [START_REF] Theret | AMPKα1-LDH pathway regulates muscle stem cell selfrenewal by controlling metabolic homeostasis[END_REF].

Rapid proliferation and survival of some cancer cells is dependent on high glutamine uptake from exogenous supply [START_REF] Cluntun | Glutamine Metabolism in Cancer: Understanding the Heterogeneity[END_REF]. By contrast, other tumor cells exhibit upregulated glutamine synthesis [START_REF] Cluntun | Glutamine Metabolism in Cancer: Understanding the Heterogeneity[END_REF]. Tissue origin of cancer cells is one possible explanation of this metabolic heterogeneity between tumors. Very recently, it was shown that Lkb1 loss confers glutamine dependency during kidney development and was associated with kidney polycystic disease [START_REF] Flowers | Lkb1 deficiency confers glutamine dependency in polycystic kidney disease[END_REF]. We also observed during testis development that Lkb1 deficiency is associated with glutamine accumulation along with alanine increase.

Exploring how LKB1 regulates both lactate and glutamine by measuring their levels, uptake and biosynthesis in lung cancer cells is therefore very important.

(ii) As previously presented, N. Bardeesy lab showed that LKB1 regulates pancreatic cancer cell growth by downregulating PSAT1 expression and activity leading to serine metabolism inhibition and DNA methylation loss (Table 1) (Kottakis et al., 2016). Coloff and his colleagues analyzed aminotransferases mRNA expression in breast cancer using The Cancer Genome Atlas (TCGA) breast cancer RNA-sequencing dataset. They identified that PSAT1, ASAT1 and ALAT2 expression positively correlated with highly proliferative breast tumors. Further, by using the TCGA reverse-phase protein array (RPPA) database, they underlined a correlation between the expression of previously detected aminotransferases and mTOR activity (Coloff et al., 2016). These data coupled with our own observation that LKB1 negatively regulates ALAT which controls mTOR pathway during embryonic development, prompts us to investigate if this function of LKB1 is also part of its tumor suppressor activity.

LKB1 mutants associated with tumors are often truncating mutations leading to LKB1 loss. LKB1 mutations strongly potentiate tumor progression due to KRas G12D oncogenic mutations. Molecular profiling performed by combining genetic and histological studies of an international lung tumors biobank collected in part at Grenoble by E. Brambilla lab, revealed that LKB1 and KRas mutations and consequent mTOR upregulation are associated with 30% of lung adenocarcinomas (Cancer Genome Atlas Research Network, 2014). However, patients harboring triple mutations in KRas, LKB1 and p53 are very rare although p53 is also very frequently mutated in lung adenocarcinomas. This observation supports the idea that LKB1 loss in a context of KRas mutation could modify p53 signaling endogenously present in tumor cells. Moreover, LKB1 has been shown to promote metabolic flexibility in lung cancer cells upon energy stress [START_REF] Parker | LKB1 promotes metabolic flexibility in response to energy stress[END_REF].

The lab is thus interested in further exploring LKB1 metabolic regulations and LKB1-p53 crosstalk in lung cancer cells. To these purposes, the lab developed lung cancer cells that express KRas G12D oncogenic mutant and are null for LKB1 and p53 (collaboration C Thibert with L Attardi and M. Winslow, Stanford, CA, USA). Several cell lines of lung tumors from patients are also available to the lab. Therefore, Marie Mevel, a second year Master student under the supervision of Chantal Thibert, will study metabolic regulations by LKB1 in lung cancer cells. The cell models described above will allow to study lung cancer cells null for LKB1 with rescued LKB1 expression (either wildtype or mutants) along with or without p53 using retroviral infections to re-express p53 wild type. With this model, the group will also study the role of LKB1 acetylated at K48 in lung cancer cells and its link to p53. The tumor suppressor LKB1 codes for a serine/threonine kinase. It acts as a key regulator of cell polarity and energy metabolism partly through the activation of the AMP-activated protein kinase (AMPK), a sensor that adapts energy supply to the nutrient demands of cells facing situations of metabolic stress. To achieve metabolic adaptations, AMPK phosphorylates numerous substrates which inhibit anabolic processes while activating catabolic reactions. In particular, AMPK inhibits the mammalian target of rapamycin (mTOR).

During my PhD, based on genetically engineered mouse models, I uncovered that Lkb1 signaling is essential for neural crest cells (NCC) formation. NCC are multipotent cells that originate from the neural tube and give rise to various derivatives including bones and cartilage of the face, pigmented cells in the skin and glial and neural cells in peripheral nerves and the enteric nervous system. I demonstrated that Lkb1 is essential for vertebrate head formation and for the differentiation and maintenance of NCC-derivatives in the peripheral nervous system. I also emphasized that LKB1 is acetylated on lysine 48 by the acetyltransferase GCN5 and that this acetylation could regulates cranial NCC ontogeny and head formation. Furthermore, I discovered that Lkb1 controls NCC-derived glial differentiation through metabolic regulations involving amino acid biosynthesis coupled to pyruvate-alanine cycling upstream of mTOR signaling.

Phenotypes due to Lkb1 loss in NCC recapitulate clinical features of human disorders called neurocristopathies and therefore suggest that aberrant Lkb1 metabolic signaling underlies the etiology of these pathologies. Abnormal activation of the tumor suppressor p53 has been described in some NCC disorders and p53 inactivation in neurocristopathy mouse models rescues the pathological phenotype. By using a NCC line that can be cultivated as progenitors or differentiated in glial cells in vitro, I demonstrated that Lkb1 expression in NCCderivatives controls p53 activation by limiting oxidative DNA damage and prevents the formation of lysosomes filled with oxidized proteins and lipids called lipofuscin granules. Interestingly, activation of mTOR and LKB1/AMPK pathways is governed by amino acid sensors and takes place at the lysosome surface. Lysosomes have been proposed as a signaling hub controlling proteolysis and aging. Thus Lkb1 and p53 signaling could converge especially through lysosome homeostasis thereby potentially impacting cellular aging.

Strikingly, Sertoli cells, that are epithelial somatic cells, located in seminiferous tubules in testes, and which govern germ cells maturation and whole testis homeostasis, share similar metabolic functions with glial cells. For example, they secrete lactate and alanine to fuel mitochondria of neighboring cells (germ cells or neurons respectively) to control their survival and maturation. During my PhD, we highlighted that Lkb1 is essential for testis homeostasis and spermatogenesis by regulating Sertoli cell polarity and, as observed in glial cells, energy metabolism through pyruvate-alanine cycling. These data suggest that this particular Lkb1 metabolic regulation is conserved in tissues with similar function.

Taken together, these studies reveal the underlying molecular mechanisms that coordinately regulate energy metabolism and cell fate. They provide new insights into NCC development and expand our understanding of the role of LKB1 as an energy metabolic regulator. Finally, my PhD projects uncover the existence of a crosstalk between Lkb1 and p53 and underline its importance in NCC disorders.

Résumé

Le suppresseur de tumeur et sérine/thréonine kinase LKB1 est un régulateur clé de la polarité cellulaire et du métabolisme énergétique en partie grâce à l'activation de sa kinase substrat AMPK. Cette protéine est un senseur métabolique pour adapter les apports énergétiques aux besoins nutritionnels des cellules confrontées à un stress. Pour cela, AMPK phosphoryle divers substrats qui activent les réactions cataboliques et inhibent les processus anaboliques dont la kinase mTOR.

Au cours de ma thèse, via l'utilisation de modèles murins d'inactivation conditionnelle, j'ai découvert que Lkb1 est crucial pour la formation des cellules de crête neurale (CCN). Ces cellules multipotentes, originaires du tube neural, donnent naissance à divers dérivés, comme les cellules des os et cartilage de la face, les cellules pigmentées de la peau et les cellules gliales et neurales des nerfs périphériques et du système nerveux entérique. J'ai démontré que Lkb1 est essentiel pour la formation de la tête des vertébrés et pour la différenciation et le maintien des dérivés des CCN dans le système nerveux périphérique. J'ai également mis en évidence l'acétylation de LKB1 sur la lysine 48 par l'acétyltransférase GCN5 et son rôle dans l'ontogenèse des CCN céphaliques et la formation de la tête. De plus, j'ai découvert que Lkb1 contrôle la différenciation des cellules gliales en réprimant un programme de biosynthèse d'acides aminés couplé à la transamination du pyruvate en alanine, en amont de la voie de signalisation mTOR.

Les phénotypes dus à la perte de Lkb1 dans les CCN récapitulent les caractéristiques cliniques de maladies humaines appelées neurocristopathies. L'activation anormale du suppresseur de tumeur p53 est également associée à certaines neurocristopathies et l'ablation de p53 sauve le phénotype pathologique. Ainsi, j'ai montré que Lkb1 dans les cellules gliales contrôle p53 en limitant les dommages à l'ADN. Lkb1 est aussi essentiel pour maintenir l'homéostasie lysosomale et le recyclage des protéines et ainsi empêcher la formation de granules nommés lipofuscine, chargés en protéines et lipides oxydés. De façon intéressante, les voies mTOR et LKB1/AMPK sont activées à la surface des lysosomes de façon dépendante des niveaux d'acides aminés. Des données récentes de la littérature suggèrent que les lysosomes constitueraient une plateforme de signalisation pour contrôler la protéolyse et le devenir cellulaire. Ainsi, nos données proposent que les signalisations Lkb1 et p53 pourraient réguler l'homéostasie lysosomale et en conséquence le vieillissement cellulaire.

De façon intéressante, les cellules de Sertoli, des cellules somatiques épithéliales, localisées dans les tubes séminifères des testicules, et qui régissent la maturation des cellules germinales et l'homéostasie testiculaire, partagent des fonctions métaboliques similaires avec les cellules gliales. En effet, ces cellules sécrètent le lactate et l'alanine qui alimentent les mitochondries des cellules voisines (cellules germinales ou neurones respectivement) contrôlant ainsi leur survie et leur maturation. Au cours de ma thèse, nous avons observé que Lkb1 est requis pour l'homéostasie testiculaire et la spermatogenèse en régulant la polarité des cellules de Sertoli et leur métabolisme énergétique par le cycle pyruvate-alanine. Ces résultats suggèrent une conservation des régulations métaboliques par Lkb1 dans divers tissus.

Dans leur ensemble, mes travaux de thèse ont apporté une meilleure connaissance des mécanismes sous-jacents des régulations métaboliques lors du devenir cellulaire. Ces résultats fournissent de nouvelles perspectives sur le développement des CCN et élargissent notre compréhension du contrôle métabolique exercé par LKB1. Enfin, mes projets de doctorat ont mis en évidence l'existence d'une communication entre les voie de signalisation Lkb1 et p53 et suggèrent l'importance de cette communication dans les pathologies humaines dues à des défauts des CCN.

Figure 1 :

 1 Figure 1: Energy metabolism Glycolysis: one molecule of glucose forms 2 molecules of pyruvate. This mechanism is cytoplasmic and generates 2 molecules of ATP and 2 NADH. Gluconeogenesis is the generation of glucose from pyruvate. Krebs cycle or TCA (tricarboxylic acid cycle) is happening inside the mitochondria. Pyruvate enters into the mitochondria and forms AcetylCoA and NADH. 2 molecules of Acetyl CoA (per glucose molecule) are oxidized to CO2, releasing different chemical energy during the process such as: 2 ATP, 6 NADH, 2 FADH2. Oxidative phosphorylation (OXPHOS) is also a mitochondrial process performed by the electron transport chain. It uses the NADH and FADH2 produced by TCA cycle and generates up to 34 ATP molecules. ROS, Reactive Oxygen Species.

Figure 3 :

 3 Figure 3: Amino acid metabolism A. Metabolic pathways essential for amino acid synthesis. B. Amino acids degradation. Glucogenic amino acids are the amino acids whose degradation form metabolic intermediates give rise to glucose via gluconeogenesis. Amino acids which are forming ketone bodies such as acetoacetate and are not involved in gluconeogenesis are named ketogenic. All amino acids are glucogenic, except leucine and lysine which are only ketogenic.

Figure 4 :

 4 Figure 4: Lactate oxidation and malate-asparate shuttle

Figure 6 :

 6 Figure 6: Systemic activity of amino acids

Figure 7 :

 7 Figure 7: Amino acids metabolism coordinates the balance between proliferation and quiescence.

  As a general conclusion to this first chapter, metabolism exploration is recently experiencing a renewed interest. First, because there are striking similarities between the metabolic shifts observed in normal development and those characterized in tumors. Second, amino acids regulate intrinsic energy metabolism and control energy metabolism of neighboring cells or distant organs due to their paracrine and/or systemic activities as signal molecules during cell-cell communication. Third, through all their actions as signal molecules, amino acids regulate stem cell development, cell fate and tumorigenesis. However, metabolic reprograming during development remains poorly identified and there is still a lot to explore concerning the drivers and regulators. Particularly the role of metabolic remodeling in neural crest cells (NCC) is poorly understood. Thus, during my PhD, I studied metabolic regulations during NCC fate. Therefore, in the next Chapter, I will describe the NCC *** formation and differentiation and I will present some human pathologies due to NCC defects.

A

  . During neurulation, neuroectoderm forms the neural plate. Jonctional tissue between neural plate and ectoderm corresponds to the neural plate border. B. During the process of neural tube closure, neural plate borders are specified into neural crest. C. After neural tube formation, neural crest cells undergo an epithelialto-mesenchymal transition and delaminate from the neural tube. D. Neural crest cells then migrate towards their target organs where they differentiate into several derivatives. E. Non exhaustive list of neural crest derivatives (Simões-Costa and Bronner, 2015).

Figure 8 :

 8 Figure 8: Neural crest formation and derivatives.

Figure 9 :

 9 Figure 9: Molecular mechanisms which control neural crest ontogeny

Figure 10 :

 10 Figure 10: Time course of Schwann cell lineage & protein expression profiling Neural crest cells committed to form Schwann cell precursors (SCP) are present in peripheral nerves at embryonic day 8.5 (E8.5). These cells differentiate into SCP at E12.5 and into immature Schwann cells (iSC) around E15.5. Only after birth immature Schwann cells maturate into myelinating or nonmyelinating Schwann cells. The radial sorting starts before birth and finishes 10 days after birth (P10).

Figure 14 :

 14 Figure 14: Multipotency of Schwann cell precursors Neural crest cells (NCC) are multipotent cells which migrate and differentiate into many cell types such as melanocytes, neurons, ectomesenchymal derivatives and Schwann cell precursors (SCP). SCP are conserving the multipotency of NCC and therefore are giving rise to several cell types. By contrast to NCC, SCP do not have migratory properties, they are transported throughout the embryo by the nerves (Modified from Petersen and Adameyko, 2017).

Figure 13 :

 13 Figure 13: Mouse enteric nervous system developmentEnteric nervous system is formed during embryogenesis. The myenteric plexus is formed before the submucosal plexus. Vagal neural crest cells are present into the foregut at embryonic days 7-9.5 and are forming the myenteric plexus. These cells migrate and differentiate in neurons at around E10 -E10.5 and in glial cells at E12-E12.5. At E13.5 they finish their migration and sacral neural crest cells are undergoing an inward migration in order to form the submucosal plexus[START_REF] Lake | Enteric nervous system development: migration, differentiation, and disease[END_REF].

Figure 15 :

 15 Figure 15: Examples of neurocristopathies and ribosomopathies In blue are examples of neurocristopathies which are only due to Neural Crest Cells (NCC) defects.In yellow are examples of ribosomopathies due to ribosomal biogenesis that have several defects, among which NCC defects (except for 5q deletion syndrome). In green are pathologies which can be classified in both categories because they are due to both NCC defects (without other cell type defects) and ribosomal biogenesis defects.

Figure 16 :

 16 Figure 16: Ribosomal biogenesisRibosomal synthesis requires 47S ribosomal RNA formation which is cleaved in 18S, 5.6S and 28S rRNA. The 40S small ribosomal subunit is composed of 18S rRNA and ribosomal proteins RPS. The 60S large ribosomal subunit is composed of one 5.8S rRNA and one 28S rRNA as well as ribosomal proteins RPL. Ribosomal subunits 40S and 60S are exported into the cytoplasm where they assemble with mRNA and form the 80S ribosome necessary for protein synthesis[START_REF] Van Riggelen | MYC as a regulator of ribosome biogenesis and protein synthesis[END_REF].

NUCLEUSFigure 17 :

 17 Figure 17: Structure of human LKB1 isoforms Example of three different isoforms of LKB1 protein generated by alternative splicing. Human fulllength LKB1 form (LKB1 L) has 433 amino acids. Human short LKB1 form (LKB1 S) has only 404 amino acids and amino acids from 372-404 are not the same as in LKB1 L isoform. Human ΔN-LKB1 isoform has only 312 amino acids. It is characterized by a N-terminal truncation of Lkb1 and is catalytic inactive due to the absence of a part of its catalytic domain. NLS, nuclear localization signal; CRD, C-terminal regulated domain (Modified from Dahmani et al., 2015).

Figure 18 :

 18 Figure 18: The active heterotrimeric complex LKB1-STRAD-MO25STRAD in association with ATP and MO25, adopt an active like conformation similar to active protein kinases. This change of conformation promotes STRAD interaction with LKB1 as a pseudosubstrate and subsequently leads to LKB1-conformational changing from an inactive kinase conformation to an active one. MO25 stabilizes LKB1 activation by binding to its A-loop and promotes an extended active A-loop conformation of LKB1 (Rajakulendran and Sicheri, 2010).

Figure 19 :

 19 Figure 19: LKB1 post-translational modifications A. Mouse Lkb1 phosphorylation and prenylation. In red are represented the autophosphorylation residues, in black are the amino acids phosphorylated by others kinases and in green is represented the prenylation site. Amino acids T366, S404, S431 and C433 in mice correspond to T363, T402, S428 and C430 in humans. S, Serine; T, Threonine; C, Cysteine; AMPK, AMP-activated protein kinase; PKC-ɣ, Protein kinase C gamma; ATM, Ataxia-telangiectasia mutated kinase; PKA, Protein kinase A; ERK, Extracellular signal-regulated kinases; RSK, Ribosomal S6 kinase. (Adapted fromAlessi et al., 2006). B. Human LKB1 acetylation sites identified by Ido's team[START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation[END_REF] (represented here in bleu), by our teamunpublished data (in red), and common to both studies (in black)(Ghawittian, 2015).

Figure 20 :

 20 Figure 20: LKB1 phosphorylates the 14 members of the AMPK-related kinase family

Figure 22 :

 22 Figure 22: Natural or Pharmacological regulators of AMPK activity

Figure 24 :Figure 23 :

 2423 Figure 24: mTOR pathway and complexes mTORC1 complex controls several cellular processes such as ribosomal biogenesis, transcription and metabolism and thus regulates cell growth. mTORC2 complex regulates AKT (Protein kinase B) and Rho pathway being therefore involved in cell survival and proliferation. Modified from http://slideplayer.it/slide/10665517/

Figure 25 :

 25 Figure 25: Evolutionary conservation in LKB1 role as a regulator of cell polarityExamples of LKB1 functions in regulation of cell polarity. In green are some examples of kinases involved in these processes. Of note is that the list of enzymes is not exhaustive. Modified from[START_REF] Partanen | Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4[END_REF]

Figure 26 :

 26 Figure 26: Molecular mechanisms underlying regulation of cell polarity by LKB1

Figure 27 :

 27 Figure 27: Lkb1 involvement in embryonic development Left panel: Light microscopy of normal, wild type mouse embryo (+/+) at embryonic day 9.25. Right panel: Lkb1 knockout mouse embryos showing unfold embryos with open neural folds and a missing branchial arch[START_REF] Ylikorkala | Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice[END_REF]

Figure

  Figure 28: Clinical characteristics of Peutz-Jegers Syndrome

Figure 29 :

 29 Figure 29: p53 has an essential function in tumor suppression[START_REF] Brady | p53 at a glance[END_REF]

Figure

  Figure 30: p53 protein structure

Figure 33 :

 33 Figure 33: Regulatory feedback loops between p53, AMPK and mTOR pathways p53 regulates AMPK and mTORC1 through many downstream targets. AMPK and mTORC1 are also able to activate or inhibit p53 via different factors (indirectly) (Modified from Berkers et al., 2013).

Figure 34 :

 34 Figure 34: p53 controls energy metabolismp53 controls aerobic glycolysis by regulating several enzymes like: HK2, Hexokinase 2; PKF1, Phosphofructo kinase 1; TIGAR, p53-induced glycolysis and apoptosis regulator; PGAM, phosphoglycerate mutase. It also regulates gluconeogenesis through inhibition of G6PC, Glucose-6-phosphatase and PCK1, Phosphoenolpyruvate carboxykinase 1 via activation of Sirt6; Sirtuin 6. The mitochondrial respiration is positively regulated by p53 through the activation of PDH, pyruvate dehydrogenase; GLS2, Phosphate-activated glutaminase and electron transport chain complexes in the mitochondria. The fatty acid oxidation (FAO) which takes place into the mitochondria is also induced by p53.

Figure 35 :

 35 Figure 35: p53 dual role in autophagy regulation Left panel: Inhibition of p53 upon starvation or ER stress leads to autophagy activation. p53 absence promotes ER stress-induced autophagy. p53 loss leads to homeostatic imbalance (for example reactive oxygen species generation) which triggers autophagy. Dotted lines represent a speculative pathway by which p53 depletion may also result in autophagy. Right panel: Positive regulation of autophagy by p53 upon oncogenic stress. p53 induces autophagy through both transcription dependent or independent functions (Levine and Abrams, 2008).

Figure 36 :

 36 Figure 36: Both p53 accumulation and depletion are responsible for lethal phenotypes (Lozano, 2010)

  p53 downregulation is necessary to prevent cell cycle arrest and to induce EMT gene expression (Snail2, Ets1, Mycn, Sox9, Pax7) (Figure37B). Thus, Rinon et al. have shown that p53 coordinates cranial NCC growth and EMT/delamination during neural tube closure (Figure37C).

Figure 37 :

 37 Figure 37: p53 regulates cranial neural crest cell migration A: Schematic representation of a transverse section of 9-10 somite chick neural tube showing the expression of p53 (green cells) and SNAIL (cranial NCC (CNC) marker, red cells); p53 + SNAIL + CNC cells are yellow. B: p53 expression decreased in CNCs undergoing EMT/delamination. p53 regulates cell cycle genes and proliferation. C: p53 coordinates CNC growth and EMT processes during neural tube closure(Rinon et al., 2011).

Figure 39 :

 39 Figure 39: mTORC1 activation at the lysosomal surface

  its translocation to lysosomes thanks to LAPF while nuclear p53 regulates mTORC1 and AMPK lysosomal localization and activation through its transcriptional activity (Chapter III.B-2.2). For the moment, nothing is known about p53 lysosomal role regarding mTORC1/AMPK localization and activation. *** Altogether, these studies have shown that lysosomes are acidic vesicles involved in many cellular processes, the most recently discovered one being adjustment to nutrient conditions through mTOR/AMPK lysosomal translocation and activity. Lysosomal localization and activation of these pathways depend on the presence of several amino acids such as leucine, arginine and glutamine. Interestingly, both LKB1 and p53 translocate at lysosome surface where they regulate different processes: LKB1 activates AMPK and subsequent cell adaptation to energetic stress while p53 induces lysosomal membrane permeabilization and apoptosis.

Figure 42 :

 42 Figure 42: Oxidative stress-induced lipofuscin formation within lysosomes (Adapted from[START_REF] Terman | Lipofuscin: mechanisms of formation and increase with age[END_REF] Brunk et al., 1992)

Figure 41 :

 41 Figure 41: Reactive oxygen species and redox imbalance outcome

(

  NCC) development, including their migration and differentiation. However, the metabolic processes underlying NCC development remain poorly understood. Therefore, during my PhD, I explored the contribution of LKB1 in the control of polarity and energy metabolism in NCC. Along this line, in a first PhD project, I participated to the exploration of LKB1 involvement during cranial NCC polarized migration and vertebrate head development. I also contributed to the characterization of LKB1 post-translational modification by acetylation, and how acetylated LKB1 could regulates cranial NCC ontogeny. In a second PhD project which was also my main project, I investigated the metabolic regulations exerted by LKB1 during NCC fate and assessed if LKB1 controls NCC differentiation by limiting oxidative stress and p53 activation while favoring lysosomal activity, possibly in order to prevent premature aging. Finally, during my third PhD project, I analyzed energy metabolism in other models of cell polarity that are Sertoli cells and epithelial colorectal adenocarcinoma cells to better understand how LKB1 signaling coordinately regulates cell polarity and metabolism.

  Experiments performed on chick embryos were mainly done by Jean Viallet, Maya Ghawitian and Jacques Thélu from M. Billaud lab in collaboration with Sophie Creuzet lab (Neurosciences Institut, Paris-Saclay). Phenotypic characterization of the mouse model was mainly done by S. Torch and C. Thibert in M. Billaud lab. During my PhD, I contributed to the revisions required for the publication of our article at Developmental Biology on both mouse and chick models.

Figure 19

 19 Figure 19 B). Exploration of upstream acetyltransferases involved in LKB1 acetylation demonstrated that among 4 different enzymes, the acetyltransferase GCN5 was the major enzyme capable of acetylating LKB1 (N. Aznar, M. Billaud, unpublished data).

  Fig.1 GCN5 acetylates ectopically expressed LKB1 at K48. A. Bosc cells were transfected with plasmids expressing a WT-Flag-GFP-LKB1 protein or with a control plasmid (C). Cells were treated with 1µM TSA for 4h. Lkb1 was immunoprecipiated with anti-Flag antibodies and the acetylation of Lkb1 was detected by western blot with acetylated lysines antibody.B. Bosc cells were co-transfected with plasmids expressing Flag-GCN5, HA-TP60, HA-CBP, Myc-P300 and plasmids expressing WT-Flag-GFP-LKB1 or control plasmid. Immunoprecipitation of Lkb1and acetylated Lkb1 were done as previously described. C. Bosc cells were transfected with plasmids expressing WT-Flag-GFP-LKB1 or K48R-GFP-LKB1 or plasmid control (only pe-GFP) and treated with 1µM TSA for 4h. Lkb1 was immunoprecipitated with anti-GFP antibody and acetylated LKB1 K48 was analyzed by Western Blot with the home made antibody (V. Mirouse). D. Bosc cells were co-transfected with plasmids expressing WT-Flag-GFP-LKB1 or with peGFP and with plasmids expressing WT-Flag GCN5 or MUT-flag GCN5. MUT= GCN5 mutant in its catalytic domain (Y621A/F622A). Immunoprecipitation of Lkb1 and acetylated Lkb1 K48 were done as previously described. E. Bosc cells were transfected as presented in D. and Lkb1 was immunoprecipitated with anti-GFP antibody and GCN5 was immunoprecipitated with anti-GCN5 antibody.

  Fig.1 GCN5 acetylat ectopically expressed LKB1 K48. A. Bosc cells were transfecte with plasmids expressing a W Flag-GFP-LKB1 protein or with control plasmid (C). Cells wer treated with 1µM TSA for 4h. Lkb was immunoprecipiated with ant Flag antibodies and the acetylatio of Lkb1 was detected by wester blot with acetylated lysin

Further, I analyzed

  the distribution of acetylated LKB1 at K48. LKB1 is expressed in both cytoplasm and nucleus and depending of cell types, it is almost exclusively nuclear or more cytoplasmic. In HEK293T BOSC cells, endogenous LKB1 is both nuclear and cytoplasmic with a clear accumulation in the nucleus. In these cells, I analyzed if endogenous GCN5 regulates LKB1 subcellular localization. Interestingly, I observed that upon GCN5 knockdown, LKB1 was exclusively localized in the nucleus (Fig.2D). Instead, overexpressing LKB1 and GCN5 in Hela cells triggered cytoplasmic accumulation of LKB1 compare to overexpressed LKB1 alone which is mainly nuclear (Fig2E). However, GCN5 controls LKB1 cellular localization independently of its capacity to acetylate LKB1 since GCN5 mutant impaired for its acetyl transferase activity still triggers LKB1 relocalization from nucleus to cytoplasm (Fig.2E).I next assessed the localization of acetylated LKB1. Lan et al. have shown that LKB1K48 acetylation is nuclear and that deacetylation of K48 by Sirtuin1 increases the cytoplasmic translocation of LKB1 from nucleus[START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation[END_REF]. In line with their results, I observed by immunofluorescence that K48 acetylated form of LKB1 is exclusively nuclear (Fig.2Eright panels). I confirmed this result by cellular fractionation (validation shown left in Fig2F) followed by immunoprecipitation of endogenous LKB1 and western blot analyses of acetylated LKB1 K48. As seen in Figure2F(right panels) acetylated LKB1 at K48 is exclusively nuclear.

Fig. 2

 2 Fig.2 GCN5 acetylates LKB1 at K48 and regulates Lkb1 cellular functions.

  GCN5 acetyltransferase activity is essential for neural tube closure and contributes to head formation especially telencephalon morphogenesis. We next assessed whether LKB1 acetylation by GCN5 could regulate neural crest cell fate. I therefore analyzed if acetylated LKB1 K48 regulates cell migration. To do so, I used LKB1 null Hela cells transfected with empty vector, wild type LKB1 (LKB1 WT), non-acetylatable mutant of LKB1 (LKB1 K48R) or LKB1 harboring mutations in its nuclear localization sequence (LKB1 NLS) and which is therefore cytoplasmic only. As shown in figure 2I, LKB1 WT limited Hela cells migration due to its known tumor suppressor activity. LKB1 NLS also impaired cellular migration thereby suggesting that limitation of cell migration by LKB1 does not require its nuclear localization (Fig 2I).

A.

  Whole-mount in situ hybridization of chick embryos at different somite stages (ss) with the GCN5 antisense probe and control sense probe (experiments performed by Jacques Thélu) B. Chicken embryos at 4 somites stage. A-control embryo. B-representative embryo subjected to GCN5 silencing via electroporation with cGCN5 dsRNA (experiments performed by Jean Viallet).

Fig. 3

 3 Fig.3 GCN5 expression in cranial NCC is essential for vertebrate head formation.

(

  JoMa1.3 cells, see next project) cultivated as progenitors and LKB1 re-expression will be performed by retroviral infection. Viral vectors were constructed to allow the re-expression of human LKB1 (insensitive to murine Lkb1 siRNA) either wildtype or non-acetylatable mutant (collaboration C. Thibert and L. Attardi). Using classical protocols, cell survival, proliferation and migration in transwell containing a TGFβ gradient of concentration (Murphy et al., 2011) could then be assessed. Second, to evaluate in vivo contribution of Lkb1 acetylation, chick embryos at 4 somite stage could be co-electroporated with double stranded RNA targeting chick GCN5 and plasmids allowing expression of LKB1 wildtype or the non-acetylatable K48 mutant (LKB1 K48R) or acetylmimetic mutant (LKB1 K48Q). Analyzing if a correct cephalic

(

  Trichostatin A) (Sigma-Aldrich) at 37°C. Short interfering RNAs (siRNA) against the human GCN5 gene and the Non-targeting control siRNA were purchased from Fermentas /Thermo Scientific, Germany (SMARTpool ON-TARGET plus KAT2A (GCN5) human siRNA (ref. L-009722-02-0005) and ON-TARGET plus Non-targeting Pool (ref. D -001810-10-05)). siRNAs were transfected into Bosc cells by using RNAi Max reagent (Invitrogen/Life technologies) according the manufacturer's protocol.

Flag-

  LKB1 fused N-terminally to eGFP was described by[START_REF] Forcet | Functional analysis of Peutz-Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity[END_REF]. The nonacetylatable mutant of LKB1, K48R (a single point mutation of lysine 48 to arginine) or the acetyl mimetic mutant K48Q (a single point mutation of lysine 48 to glutamine) were obtained by Maya Ghawitian(Maya Ghawitian, 2015, PhD). The peB-flag and peBB-flag-GCN5 wild type vectors were obtained from E. Burstein, UT Southwestern medical center, Dallas, Texas.The GCN5 mutant in its catalytic domain (Y621A/F622A) was obtained by Maya Ghawitian. The different vectors were transfected into Bosc or Hela cells using JetPRIME transfectant (Ozyme/Polyplus) according to the manufacture's protocol. Experiments were realize 48h post-transfections. Immunoprecipitation Bosc cells were seeded in 8 dishes of 10cm of diameter at 2x10 6 cells/plate for endogenous protein immunoprecipitation or in 2 dishes per condition for overexpressed protein immunoprecipitation. Cells were lysed with NP-40 lysis buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA and 1% NP-40, supplemented with 1% proteases inhibitors (protease inhibitor cocktail /ref. P8340, SIGMA) and 10% PMSF (Phenylmethanesulfonyl fluoride/ref. P7626, SIGMA). After lysis and protein quantification, 1mg of lysates (for endogenous proteins) or 500µg of lysate (for overexpressed proteins) were completed to 1mlwith lysis buffer. They were incubated for 1h at 4°C on a wheel with protein A -coupled sepharose beads (Dutscher). After 1 mins of centrifugation at 4°C and 8000 rpm, beads were eliminated and lysates were incubated for 2h at 4°C with 4 mg of LKB1-K48 acetylated antibody developed by Vincent Mirouse team or with 4 mg of anti-Lkb1 antibody from Santa Cruz (Ley37DG-6) or with 1 µg of anti-Flag antibody from Sigma (clone M2). Further the lysates were incubated with protein A -coupled sepharose beads for 1h. The beads were then recovered by centrifugation, washed 4 times with the lysis buffer and boiled in 50uL of 2X Laemmli buffer, 5 mins at 95°C. Finally, they were analyzed by western blot.Western blot analysis10uL of protein extracts obtained after immunoprecipitation or 20µg of proteins were separated in SDS-10% polyacrylamide gels and transferred to PVDF Immobilon-P membranes (Millipore). After blocking, membranes were incubated overnight with different antibodies, as described Table1, in blocking solution at 4°C and with horseradish peroxidase-conjugated anti-rabbit or antimouse immunoglobulin G (Biorad) for 1 h at room temperature. Immunoreactive proteins were detected with Amersham ECL chemiluminescence reagents (GE Healthcare) according to the manufacturer's instructions.

  address the contribution of the Lkb1-dependent control of energy metabolism during the formation and the maintenance of NCC, our team generated another mouse model with temporal disruption of Lkb1 later in NCC development ("late" inactivation) compared to the model used in the first project ("early" inactivation). This model consists in Lkb1 Flox mice bred with Tyr::Cre transgenic mice established by L. Larue lab (Curie Institute, Orsay) thus allowing the recombinase Cre expression under the Tyrosinase (Tyr) promoter in NCC from E10.5 i.e.

  Neural crest cells (NCC) are highly invasive stem cells that originate from the dorsolateral folds of the neural tube and delaminate from the neuroepithelium through an epithelial-mesenchymal transition (LeDouarin and Kalcheim 1999). NCC migrate along stereotypical paths and give rise to a broad array of derivatives including most of the craniofacial skeleton, neuroendocrine tissues and pigment cells. Furthermore, the peripheral nervous system, comprising sensory neurons and Schwann cells (SC) of the parasympathetic and sympathetic nervous systems, as well as neurons and glial cells that constitute the enteric nervous system (ENS), arise from NCC. Defects in human neural crest development are the cause of a constellation of disorders called neurocristopathies. The most common clinical phenotypes include craniodefects and enteric ganglia agenesis (Mayor and Theveneau 2013). Although the cascade of molecular events that specify neural crest territories, orchestrate NCC delamination and guide their polarized migration throughout the embryo has been extensively explored, the contribution of metabolic regulation to NCC formation and differentiation has received less attention. In this regard, morphogenesis requires adjustment of the metabolism of embryonic cells to cope with alterations in nutrient availability and tissue oxygenation specific to their microenvironment (Simon and Keith 2008). The mapping of the metabolic pathways that are activated in early stem cells has begun to shed light on the respective contribution of glycolysis versus oxidative phosphorylation in the maintenance of pluripotency and cell fate commitment (Ochocki and Simon 2013).However, the role of metabolic remodeling in later developmental stages, including the formation of neural crest derivatives, remains poorly understood.The tumor suppressor LKB1 (also named STK11) encodes a serine/threonine kinase that has been evolutionarily conserved from invertebrates to mammals(Baas et al. 2004; Hardie and Alessi 2013).Germline mutations of LKB1 are responsible for the Peutz-Jeghers syndrome, a dominantly inherited cancer disorder and somatic mutations of this gene have been associated with various cancers including lung and cervical tumors[START_REF] Shorning | Energy sensing and cancer: LKB1 function and lessons learnt from Peutz-Jeghers syndrome[END_REF].The binding of LKB1 to the pseudokinase STRAD (STRADa or STRADb) and the scaffolding molecule MO25 allosterically controls LKB1 catalytic activity and promotes its nuclear export[START_REF] Baas | Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD[END_REF].LKB1 acts as a key regulator of energy metabolism through the activation of the AMP-activated protein kinase (AMPK), a sensor that adapts energy supply to the nutrient demands of cells facing situations of metabolic stress(Shackelford 2013). AMPK phosphorylates a large array of substrates to achieve metabolic adaptations. In particular, AMPK phosphorylates the acetyl CoA carboxylase (ACC) to inhibit fatty acid synthesis[START_REF] Davies | Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase[END_REF] Merrill et al. 1997) as well as two regulators of mammalian target of rapamycin complex 1 (mTORC1), RAPTOR and TSC2, to inhibit the mTOR pathway(Gwinn et al. 2008; Inoki et al. 2003). In addition to AMPK, LKB1 also phosphorylates 12 AMPK-related kinases that regulate cell polarization, axon branching of cortical neurons and hepatic neoglucogenesis(Alessi et al. 2006; Hezel and Bardeesy 2008; Shackelford and Shaw 2009; Shorning and Clarke 2011).We recently showed that Lkb1 governs several aspects of cephalic NCC development that are crucial during vertebrate head formation (Creuzet et al. 2016). To better understand the contribution of Lkb1 to the formation of neural crest derivatives, we disrupted this gene in a subset of vagal and truncal mouse NCC using the tyrosinase Cre (Tyr::Cre) transgene. We report here that Lkb1-deficient mice exhibit coat depigmentation as well as progressive hindlimb paralysis originating from a blockade of SC differentiation and intestinal pseudo-obstruction arising from a degeneration of the ENS. Using a neural crest cell line, we demonstrate that Lkb1 is key to establish the glial lineage. Although Lkb1 loss induces mitochondrial dysfunction, we show that mitochondrial respiration is dispensable for glial differentiation. Metabolomics profiling of peripheral nerves demonstrates that Lkb1 loss leads to alanine and glutamate accumulation. By mechanistic analyses, we identify that Lkb1 knockdown triggers a preferential transformation of pyruvate into alanine by the alanine aminotransferase (ALAT) and inhibition of this enzyme rescues glial differentiation of Lkb1-null NCC in an mTOR-dependent manner. AICAR, an analogue of AMP, normalizes mTOR signaling, promotes glial differentiation in vitro and prevents hindlimb defects and ENS degeneration in vivo. Altogether, our results provide evidence that Lkb1 signaling contributes to the setting up of several different neural crest-derived cell populations that belong to the melanocytic, enteric and Schwann cell lineages. These data also highlighted that Lkb1 signaling controls glial lineage by repressing a nonessential amino acids biosynthetic program coupled to pyruvate-alanine transamination.

  The tyrosinase promoter is active from embryonic day E10.5 in subsets of NCC, thereby allowing the targeting of migrating melanoblasts(Delmas et al. 2003) as well as NCC-derived cells of the ENS[START_REF] Puig | The tyrosinase promoter is active in a subset of vagal neural crest cells during early development in mice[END_REF])(Delmas et al. 2003;[START_REF] Puig | The tyrosinase promoter is active in a subset of vagal neural crest cells during early development in mice[END_REF]R26RF/F;Tyr::Cre mutants and wild-type control littermates in a mixed genetic background mostly C57BL6/J (FigureS1B). Since the Tyr::Cre transgene is localized on the X-chromosome, to avoid a bias due to X chromosome inactivation, our study is solely based on the analysis of males. Heterozygous Lkb1+/F;Tyr::Cre/° male mice were fully healthy and fertile (data not shown).HomozygousLkb1-deficient mice (Lkb1F/F;Tyr::Cre/° referred here as cKO) were born at the expected Mendelian ratio (FigureS1C) but showed either an almost complete lack of coat color pigmentation or white spotting (Figure1Band FigureS1D). Since a previous report demonstrated that Lkb1 function is required for the proper development of melanocytes, we did not focus our study on the physiological mechanisms underlying the depigmented phenotype. Most of the cKO mice failed

Lkb1

  in mouse intestine and colon. We observed that Lkb1 is expressed in myenteric and submucosal plexuses both in the intestine and in the colon of 21 days old mice (P21; Figure1H, left panels), and corresponds to enteric ganglia reflected by colocalization with the neural marker Tuj1 (FigureS2A). As expected, less or no Lkb1 was detected in the ganglia of cKO mice, although the retention of staining in a few ganglia indicated that Lkb1 deletion is mosaic. Analysis of intestinal expression of bgalactosidase as a reporter of Cre-mediated Lkb1 deletion revealed a staining in cells that were identified as neural and glial enteric using antibodies recognizing the RNA-binding protein HuC/D and GFAP respectively (FigureS2B). A similar LKB1 expression pattern was observed in human intestine and colon specimens (FigureS2C). Altogether, these results indicate that Lkb1 is expressed in the ENS cells of human and mouse tissues and that Lkb1 inactivation in our mouse model occurred in neural and glial enteric cells.

First, we studied

  by whole-mount staining the development of enteric neurons using the neural marker Tuj1 at embryonic day 15.5 (E15.5, not shown) and at birth (P0; Figure 1I). Four different areas of the digestive tract were analyzed: proximal and distal intestine and proximal and distal colon (PI, DI, PC and DC, respectively). No overt sign of alteration of the ENS network was observed at E15.5 or P0 suggesting that Lkb1 inactivation starting from E10.5, when Cre is produced by enteric NCC precursors, neither altered NCC migration nor their differentiation into neurons. Second, we used AcetylCholine Esterase (AChE) activity in wholemount to visualize the ENS at P21. In wild-type intestine and colon, ganglia developed correctly and the neuronal network that connects the nervous plexuses was clearly visible (Figure 1J). In contrast, in Lkb1-deficient mice the staining was either strongly reduced or undetectable in several regions of the intestine. The affected areas were variable in length and involved either the proximal or distal parts of the intestine or colon, with the distal regions being most commonly affected. Hematoxylin-eosin staining indicated that the tunica muscularis and enterocyte epithelium had normal morphology. However, we frequently observed damaged intestinal villi in the distal intestine, probably due to fecal accumulation in the digestive tract (Figure S2D). Taking advantage of the R26R allele, we visualized b-galactosidase

Figure S2G (

 S2G Figure S2G (left panels), Lkb1 loss was observed in 6 days old cKO animals (P6). At this time point, compared to WT mice, cKO animals exhibited clearly diminished numbers of Tuj1 and GFAP-positive ganglia in the distal intestine and colon and reduced staining of the remaining ganglia that were smaller in size and displayed punctiform morphology (Figure S2G right panels). These results indicated that Lkb1 deficiency is associated with a progressive postnatal degeneration.To determine whether the progressive loss of enteric neurons observed in Lkb1-deficient mice after birth at P6 and P21 was due to an increase in neuronal death, we used TUNEL assays and cleaved caspase-3 immunostaining. No apoptosis was detected in enteric ganglia of cKO animals at all stages analyzed (FigureS2Hand not shown).

  sections of sciatic nerves at P6 and P21. We observed a relatively homogeneous staining in wild-type animals (Figure2A). Lkb1 staining was confirmed by immunohistochemistry using a second antibody specific for Lkb1[START_REF] Nakada | The LKB1 tumor suppressor as a biomarker in mouse and human tissues[END_REF]) (FigureS3A).In cKO mice, Lkb1 staining was clearly reduced on sections of sciatic nerves (Figures2A and S3A).However, the loss of Lkb1 was not homogeneous as Lkb1 expression was still detected in some areas. To confirm Lkb1 loss of function, we evaluated the phosphorylation status of the LKB1 substrate AMPK. markedly decreased in absence of Lkb1 (FigureS3B). Consistent with our previous observations in the enteric nervous system, phospho-S6RP labeling was enhanced in the sciatic nerves (Figure2B). These data confirm that inactivation of Lkb1 in sciatic nerves results in the dysregulation of AMPK-mTORC1 signaling.Although expression of the Cre recombinase driven by the tyrosinase promoter has been previously observed in peripheral nerves(Delmas et al. 2003), its precise pattern of expression was unknown. Thus, in order to discriminate whether Lkb1 was inactivated in peripheral sensory axons, in SC or in both cell types, we first exploited reporter Tyr::Cre;Z/EG mice which allow labeling of neural crest-derived cells expressing the Cre recombinase with enhanced green fluorescent protein (EGFP)[START_REF] Colombo | Genomic localization of the Z/EG transgene in the mouse genome[END_REF]. Immunofluorescence on embryonic sections allowed the detection of glial cells, labeled with an antibody recognizing the intermediate filament protein GFAP that were also positives for EGFP (FigureS3C) whereas neuron--tubulin staining recognized by the Tuj1 antibody was never co-localized with EGFP (FigureS3D). We also took advantage of the presence of the R26R reporter in the Tyr::Cre;Lkb1 mice to stain semi-thin sections of sciatic nerves from wild-type animals with X-Gal.These studies revealed cytoplasmic staining in SC but not in the axons (FigureS3E). Finally, we analyzed sections of the dorsal root ganglia, which contain neuronal cell bodies and glial cells with galactosidase allowing a more sensitive detection of galactosidase activity in the dorsal root ganglia of either WT or cKO animals (Figure S3F). -galactosidase expression in dorsal root ganglia revealed a clear colocalization with glial GFAP-positive cells but not with neuronal Tuj1-positive cells (Figure S3G). Altogether, these results indicate that Lkb1 inactivation was restricted to SC and did not occur in sensory neurons. We next examined the ultrastructure of sciatic nerves at P21 by transmission electron microscopy. The sciatic nerves of control animals were composed of myelinated sensory and motor axons of medium to large diameters as well as clusters of small-diameter unmyelinated sensory axons shown on Figures 2Cac. The number of myelinated axons in cKO animals was reduced and degeneration of axons and myelinating SC was observed (Figures S3H and

  2Ci) indicating hypomyelination of axons in cKO animals. Altogether, these results indicated that Lkb1 is involved in the sorting of axons by Schwann cells, the myelination process and the maintenance of myelinated axons. As axon sorting and SC myelination in the mouse begins shortly after birth, we assessed neural and glial cell differentiation in the sciatic nerves of cKO mice at P6 and P21. Tuj1 neuron-specific staining was strongly reduced in cKO animals compared to wild-type at both ages (Figures 2D, left panels and S3I). By contrast, the staining of SC visualized with the GFAP antibody was not reduced at P6 and P21, although the subcellular localization of GFAP was disrupted at P21 (Figures 2D, right panels, insets and S3J), thus suggesting that SC polarity was disrupted.

  glial differentiation medium (without 4-OHT and supplemented with Heregulin and Forskolin), JoMa1.3 cells underwent morphological changes, acquiring a spindle shape (Figures S4B and S4C). The induction of glial differentiation was confirmed by positive staining for the glial markers GFAP and S100, and loss of expression of the progenitor marker p75 NTR (Figure 3B and S4D). We then established and validated a protocol to efficiently down-regulate Lkb1 expression by transfecting Lkb1 siRNA either in progenitor cells or during NCC-Lkb1 triggered a reduction of AMPK phosphorylation and an activation of the mTOR pathway in JoMa1.3 cells (FiguresS4E-H) as observed in vivo. Lkb1 knockdown strongly impacted cell morphology during glial differentiation as visualized with the formation of stress actin fibers labeled with phalloidin (FigureS4I). Accordingly, glial differentiation was impaired as assessed by the reduced expression of glial specific markers S100 or GFAP (Figure3C). RT-qPCR revealed that Lkb1 knockdown resulted in an increase of p75 NTR expression and a loss of GFAP, further confirming that cells were maintained as progenitors and showed impaired glial differentiation (Figure3D; data not shown).

  alanine and glutamate deregulation, we took advantage of the in vitro JoMa1.3 NCC model. We first evaluated alanine levels in JoMa1.3 cells committed to glial differentiation upon Lkb1 knockdown. As observed on sciatic nerves, alanine concentration was increased in the absence of Lkb1 (Figure5A). In addition, we confirmed the accumulation of alanine in Lkb1-null Mouse Embryonic Fibroblasts (FigureS5A). Conversely, alanine was not increased in JoMa1.3 progenitor cells upon Lkb1 knockdown (FigureS5B).Our results support a mechanistic link between Lkb1 function and pyruvate-alanine transamination via the alanine transferase (ALAT, Figure5Bupper panel), either cytoplasmic (ALAT1) or mitochondrial (ALAT2). We thus blocked ALAT enzymatic activity using the inhibitor Chloro-alanine (Figure5Blower panel)[START_REF] Beuster | Inhibition of Alanine Aminotransferase in Silico and in Vivo Promotes Mitochondrial Metabolism to Impair Malignant Growth[END_REF]) and observed normalized alanine levels upon Lkb1 knockdown, demonstrating that the Chloro-alanine is indeed an inhibitor of ALAT in glial JoMa1.3 cells (FigureS5C). We also measured lactate levels and observed that Lkb1 knockdown resulted in a decrease of lactate concentration whereas Chloro-alanine treatment led to an increase of lactate level (FigureS5D). These data indicate that Lkb1 knockdown results in an enhanced conversion of the pyruvate into alanine instead of lactate. To further test if the preferential transformation of pyruvate into alanine could also be associated with deregulation in glycolysis per se, we measured the medium acidification resulting from lactate and proton production via glycolysis using the Seahorse analyzer in the presence or not of Chloroalanine. We first observed that basal glycolysis

Multipotentphenocopying

  NCC face a limiting supply of oxygen and nutrients during both their journey through the embryonic landscape and the distant colonization of anatomical structures. The metabolic adaptation of NCC to these microenvironmental conditions is thought to constrain their migration as well as their proliferation, differentiation and survival once they reach their final destination. In agreement with this notion, we show here that Lkb1 is essential for NCC fate commitment and controls a metabolic program in NCC that is key for glial differentiation and survival of ganglion cells of the ENS.Our work reveals that the NCC-specific ablation ofLkb1 results in a progressive peripheral neuropathy stemming from an impaired differentiation of SC.These results are consistent with recent studies demonstrating the role of Lkb1 in SC-axon maintenance and myelination(Beirowski et al. 2014; Pooya et al. 2014; Shen et al. 2014). Through the combination of in vivo, metabolomic and pharmacological approaches, we find that Lkb1 deletion leads to an intracellular accumulation of glutamate and alanine. Inhibition of pyruvate-alanine transamination restores glial differentiation of Lkb1deficient NCC. Consistently, increase of the glutamate level through inhibition of GS led to an accumulation of pyruvate-alanine transamination, Lkb1 inactivation. It has been recently reported that rapidly proliferating mammary epithelial cells utilize transaminases to link NEAA synthesis to the production of a-ketoglutarate and tricarboxylic acid anaplerosis (Coloff et al. 2016). Conversely, activation of glutamate dehydrogenase that decouples carbon and nitrogen utilization promotes cell quiescence (Coloff et al. 2016). These findings fit with our observation since the unrestrained activation of the pyruvate-alanine cycling is incompatible with the transition of Lkb1-deficient NCC to quiescence and subsequent glial differentiation. Interestingly, Lkb1 loss has also been associated with an increased expression of phosphoserine amino transferase, an enzyme participating to the serineglycine one carbon network whose aberrant activation supports the malignant transformation of Lkb1-mutant pancreatic cells (Kottakis et al. 2016). These data point to Lkb1 acting as a key component of a metabolic checkpoint that controls a switch between cell proliferation and differentiation through transaminases-dependent NEAA synthesis. They also suggest that the Lkb1 pathway may act in the glutamate-glutamine cycle that participates directly in the glial-neuron metabolic dialogue. The mitochondria topology is altered in cKO myelinated fibers and although mitochondrial mass is increased, both respiration and ATP production are severely reduced in Lkb1-deficient Joma1.sorting could not be studied with the Joma1.3 model, and an active mitochondrial oxidative metabolism is probably needed for the final step of NCC differentiation into mature myelinating SC ((Pooya et al. 2014), We also report that mTORC1 hyperactivation is connected to the elevated level of alanine and downregulation of mTOR restored glial differentiation of Lkb1-deficient NCC.In accordance with our data, mTORC1 is known to drive the early steps of SC differentiation, and an adequate dosage of mTOR activity is required for the peripheral nervous system myelination[START_REF] Norrmén | mTORC1 controls PNS myelination along the mTORC1-RXRγ-SREBP-lipid biosynthesis axis in Schwann cells[END_REF][START_REF] Preitschopf | mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression[END_REF][START_REF] Sherman | Arrest of Myelination and Reduced Axon Growth When Schwann Cells Lack mTOR[END_REF] Beirowski et al. 2017). Although leucine and arginine are major activators of mTORC1[START_REF] Meijer | Regulation of autophagy by amino acids and MTOR-dependent signal transduction[END_REF], additional amino acids are suspected to be involved in this process[START_REF] Bar-Peled | Regulation of mTORC1 by amino acids[END_REF][START_REF] Hara | Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism[END_REF], and it will be relevant to investigate how alanine sensing is tied to the mTOR signaling machinery. We further find that treatment of Lkb1-silenced NCC with the cellpermeable nucleoside AICAR normalizes mTOR signaling without restoring AMPK phosphorylation and promotes glial differentiation. This result is unexpected since AICAR is known to exert most of its biological effects through AMPK, although it has become progressively evident that this metabolite has additional cellular targets (Daignan-Fornier and Pinson 2012). The rescue of hindlimb paralysis and of ENS degeneration obtained with AICAR indicates that this compound or molecules upregulating its intracellular level (Asby et al. 2015) may represent a viable therapeutic intervention to target the metabolic defects induced by LKB1 inactivation. Our work unveils a critical function of Lkb1 in the postnatal maintenance of myenteric and submucous plexi. If our data clearly establish a role for Lkb1 in cell survival of the ENS, we cannot exclude that Lkb1 activity is required at earlier stage of vagal NCC migration, since NCC invade the rostral foregut at E8.5-E9 and the tyrosinase promoter in turned on at E10.5. TUNEL assays and immunostaining of active caspase 3 do not show an increase of apoptosis in the ENS of cKO mice. A similar unconventional death of postmigratory enteric neurons has been describedin mice with a conditional ablation of each subunit of the GDNF receptor, GFRa1 and RET[START_REF] Uesaka | Conditional ablation of GFRalpha1 in postmigratory enteric neurons triggers unconventional neuronal death in the colon and causes a Hirschsprung's disease phenotype[END_REF][START_REF] Uesaka | Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice[END_REF]. Interestingly, SC precursors (SCP) that display NCC stem-like properties and depend on GDNF signalling populate the gut through extrinsic nerves to contribute to the post-natal integrity of the ENS[START_REF] Green | Ancient evolutionary origin of vertebrate enteric neurons from trunkderived neural crest[END_REF] Uesaka et al. 2015). It is thus plausible that the post-natal degeneration of the ENS occurring in cKO mice reflects an attrition of this pool of cells. Since SCP give rise to melanocytes(Adameyko et al. 2009), the coat depigmentation observed in Lkb1 cKO mice may also result from the compromised competence of SCP to generate pigmented cells.

  experimental procedures were conducted according to the standard operating procedures of the lab animal facility and were approved by the Animal Ethics Committee of Grenoble (Permit number 261_ IAB-U823-CT-01).Human samplesNormal Human intestine and colon tissue samples were obtained from biopsies of patients with peritoneal intestinal carcinosis. All samples collected were taken at distance from the bulk of the tumor, stored and used with the informed consent from the patients.Neural crest cell culture conditions, siRNA transfection, metabolic drug treatmentsMouse immortalized neural crest cells (JoMa1.3) were cultured as described previously(Maurer et al. 2007). Differentiation of these cells into glial cells was achieved by adding Heregulin (50 ng/mL; Peprotech,120-02) and Forskolin 5 µM (Sigma) to the JoMa1.3 culture medium during 6 days. Short interfering RNAs (siRNA) against the mouse Lkb1 gene and the Non-targeting control siRNA were purchased from Dharmacon (ON-TARGET plus mouse Stk11 SMART pool and ON-TARGET plus Non-targeting Pool, Dharmacon). siRNAs were transfected (11 nM) into JoMa1.3 cells by using JetPrime (Ozyme) according the manufacturer'72h post-transfection. For differentiated JoMa1.3, two cycles of 96h of Lkb1 knockdown were realized, as described in Figure S4E. During the 6 days of cell differentiation and the second cycle of Lkb1 inactivation, cells were preincubated, or not, at 37°C in NCC medium every two days from D4 to D9 with different metabolic inhibitors: 100 µM b-Chloro-L-alanine (Chloro-ala, ALAT inhibitor, Sigma), 2 mM MSO (Glutamine Synthetase irreversible inhibitor) and 50 µM AICAR (a metabolic regulator and AMPK agonist).

  were dried overnight at room temperature and stored at -20°C. For E-cadherin immunostaining, the teased fibers were permeabilized in cold aceton for 10 minutes, washed with PBS, incubated 1h at room temperature in blocking solution (10% goat serum and 0.3% Triton X-100 in PBS) and then incubated with primary antibodies in blocking solution overnight at 4°C. Samples were washed in PBS and incubated for 1h at room temperature with secondary antibodies. Finally, samples were washed in PBS incubated in Hoechst to stain nuclei and mounted in Mowiol. Images were acquired using a Zeiss biphoton confocal microscope LSM510. To label mitochondria, teased fibers were incubated with MitoTracker® Red CMX-ROS (Invitrogen) 30 minutes at 37°C (500 nM), washed in PBS, fixed 10 minutes in 4% paraformaldehyde, incubated in Hoechst 10 minutes and mounted in Mowiol. Images were acquired using a Zeiss biphoton confocal microscope LSM510.

  Seahorse XF-Analyzer (Seahorse Bioscience) and 3×10 4 cells were seeded in 96-well Seahorse plates in DMEM with 10% FCS 16 h before assay. Cells were equilibrated with DMEM lacking bicarbonate at 37°C for 1h in a custom incubator without CO2. OCR and ECAR were measured at baseline and following addition of reagents for indicated times. For the mitochondrial respiration, the spare respiratory capacity was calculated by subtracting the maximal respiration values by the basal respiration values. For both respiration and glycolysis, quantifications were performed by calculating for each category the mean and standard deviation from the values obtained from several independent experiments. mice WT and cKO mice were treated with AICAR (0.5 mg/g bw) or vehicle by i.p. injection (100 ml/mouse) every two or three days from P10 to P21. Mice received 5 injections and were sacrificed at P21 and analyzed as described previously. Graphical representations and Statistical analyses All cell culture experiments were realized at least by three independent experiments and for one experiment, each condition was performed in triplicate. In vivo analyses were conducted with at least three animals per condition. The exact number of experiments is indicated in the figures or in the legends. Values are given as mean with standard deviation. Normality of the data was checked with the F-Test. The unpaired Student's t-test was used to generate p-values for selected pairwise comparisons and p < 0.05 was considered significant. Nerve metabolomic profiling by HRMAS NMR Sciatic nerves from 16 WT mice and 13 cKO animals were rapidly dissected at p21, cleaned of surrounding fat and connective tissues and immediately frozen in liquid nitrogen. Left and right nerves of each animal were inserted in a disposable insert for HRMAS NMR spectroscopy in a -20°C cooling chamber. Sample preparation for HRMAS NMR 7 µL of D2O containing trimethylsilylpropionate (TSP, 1 mM) as chemical shift internal standard (0 ppm) was added to the sample in the disposable insert, which was sealed and inserted in a 80 µL zirconium rotor. 1 H HRMAS NMR acquisition 1 H-NMR spectra were acquired with a Bruker Avance III 500 spectrometer (BrukerBiospin, Wissembourg, France) (CEA-Grenoble and IRMaGE facility). Samples were spun at 4000 Hz and temperature maintained at 4°C for all experiments. 1D spectra were acquired with a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence synchronized with the spinning rate (inter-pulse delay 250 μs, total spin echo time 30 ms). The acquisition of one 1 H-NMR spectrum consisted of 512 scans lasted 32 min.

  e., weighted by 1/standard deviation for a given variable) and submitted to a principal component analysis (PCA) to ensure good homogeneity of data and possibly to exclude outliers. For this aim, data were visualized by score plots, where each point represents a NMR spectrum and thus a sample. Supervised analyzes like orthogonal partial least square discriminatory analyses (OPLS-DA) were thus performed, using the group belonging (control or exposed) as Y matrix. The number of components was determined using the cross-validation procedure that produces R 2 Y and Q 2 factors (> 0.5). Moreover, the reliability of our OPLS-DA model was assessed by a CV-ANOVA test. The results were visualized by plotting the score of individuals relative to two first components of the model. In order to highlight metabolites that are the most discriminating between controls and exposed, S-line were examined. This plot allows the visualization in a single graph, which mimics a NMR spectrum, the covariance (peak intensity) and correlation (peak color) between cKO and WT animals. Moreover, the signs of peaks in S-line give indications about the direction of change of metabolites increased (positive peaks) or decreased (negative peaks) relative to WT.

Figure 1 .

 1 Figure 1. Lkb1 is crucial for neural crest cells lineage and enteric maintenance. (A) Schematic representation of the conditional ablation of Lkb1 specifically in neural crest cells (NCC) with the Tyr::Cre driver after they migrated to their target organs. NCC derivatives targeted for Lkb1 inactivation with this model are represented on the right and below with indications about their origin, their normal timing of differentiation and the stage of genetic ablation. (B) Representative picture of a male animal with homozygous Lkb1 ablation (Lkb1 cKO) which typically displays coat color hypopigmentation. (C) Kaplan-Meier graph comparing the survival of Lkb1-knockout (cKO), heterozygous (Htz) and wildtype (WT) male littermates. The number of animals is indicated above the curves. (D) Lkb1 cKO mice rapidly loosed weight compare to WT littermates (weight curves for cKO indexed to WT). (E) cKO mice develop a progressive intestinal pseudo-obstruction as shown at postnatal day 21 (P21) (middle panel) by empty colon (Co) and atrophy of the cecum (Ce) and at adulthood (lower panel) with atrophy of the stomach (St, dotted arrow), dilation of the small intestine (Int, arrow head), hypertrophy of the cecum (arrow) and colon constriction (empty arrow) compare to WT (upper panel) (n≥8 mice per group). (F) Loss of hind limb extension reflex in Lkb1 cKO P21 mice compared to control animal (WT) manifested by hind limb clenching when lifted by the tail. (G) Appearance of sciatic nerves from control and Lkb1 cKO mice at P21 showing myelinization defects in the mutant. (H) Staining with Lkb1 Cell Signaling D60C5 antibody of myenteric ganglia in P21 mice in WT (arrows) and cKO animals. (I) Wholemount Tuj1 labeling at birth (P0) on digestive tract from WT or Lkb1 cKO animals. No obvious difference in proximal and distal intestine (PI, DI) and proximal and distal colon (PC, DC) was observed in cKO animals compare to WT. (J) Whole-mount Acetylcholinesterase (AChE) staining of enteric nervous system at P21 showed marked loss of enteric ganglia. (K) Neural (Tuj1) and glial (GFAP) staining on roll sections of intestine and colon (WT

Figure 2 .

 2 Figure 2. Defective myelination of sciatic nerves in Lkb1 cKO mice correlates with defects in maturation of Schwann cells (A) Longitudinal sections of sciatic nerves of WT and Lkb1 cKO mice at P6 and P21 immunostained with Lkb1 antibodies. (B) Increased phospho-S6RP staining was observed in cKO animals in transverse sections of sciatic nerves (n=5 WT, n=4 cKO). (C) Electron microscopy of sciatic nerves of P21 WT (ac) and Lkb1 cKO mice (d-h). Transverse sections of WT nerves show normally myelinated nerve fibers (a,b) and regular unmyelinated axons in Remak bundles (c) whereas sections of cKO nerves show disorganization of the tissue (d) with neurodegenerative figures (e), aberrantly unsegregated axons of small to large diameter (f) and axonal sorting defects (g, h). (i) Scatter plot of g ratio values with linear regressions in sciatic nerves of WT (blue, n=410 axons, 2 animals) and cKO (red, n=473 axons, 3 animals). Scale bars represent 10 mm (Ja and d), 5 mm (Jb), 2 mm (Je, g, h) and 1 mm (Jc and f). (D) Tuj1 and GFAP immunostaining on transverse sections of P21 sciatic nerves. Insets show higher magnification of images with neural loss (Tuj1) and glial marker mislocalization (GFAP) in cKO animals (WT n=5, cKO n=3). (E) E-cadherin (E-cad) staining on isolated nerve fibers from sciatic nerve of P21 mice showing aberrant E-cad localization. (F) Immature Schwann cell marker Sox2

Figure 3 .

 3 Figure 3. Lkb1 knockdown impairs NCC-derived glial differentiation. (A) Schematic representation of the Schwann cell lineage depicting first steps involved in glial commitment and later steps in Schwann cell differentiation. (B) Transformed mouse NCC JoMa1.3 cells were maintained either as progenitors or differentiated into glial cells (first steps of the lineage) as validated by staining against glial markers GFAP (left panels, n=3) or S100 (right panels, n=3). (C) Transfected JoMa1.3 cells during glial commitment with Lkb1-targeting siRNA (siLKB1) showed decrease of glial markers compared to nontargeting control siRNA (scramble, siScr); GFAP n=3; S100 n=4. (D) RT-qPCR showing expression of Sox10 and p75 NTR genes in glial JoMa1.3 cells upon Lkb1 silencing. Data are expressed as relative expression in control cells compare to siLkb1 cells (siCont./siLkb1, n=4). Expression level of endogenous mouse Lkb1 in JoMa1.3 cells is also shown (n=4). (E) Mitochondrial mass was quantified by FACS analyses with Mitotracker Green staining (n=3). (F) Mitochondrial topology of glial JoMa1.3 cells stained with Mitotracker Red was analyzed by confocal microscopy (n=3). (G) Mitotracker staining on isolated myelinated nerve fibers of wildtype (WT) or Lkb1-deficient mice (cKO). (H) Representative electron microscopy images of mitochondria from control (siCont.) and Lkb1 knockdown (siLkb1) glial JoMa1.3 cells showing increased cristae width in the absence of Lkb1. Quantification was performed on 429 and 336 cristae from mitochondria of control and Lkb1-silenced cells respectively. (I) A representative trace showing change in oxygen consumption rates (OCR) during a mitochondrial stress assessed by Seahorse analyses on control (siCont.) and Lkb1silenced (siLkb1) glial JoMa1.3 cells (n=12; quantification is shown in S4J).

Figure 4 .

 4 Figure 4. Lkb1 loss in sciatic nerves triggers a metabolic reprogramming.

Figure 5 .

 5 Figure 5. Increased levels of the alanine and glutamate are associated with impaired glial differentiation. (A) Intracellular alanine concentration of control (siCont.) or Lkb1 knockdown (siLkb1) glial JoMa1.3 cells (n=3). (B) Schematic representation of metabolic reprogramming triggered by Lkb1 silencing (siLkb1, up) or by ALAT inhibition with Chloro-alanine inhibitor (Cl-ala, down). (C) Representative curves (left) of pH medium acidification (ECAR) of JoMa1.3 cells control (siCont.) or with Lkb1 knockdown

Figure 6 .

 6 Figure 6. Normalizing alanine and glutamate levels or mTOR activity rescues glial differentiation. (A) Schematic representation of glutamine synthetase (GS) inhibition by Methionine Sulfoximine (MSO). (B) Glutamate concentration upon dosedependent MSO treatment of glial JoMa1.3 cells compare to non-treated cells (Cont.) (n=4). (C) Alanine concentration of MSO-treated glial JoMa1.3 cells (n=2). (D) Cytoskeleton organization was assessed by phalloidin staining of control (Cont.) or MSO-treated cells. Nuclei were counterstained with Dapi (n=4). (E) Glial differentiation assessed by S100 immunostaining of JoMa1.3 cells upon MSO treatment (n=3). (F) Western blot (left) of glial cells after MSO treatment showing the relative level of phosphorylated S6RP with quantitative analysis (right) (n=4). (G) Impaired glial differentiation visualized with the S100 glial marker due to Lkb1 knockdown was rescued by AICAR treatment. Graph on the right side shows the percentage of S100positives cells (n=3). (H) AICAR addition also

Figure 7 .Figure S1 .

 7S1 Figure 7. AICAR treatment of Lkb1-deficient mice prevents the peripheral neuropathy and the enteric nervous system degeneration. (A, B, C) Injection of Lkb1-deficient animals by i.p. from P10 to P18 with the AMP analogue AICAR partly rescued weight loss (A), improved hind limb extension reflex (B) and prevented intestinal dilation (C). (D, E) AICAR treatment of cKO mice rescued mTOR inhibition compare to vehicle-treated cKO animals both in sciatic nerves (D) and enteric nervous system (E). Myenteric ganglia are surrounded by dotted lines. Graph in E represents the percentage of phospho-S6RP stained ganglia in the distal intestine (DI). (F) The integrity of the intestinal villi and the number of enteric neurons (red) and glial cells (green) are rescued in AICAR-treated mice. Representative pictures from two different animals are shown (upper and lower pictures). n=5 mice per condition.

Figure S2 .

 S2 Figure S2. Lkb1 inactivation in neural crest derived enteric cells triggers degeneration. Related to Figure1. (A) Lkb1 immunostaining on Swiss-roll sections of wild-type mice intestine and colon. Lkb1 expression in myenteric and submucosal ganglia colocalized with the neural marker Tuj1. Lkb1 immunostaining of cKO mice shows a strong reduction both on intestine and colon Swiss-roll sections (n=6 mice/group). (B) Confocal analysis of bGalactosidase-labeled cells (bGal) of an intestinal myenteric ganglion (left) or an isolated glial cell (right) showing co-staining with neural (HuC/D) or glial (GFAP) markers. (C) LKB1

Figure S3 .

 S3 Figure S3. Defective myelination of sciatic nerves inLkb1 cKO mice correlates with defects in maturation of Schwann cells. Related to Figure2.

Figure S4 .

 S4 Figure S4. Lkb1 is crucial for mitochondrial respiration during in vitro NCC-derived glial differentiation. Related to Figure 3. (A) Although inducible c-Myc expression is not significantly different in glial cells (G) compare to progenitors (P) (left qPCR results, n=3), c-Myc ER protein translocates into the nucleus (western blot, right panels) and its further activation maintains the progenitor status of NCC in vitro. Histone 4 (H4) and the chaperone protein HSP90 are used to validate the fractionation and are associated with nuclear and cytoplasmic fractions respectively. (B) Cultivating NCC in specific medium for glial differentiation triggers cell morphology changes as assessed by phase contrast. (C) The morphologic reorganization during glial differentiation was also visualized by staining of cytoskeleton components with phalloidin for actin filaments (left pictures) and tubulin antibody for the microtubules (middle pictures) (n=3). (D) Progenitor cells express more of the stem cell marker p75 NTR whereas cells committed to glial differentiation express more of the glial marker GFAP as assessed by RT-qPCR (n=5). (E) Experimental protocol performed in order to knockdown Lkb1 expression during glial differentiation and performed treatment as mentioned throughout the study. (F) Western blot to validate Lkb1 knockdown at the protein level. Phosphorylation of AMPK and S6RP (as an indicator of mTOR activity) are also shown compare to total protein level. Actin was used as a loading control. (G) The relative Lkb1 protein level observed by western blot has been quantified in progenitors (P) and glial (G) cells (n=4). (H) Fold changes have been quantified for phospho-AMPK to total AMPK protein (left graph, n=4) or phospho-S6RP to total S6RP protein (right graph) (n=4 in glial cells (G); n=3 in progenitors (P)). (I) Lkb1 knockdown during glial differentiation triggers the occurrence of actin stress fibers as visualized by phalloidin staining (n=6). (J) Quantification of mitochondrial respiration (OCR) assessed with the metabolic analyzer

Figure S5 .

 S5 Figure S5. Normalizing NEAA levels. Related to Figures 5 and 6. (A) Intracellular alanine concentration of control (Rescue Lkb1 WT) or Lkb1 knockout (Lkb1 -/-) MEF cells (n=3). (B) Intracellular alanine level of control (siCont.) or Lkb1 knockdown (siLkb1) progenitor JoMa1.3 cells expressed as the percentage relative to control cells (siCont) (n=5). (C) Percentage of intracellular alanine relative to control cells (siCont.) in glial JoMa1.3 cells upon Lkb1 knockdown and ALAT inhibition with Chloro-alanine (Cl-ala) (n=2). (D) Intracellular lactate level of control (siCont.) or Lkb1 knockdown (siLkb1) glial JoMa1.3 cells with Clala treatment expressed as the percentage relative to control cells (siCont). P: progenitors; G: glial cells (n=3). (E) Glycolytic activity of one representative experiment evaluated by pH extracellular acidification of glial JoMa1.3 cells either control (siCont.) or upon Lkb1 knockdown (siLkb1) (n=4). Right graph: Seahorse quantification of medium acidification (ECAR; n=4). (F) Expression of some metabolic genes assessed by RT-qPCR on progenitor and glial cells. GPT1, 2: Glutamic pyruvate transaminase (encoding ALAT1, 2); LDHA, B: Lactate dehydrogenase A, B; PKM1, 2: Pyruvate kinase M1, 2; GLUL: Glutamate ammonia Ligase (encoding GS). LDHB expression is enriched in glial cells whereas PKM2 and GLUL expression is enriched in progenitor cells (n=5). (G) Expression of the same metabolic genes as in (F) in glial JoMa1.3 cells either control (siCont) or silenced for Lkb1 (siLkb1). Only GPT2 expression is slightly enriched in siLkb1-treated cells (n=4). (H) Glutamine synthetase (GS) protein levels illustrated from a representative experiment (upper panels). Quantification of GS quantity normalized to actin is shown on the graph (n=4). (I) GS levels upon Lkb1 silencing (western blot, left; quantification graph, right, n=3). (J) Mitochondrial topology of MSO-treated JoMa1.3 cells stained with Mitotracker Red analyzed by confocal microscopy (n=3). Scale bar represent 10 mm. (K) Representative curves (top graph) of

Figure S6 .

 S6 Figure S6. Glial differentiation in Lkb1-silenced cells is rescued by normalizing NEAA levels or the mTOR pathway. Related to Figures 6. (A) Confocal imaging illustrating cytoskeleton reorganization assessed by phalloidin staining of control (siCont.) or Lkb1 knockdown (siLkb1) cells with or without AICAR treatment. Nuclei were counterstained with Dapi (n=4). Scale bars: 50 mm; 10 mm for the inset pictures. (B) Phalloidin staining of glial JoMa1.3 control or MSO-treated cells in the presence or absence of AICAR treatment (n=3). Scale bar: 20 mm. (C) Percentage of intracellular glutamate relative to control cells (siCont.) in glial JoMa1.3 cells treated with MSO in the presence (+) or absence (-) of AICAR (n=4). (D) Same as in (C) with MSO-treated glial JoMa1.3 cells (+) in the presence (+) or absence (-) of AICAR (n=2). (E) Percentage of intracellular alanine relative to control cells (siCont.) in glial JoMa1.3 cells upon Lkb1 knockdown and AICAR treatment (n=3). (F) Quantification of oxygen consumption (OCR) in control or siLkb1 JoMa1.3 cells with or without AICAR treatment (n=5). (G) Representative curves showing medium acidification (ECAR) in control or siLkb1 JoMa1.3 cells with or without AICAR treatment (n=4). (H) Torin1 is a potent mTOR inhibitor in JoMa1.3 glial cells as illustrated by a representative western blot (n=2).

  and rehydrated, rinsed in PBS and boiled for 20 minutes at 95°C in a decloacking chamber (Biocare Medical) in 10 mM Sodium citrate antigen retrieval buffer (pH 6). Sections were then treated with 1% H2O2 for 20 minutes. After 1 hour incubation in a blocking buffer (PBS-3% BSA-10% Goat preimmune serum for polyclonal antibodies or ABC MOM kit from Vector laboratories for monoclonal antibodies according to the supplier's protocol), sections were incubated overnight at 4°C with the primary antibody (see Table3for dilutions and antibodies references). Secondary antibodies used for immunohistochemistry were anti Mouse-For phospho-AMPK, tissues were blocked with horse serum and ImmPRESS anti-Rabbit-HRP conjugated secondary antibodies were used to amplify the staining, according to the supplier's protocol (Clinisciences). DAB peroxidase substrate kit was used for detection of the antibodies (Vectors laboratories). Sections were then counterstained with Harris Hematoxylin (Sigma-Aldrich), dehydrated and mounted in Merckoglas (Merck).

  anti-Mouse Alexa-488, Donkey anti-Mouse Cya3, Donkey anti-Rabbit Alexa-488, Donkey anti-Rabbit Cya3, Donkey anti-Rabbit Cya2 and Goat anti-Chicken A488). For tissue sections, nuclei were stained with Hoechst 33342 (10 mg/ml, Invitrogen). For JoMa1.3 cells, nuclei were stained with Dapi. Sections and cells were mounted in mowiol. Actin cytoskeleton was stained with phalloidin conjugated to TRITC (1/400, Sigma). TUNEL staining was performed according to the supplier's protocol (Roche). Analyses and acquisitions were performed using a Zeiss AxioImager Z1 microscope equipped with an AxioCam MRm camera, a Zeiss AxioImager M2 microscope, equipped with a Hamamatsu Orca R2 camera or for confocal microscopy a Zeiss AxioObserver Z1 multiparameter microscope LSM710 NLO -LIVE7 -Confocor3.

4°C

  Whole mount detection of b-galactosidase activity was performed on P21 stages. Dissected guts were fixed on ice for 2 hours in phosphate buffer saline containing 0.2% glutaraldehyde. Guts were stained overnight at 37°C in 5-Bromo-4-chloro-3-indolyl-b-Dgalactopyranoside (X-Gal) solution, containing 1 mg/ml X-Gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% nonidet P-40 (NP-40) in phosphate buffer saline. After staining, guts were washed in phosphate buffer saline containing 2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% NP-40, postfixed in 4% paraformaldehyde and examined under a stereomicroscope. DRG Salmon gal staining and Immunofluorescence Spinal cords from L1 to L6 lumbar vertebrae level were dissected from freshly collected E18.5 male embryos and fixed in 1% PFA/0.2% Glutaraldehyde in Phosphate buffer pH7.4 for 10 minutes. Tissues were rinsed 3 times with PBS and then prestained overnight at 37°C in a pre-stain solution without substrate (200mM ferricyanide potassium, 200mM ferrocyanide potassium, 4mM MgCl2, 0.04% NP40, in PBS) to reduce endogenous ß-galactosidase activity. Staining was performed for 30 minutes at 37°C, in a stain solution with 2mM MgCL2, 0.04% NP40, Salmon Gal 1mg/ml, NBT (4-nitro blue tetrazolium chloride) 0.33mg/ml in PBS. Spinal cords were then rinsed and postfixed in PFA 4% in PBS, transferred to 20% sucrose cryoprotective solution overnight and OCT-mounted for cryosections and treated for immunofluorescence with standard protocols (See Table

  coverslips coated with Fibronectine (10μg/μL, 1h at 37°C) and Poly-L-Lysine (10μg/μL, 1h at 37°C) and re-transfected as previously described. After 96h of the second cycle of transfection, cells were harvested and stained with 200 nM MitoTracker Red CMX-ROS (Invitrogen) or with MitoTracker Green (Invitrogen) for 30 min at 37°C in DMEM medium. For mitochondrial mass quantification, cells were directly analyzed by FACS (Accuri-C6, BD-Biosciences). To assess the mitochondrial network morphology, JoMa1.3 cells were rinsed 3 times in PBS. Cells were fixed in 4% paraformaldehyde and analyzed by biphoton confocal microscopy (LSM510, Zeiss).

Figure 1 .

 1 Figure 1. Cre drivers used for Lkb1 conditional depletion in NCC or Schwann cell lineage

Furthermore

  , some ribosomopathies (pathologies due to defective ribosome biogenesis, also described in Introduction, Chapter II.3) harbors similar NCC defects as neurocristopathies. Several of these pathologies involve aberrant p53 activation and the pathological phenotype of several mouse models of neurocristopathies and ribosomopathies can be rescued by p53 inactivation (Introduction, Chapter III.B.4). Therefore, in the continuity of these results I analyzed the levels and activity of p53 in our NCC cell model and studied the Lkb1/p53 crosstalk in NCC development. 2. LKB1 regulates NCC differentiation by limiting p53 signaling and controlling lysosomal activity. Article Nb3: Radu et al. in preparation 2.1 Scientific context and results summary p53 is a transcription factor known as the guardian of the genome. Unrestrained signaling of p53 has been described in both human neural crest disorders and mouse models of neurocristopathies and ribosomopathies (see Introduction Chapter II.3). The transcription factor p53 is activated partly by phosphorylation on serine 15 in response to cellular stress such as accumulation of reactive oxygen species (ROS) and DNA damage. Activated p53 then modulates cellular adaptation to stress by acting on cell cycle, autophagy and metabolism and,

  By using the NCC line and in vitro glial differentiation as in the previous study, I have shown that Lkb1 loss triggers increased p53 phosphorylation on serine 15 during NCC-derived glial differentiation. This hyperphosphorylation in Lkb1-silenced NCC is promoted by oxidative DNA damage induced by increased ROS production. Treatment of NCC with the antioxidant N-acetyl cysteine, prevented ROS accumulation, DNA damage and p53 phosphorylation. p53 hyperphosphorylation was validated in vivo on sciatic nerve sections of Lkb1 knockout animals from the "late" inactivation model (Tyr::Cre driver). Moreover, during this study I underlined a link between Lkb1 signaling and lysosomal homeostasis. mTOR and LKB1/AMPK signaling are activated at the lysosome surface by the same protein complexes and their activation is highly dependent on amino acid levels (Introduction, Chapter IV.2). Interestingly, I demonstrated that Lkb1 loss during glial differentiation leads to aberrant lysosomal topology and massive accumulation of autofluorescent granules with characteristic spectral emission. The granules, known as lipofuscin granules correspond to defective lysosomes which are filled with unsorted oxidized proteins, lipids and metal ions and they accumulate with aging in cells of various tissues. Increase of oxidative damage of DNA and accumulation of oxidized proteins and lipids represent a typical feature of premature aging resulting from imbalanced prooxidant and antioxidant activities. This recent study thus provides new insights into NCC development and expand our understanding of the role of LKB1 on oxidative stress regulation, partly via its crosstalk with p53 signaling and lysosomal homeostasis in neural crest disorders. Introduction The neural crest is composed of highly invasive multipotent cells called neural crest cells (NCC), that delaminate from the dorsal part of the neural tube and migrate along stereotypical paths. Once they reach their target organs, they give rise to a broad range of derivatives such as osteocytes and chondrocytes of the craniofacial skeleton, pigment cells, enteric neurons and glial cells and sensory neurons and Schwann cells of the peripheral nerves (Le Douarin

that

  Lkb1 limits oxidative DNA damage during glial differentiation. Mechanistic analyses show that oxidative DNA damages upon Lkb1 knockdown trigger p53 hyper-phosphorylation both in vitro and in vivo. Concomitant to aberrant p53 signaling, Lkb1 loss triggers increased autophagy and defective lysosomal homeostasis leading to accumulation of oxidized lipids and proteins as well as metals. Strikingly, AICAR treatments, which rescue glial differentiation upon Lkb1 loss, prevented p53 hyper-phosphorylation. These results provide evidence that Lkb1 limits oxidative DNA damage of NCC progenitors and derivatives, therefore preventing activation of the p53 signaling.

  Lkb1 signaling protects cells committed to glial lineage from oxidative DNA damage. Oxidative DNA damages upon Lkb1 loss cause p53 hyper-phosphorylation Next, we explored if the increased oxidative stress observed upon Lkb1 loss could trigger p53 signaling. We therefore explored p53 expression, stability and post-translational modifications upon Lkb1 knockdown either in progenitors or glial derivatives NCC. Expression of p53 gene was not modulated upon Lkb1 knockdown as quantified by RT-qPCR onJoMa1.3 cells compared to control cells (Fig.2A).Immunoblot analyses over several independent experiments did not show any reproducible p53 stabilization (Fig.2B). However, they highlighted a Serine 15 upon Lkb1 knockdown either in progenitors or glial cells (Fig.2C). Staining of phosphorylated p53 at serine 15 on sciatic nerve sections upon conditional ablation of Lkb1 in neural crest cells using the Tyr::Cre;Lkb1 F/F mice(Radu et al., submitted) confirmed in vivo the increased of p53 phosphorylation upon Lkb1 loss (Fig.2D). To assess if p53 induction was due to oxidative DNA damage during glial differentiation, we visualized phosphorylated p53 at serine 15 in JoMa1.3 cells with or without NAC treatment. We observed that NAC effectively suppressed p53 hyperphosphorylation (Fig.2E).

JoMa1. 3

 3 cells by assessing the expression level of classical p53 target genes corresponding to several cell functions such as cell cycle, DNA repair, stem cell, maintenance or autophagy. None of them were upregulated upon Lkb1 loss during glial commitment (Fig. 2F), suggesting that increase of p53 phosphorylated on serine 15 was not associated with upregulation of the transcriptional activity of p53.

  cells compare to cells with Lkb1 knockdown exhibit very few of these structures (Fig 3D, pictures) and their spectral properties are distinct to those in cells with Lkb1 knockdown (Fig. 3D, graph). Strikingly, electron microscopy analyses of JoMa1.3 glial cells highlighted strongly aberrant late endosomal/lysosomal compartment upon Lkb1knockdown whereas these structures where seen in control cells to a much lower extent and never as large as in the absence of Lkb1 (Fig.3E)electron microscopy strongly suggest that these autofluorescent inclusions correspond to lipofuscin granules known to accumulate in cells over time and use as markers of premature aging.

  Neural crest cells are very sensitive to oxidative stress accumulated upon genetic mutations disturbing endogenous signaling or upon exposure to environmental factors such as alcohol or high glucose levels. Along this line, our work reveals that LKB1 controls metabolic pathways essential to limit the production of reactive oxygen species (ROS), and consequently DNA damages and p53 signaling, during NCC differentiation. Our study also uncovers a major regulation exerted by the LKB1/AMPK pathway on lysosomal homeostasis. Indeed, we observed massive defective lysosomal activity upon Lkb1 loss either in vivo in myelinating Schwann cells of sciatic nerves or in vitro either in progenitors or in cells committed to the glial lineage. Under conditions of metabolic stress, LKB1/AMPK are activated by protein complexes (a vATPase and the Ragulator complex) localized at the lysosomal surface. These proteins are the same that activates mTOR signaling when the cells are in high nutrients conditions. Therefore, lysosomal membrane surface assembles and coordinates the activities of the antagonistic kinase hubs mTOR complex 1 and AMPK. Our previous work has shown the important link between

  well as rescue lysosomal function. One hypothesis to explain this striking result is that AICAR refurnishes the ATP pool of the cell. Indeed, AICAR is an IMP analog and therefore an AMP and then ATP precursor through several enzymatic reactions belonging to the late steps of the pentose phosphate pathway. Therefore, AICAR treatment of cells could refurnish the ATP pool, which in turn could contribute to various cellular processes required to adjust cell homeostasis upon LKB1 loss. One example among several concerns kinesine-1 activity since to direct lysosome motility on microtubules, this motor protein requires ATP. Altogether, emerging knowledge highlights that lysosomes remodeling is essential for cell adaptation in response to various stresses. Exploring the molecular mechanisms linking amino acid levels, LKB1/mTOR pathways and lysosomes motility on cell cytoskeleton is therefore of critical interest.

  were from Jackson ImmunoResearch (Donkey anti-Mouse Alexa-488, Donkey anti-Mouse Cya3, Donkey anti-Rabbit Alexa-488, Donkey anti-Rabbit Cya3, Donkey anti-Rabbit Cya2 and Goat anti-Chicken A488). For tissue sections, nuclei were stained with Hoechst 33342 (10 g/ml, Invitrogen). For JoMa1.3 cells, nuclei were stained with Dapi. Sections and cells were mounted in mowiol. Analyses and acquisitions were performed using a Zeiss AxioImager Z1 microscope equipped with an AxioCam MRm camera, a Zeiss AxioImager M2 microscope, equipped with a Hamamatsu Orca R2 camera or for confocal microscopy a Zeiss AxioObserver Z1 multiparameter microscope LSM710 NLO -LIVE7 -Confocor3. Confocal spectral microscopy Spectral imaging was performed using a Zeiss LSM710 spectral confocal (Carl Zeiss, Jena, Germany) with 2-photon excitation at 840 nm on stain-free samples. Lambda stack acquisition was collected from 421 to 721 nm in a spectral resolution of 9?7 nm (QUASAR-PMT with 32 channels). Emission spectra were measured in Lkb1knockdown cells for 39 inclusions from 4 different fields and for 6 inclusions in control cells from 5 different fields in ZEN 2010 software (Carl Zeiss, Jena, Germany). Fluorescence intensity was measured. Fluorescence spectra represent mean fluorescence normalized to the value of the emission bandwidth with the highest intensity. Cell sorting and ROS quantification with DCF-DA probe Control or Lkb1 knockdown JoMa1.3 cells were differentiated into glial cells and treated daily with various concentrations of N-acetyl cysteine (NAC 10, 12 and 15 µM). One hour prior to the end of the treatment, 50 µM of 2',7'-dichlorofluorescin diacetate (DCFDA) was added to the medium and incubated for 30 minutes at 37°C. The cells were then washed with PBS and kept in culture media (with 10% SVF and without red phenol) for 30 minutes at 37°C. Cells were harvested, washed, and analyzed by flow cytometry with the red laser channel (FL-3) using a C6 BD-Accuri analyzer.

  three independent experiments and for one experiment, each condition was performed in triplicate. In vivo analyses were conducted with at least three animals per condition. The exact number of experiments is indicated in the figures or in the legends. Values are given as mean with standard deviation. Normality of the data was checked with the F-Test. The unpaired Student's t-test was used to generate p-values for selected pairwise comparisons and p < 0.05 was considered significant.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .(

 2 Figure 2. Oxidative DNA damage upon Lkb1 loss triggers p53 hyper-phosphorylation. (A) p53 mRNA level are not change upon Lkb1 knockdown during glial differentiation of JoMa1.3 cells. (B) Representative imagines of western blot showing accumulation of phospho-p53 Ser15 in progenitors (left) and glial (right) JoMa1.3 cells. Quantification of 3 independent experiments is shown (graphs in right). (C) Immunofluorescence staining showing the increase number of positive cells for phospho-p53 Ser15 . Quantifications are shown below (n=3). (D) Immunofluorescence staining for phospho-p53 Ser15 on sciatic nerve sections of wildtype animals (WT) or upon inactivation of Lkb1 (cKO). (E) Representative images of immunofluorescence staining for phospho-p53 Serine15 upon NAC treatment of glial cells. Quantification is shown below. (F). RT-qPCR showing expression of p53 gene in glial JoMa1.3 cells upon Lkb1 silencing (siLkb1) compare to control cells (siCont.) (n=4).

Figure 3 .

 3 Figure 3. Lkb1 sustains lysosomal activity. (A) Western blot analysis showing accumulation of LC3-II staining upon Lkb1 knockdown associated

Figure 4 :

 4 Figure 4: AICAR treatment limits p53 hyperphosphorylation and defective lysosomes upon Lkb1 loss. (A) S100 immunofluorescence of glial JoMa1.3 cells upon AICAR treatment showing the disappearance of the inclusions. (B) Representative western blot images illustrating the ability of AICAR to prevent phospho-p53 Ser15 accumulation upon Lkb1 knockdown. (C) Immunofluorescence staining for phospho-p53 Ser15 in control or Lkb1-silenced glial JoMa1.3 cells illustrating the decrease of phospho-p53 positive cells upon AICAR treatment.

Furthermore I uncoveredFigure 5 .

 5 Figure 5. Lkb1/p53 crosstalk to control cell fate

  Secondly, in collaboration with R. Almeida lab (IPATIMUP, Porto, Portugal), I studied the energy metabolic regulations exerted by MEX-3A, a putative downstream effector in LKB1 signaling, in Caco2 (Colorectal adenocarcinoma) cells which are intestinal epithelial cells characterized by polarity and tight junctions formation at postconfluency. 1. Lkb1-metabolic regulations in Sertoli cells are essential for testis homeostasis and germ cells maturation. Article in preparation, Torch S.*, Radu AG.*, Lucas A., Fauvelle F., Hainaut P.,Billaud M. and Thibert C, * equally contributing authors 1.1 Scientific context and results summary LKB1 role in Sertoli cells has been already described: Lkb1 inactivation in Sertoli cells was performed by expressing Cre recombinase under the promoter of the receptor of Anti

  better characterize the capacity of Lkb1 to regulate Sertoli cell metabolism and to analyze the underling molecular mechanism, I used a Sertoli cell line (TM4 cells). First, I confirmed that Lkb1 loss promotes an increase of mTOR pathway and a decrease of AMPK phosphorylation. Second, I demonstrated that glycolysis was not affected in the absence of Lkb1 whereas oxidative phosphorylation (OXPHOS) was clearly augmented, thus leading to an increase of energy production. Finally, I have shown that Lkb1 controls phagocytosis, one of the most characterized Sertoli cell function. Altogether, these findings highlight a novel function of LKB1 as a crucial metabolic regulator in Sertoli cells, necessary to control their functions and whole testis development. Lkb1 inactivation in Sertoli cells influences germ cell localization and maturation. As a reminder, Sertoli cells are located in the seminiferous tubules from their basal part to their lumen, being in contact with germ cells (Figure 1A, left panel). Germ cells undergo progressive maturation from the basal part of the tubule, where they are called spermatogonia, to the lumen, were they are differentiated into spermatozoa. Mouse spermatogenesis starts with mitotic proliferation of type A and type B spermatogonia. Type B spermatogonia are forming spermatocytes through symmetric divisions. This step represent the beginning of a meiotic phase. Spermatocytes I are diploid cells which undergo meiosis and generate two haploid

Figure. 1

 1 Figure.1 Sertoli cells-LKB1 regulates germ cells proliferation, localization and maturation.A. Left panel: Schematic representation of seminiferous tubules organization. Right panel: Schematic representation of different types of spermatogonia. Spermatogonia Ad (dark) are germ stem cells, Spermatogonia Ap (pale) and B undergo a symmetric division. B. Quantification of seminiferous tubules area from 4 WT and 4 LKB1 cKO at P21. C. Quantification of germ cells on transversal sections of testes of 3 WT and 3 LKB1 cKO at P21. D. Transverse section of WT and LKB1 cKO testis at P21 subjected to immunohistochimisty (IHC) with VASA antibody. VASA is a marker of germ cells from spermatogonia to round spermatids. Scale bars: 100 µm the top panels and 50µm botom panels. Quantification of spermatogonia (right panel) were performed on only 2 WT and 2 LKB1 cKO mice and 8 seminiferous tubules/animal were used for counting. E and F. SCP3 immunofluorescence on transevrse section of WT and LKB1 cKO testes at E. P21 and F P90. SCP3 is a marker of spermatocytes I and II. This experiments were done on 3 WT and 3 LKB1 cKO animals.

Figure 2 :

 2 Figure 2: Lkb1 is a novel metabolic regulator of Sertoli cells and controls their phagocytic function. A. Western blot to validate Lkb1 knockdown at the protein level. Phosphorylation of AMPK and S6RP (as an indicator of mTOR activity) are also shown compare to total protein level. Actin was used as a loading control. The right panel is showing the percentage of phospho-AMPK to total AMPK protein (experiments realized 2 times (n=2)) or phospho-S6RP to total S6RP protein (n=2) relative to siControl. B. Representative curves showing changes in medium acidification (ECAR, top) obtained by Seahorse analyzer and quantification of ECAR (bottom) (n=3).C. Representative curves showing change in oxygen consumption (OCR, top) analyzed by Seahorse and quantification of % OCR relative to siControl (n=3).D. siCont and siLKB1 TM4 cells were incubated 2h with fluospheres, then harvested, washed and analyzed by flow cytometry. Phagocytotic cells can be detected by florescence spectroscopy at 645nm. M4 represents the cells considered non phagocytotic, M1 represents the cells which ingested n fluospheres, M2 ingested n+1 fluospheres and M3 ingested n+2 fluospheres. Quantification of the % of phagocytotic cells is presented in the right panel.

2 .

 2 Characterization of MEX-3A-associated metabolic profile Collaboration with R. Almeida and B. Pereira (Institute of Molecular Pathology and Immunology of the University of Porto, IPATIMUP, Portugal) 2.1 Scientific context and results summary

Figure1:

  Figure1: MEX-3A a novel metabolic regulator involved in cell fate control. Caco2 cells overexpressing MEX-3A or Caco2 control cells (MOCK) were cultured for 3 days (pre-confluence) or 18 days (post-confluence) and analyzed by Seahorse. A. (pre-confluence) & D.(post-confluence).Representative curves showing change in oxygen consumption (OCR) analyzed by Seahorse (top) and quantification of % OCR relative to the control (MOCK)(Bottom). B (pre-confluence) & E (postconfluence). Representative curves showing changes in medium acidification (ECAR) obtained by Seahorse analyzer and quantification of ECAR (bottom). C. Comparison between the MOCK cells in pre and post-confluence conditions regarding OCR (top) and ECAR (bottom) quantifications. F.Quantification of MEX-3A regulation of OCR relative to MOCK in pre and post confluence conditions. All the experiments were performed three times, with three replicates in each experiment.

Figure 1 .

 1 Figure 1. Schematic representation of my PhD projects

  aminotransferase (ALAT) activity and alanine-pyruvate cycling. Further it will be interesting to analyze ApoD in patients with neurocristopathies.Such data would expand our understanding of the role of LKB1 on oxidative stress regulation, partly via its crosstalk with p53 signaling, lysosomal homeostasis and premature aging in neural crest disorders. *** Overall, our projects will provide relevant information on the pathological underpinning of neural crest diseases as well as on mechanisms linking metabolism and cell fate such as differentiation but also premature aging. However, it remains difficult to transpose these knowledges in patients with neurocristopathies. Therefore, it will be interesting to analyze the role of Lkb1 in other human pathologies related to NCC-derivatives defects like mesenchymal stem cellsderived-Schwann cell precursors defects.

Figure 1 .

 1 Figure 1. Serine glycine one carbon network promotes DNA methylation in absence of Lkb1.(Kottakis et al., 2017)

  Recently C. Perret team (Paris, Institut Cochin) has shown that hepatocellular Lkb1 negatively regulates Alanine Glycoxylate AminoTransferase (AGXT) expression at the transcriptional level (Table1) (data not published yet). AGXT is highly expressed in liver and has a double function: it detoxifies glycoxylate and is involved in gluconeogenesis from serine (Ichiyama, 2011). C. Perret team demonstrated that Lkb1 regulates hepatic gluconeogenesis by downregulating AGXT aminotransferase expression and therefore limiting the availability of amino acids such as alanine, glutamine and serine as gluconeogenic precursors (Charawi S et al., data not published yet). -In addition to these studies, I have shown that Lkb1 regulates ALAT although the underlying mechanisms are still unknown (Table1). I observed that neither the expression nor the protein stability of ALAT and other metabolic enzymes were drastically impaired upon Lkb1 loss. Measurement of ALAT activity in control cells or Lkb1 knockdown cells are necessary to further confirm our first results.

Figure 2 :

 2 Figure 2: Serine and alanine regulate the generation of bioactive sphingolipids and toxic deoxy-sphingolipids. A. Formation of bioactive sphinganine and toxic deoxy-sphinganine (modified from Kowlurum A, 2014). B. Sphingolipid de novo synthesis is catalyzed by the SPT enzyme through the condensation of palmitoyl-CoA and L-serine to form sphinganine in the endoplasmic reticulum (ER).In HSAN1, mutant SPT prefer alanine or glycine instead of serine and form deoxy-or deoxymethylsphingolipids, which can no longer be metabolized to complex sphingolipids or degraded by the canonical degradation pathway, thus leading to toxic accumulation(Klein, 2015).

  using both in vivo and in vitro approaches as well as the crosstalk between Lkb1 and p53 signaling pathways. So far, we focused on exploring Lkb1 contribution to developmental steps and how defects in Lkb1 signaling could be part of developmental diseases. Transposition of these findings observed during development to cancer cells is crucial especially regarding recent data from the literature highlighting (i) the important role of lactate and glutamine in the metabolism of tumor cells and (ii) the role of transaminases in cancer cell proliferation (see Introduction, Chapter I.3.3).

Finally

  , M. Billaud lab identified a potential antitumoral drug, named L6, by a screen on LKB1 null cells compared to LKB1 wildtype cells. This drug belongs to the family of biguanides, such as Metformin, widely used in type 2 diabetes treatment and known to control energy metabolism of the cells. Using Metformin for several years to limit diabetes, an antitumoral role was suspected and more than hundred clinical trial are currently ongoing. M. Billaud lab observed that the biguanide L6 acts with much lower doses than Metformin and could have a mode of action on energy metabolism different than the one of Metformin. Therefore, the lab will analyze if L6 treatment of lung cancer cells target deregulated metabolic pathways due to LKB1 loss such as amino acid levels and transaminases activity. Exploring antitumoral properties of L6 on lung cancer progression in vivo will be explored using chick models of cancer cells grafts, either on the chorioallantoidian membrane (InOvotion startup, Grenoble) or in the proper tumoral microenvironment (OncoFactory startup, Lyon) as well as in mice (Optimal, IAB, Grenoble). *** Altogether, these projects open new perspectives of translational research based on the use of aminotransferase inhibitors, AICAR or compounds such as biguanides acting on metabolic pathways that might be effective in certain forms of neuropathies, metabolic syndromes and in LKB1-deficient tumor cells. In this context, the research conducted by the lab will bring new insights for future therapeutic applications.

Table 1 : Aminotransferases and their activities.

 1

Table 3 : Description of some human neurocristopathies

 3

Table 8 (

 8

Introduction, Chapter III.B.4

) to be mated to p53 null mice to study if p53 loss rescues the phenotype.

Table 4 : Mouse models reminiscent to human neurocristopathies

 4

  pathway through PI3K-Akt and mTOR signaling, the receptor tyrosine kinase pathway through Ras/MEK/ERK and Myc, etc… Among them, the LKB1/AMPK pathway is able to sense ATP levels and in a context of low ATP versus high AMP, it consequently regulates energy metabolism. Several data from the literature suggest that the master kinase LKB1 could be a

regulator of neural crest cells (NCC) ontogeny. First, LKB1 is essential in early embryonic development since Lkb1 knockout mice are embryonic lethal. Lkb1 null mice exhibit, among other defects, an absence of the first branchial arch which is mainly composed of NCC and therefore suggest an agenesis of NCC. Second, germline mutations of LKB1 in human are responsible for the Peutz-Jeghers syndrome, an autosomal dominant disease characterized, among other symptoms, by defects in melanocytes which are NCC derivatives. Third, LKB1 is

Table 7 : LKB1 localization in adult and embryonic normal tissues

 7

(Modified from Sanchez-Cespedes, 2007)

Table 8 : Rescue of the neurocristo/ribosomopathy phenotype by p53 inactivation.

 8

  Bosc cells were treated with TSA 1µM for 4h and Lkb1 was immunoprecipitated with anti-LKB1 antibody.Mouse IgG were used as control immunoprecipitation. Acetylated Lkb1 K48 was analyzed by western blot with anti-acetylated LKB1 K48 antibody. B. Bosc cells were transfected with siRNA targeting GCN5 mRNA (siGCN5) for 48h. Non-targeting siRNA were used as control. LKB1 was immunoprecipitated as in A. Lkb1 interaction with GCN5 was analyzed by western blot with anti-GCN5 antibody. C. Bosc cells were co-

	transfected with plasmids expressing WT-Flag-GFP-LKB1, K48-Flag-LKB1 or K48Q-Flag-LKB1 proteins and
	with plasmids expressing WT-HA-GCN5 or control plasmid (pSG5-Flag). LKB1 immunoprecipitation and
	interaction with GCN5 were done as in B. D. Bosc cells were transfected with siRNA as in B and
	immunofluorescence was used to detect Lkb1 (green) and GCN5 (red). Nuclei were labeled with Dapi (blue).
	E. Hela cells were co-transfected with plasmids expressing WT-Flag-GFP-LKB1 protein and plasmids
	expressing GCN5 wildtpe (WT) or mutant in its catalytic domain Y621A/F622A (GCN5mut) or control (peBB-
	HA). Acetylated LKB1 K48 (in magenta) was detected by immunofluorescence with anti-acetylated LKB1 K48
	antibody. Lkb1-GFP (in green) was visualized with an epifluorescent microscope. Nuclei were labeled with
	Dapi (blue). F. Cellular fractionation was performed on Bosc cells to separate cytoplasmic and nuclear
	fractions. LKB1 was immunoprecipitated in both fractions with anti-LKB1 antibody. Acetylated-LKB1-K48 was
	analyzed by western blot (left panel). Cellular fractionation was also verified (right panel) by western blot using
	anti-Hsp90 antibody for the detection of cytoplasmic fractions and anti-H4 antibody for nuclear fractions .LKB1
	was detected with anti-LKB1 antibody from Santa Cruz (Ley37DG6) which recognize the C terminal part of

A.

LKB1 and therefore it detects also the LKB1 ΔN isoform. G. The cellular fractionation and LKB1 immunoprecipitation were done as decribed above. LKB1 interaction with GCN5 was analyzed in both fractions by western blot with anti-GCN5 antibody. H. Hela cells were transfected with plasmids expressing WT Flag-GFP-LKB1 proteins and/or WT-Flag GCN5 or MUT-Flag-GCN5 proteins for 48h. AMPK phosphorylation was analyzed by western blot. I. The migration of Hela cells was analyzed by wound healing assay using ibidi.

Cells were transfected with plasmids expressing WT-Flag-LKB1 or K48R-Flag-K48R or NLS-Flag-LKB1 for 48h. Representative pictures of the size of the wound 48h and 72h after wounding are shown. The percentage of closure for each wound was evaluated from microscopy images at 48h after wounding relative to Control from 2 independent experiments.

Table 1 -

 1 Primers used for genotyping and qPCR

Table 2 -

 2 Antibodies used throughout the study.

Table 1 -

 1 Primers used for genotyping and qPCR.

Table 2 -

 2 Antibodies used throughout the study.

  p53 rescues in vivo and in vitro impaired NCC differentiation and maintenance. For in vitro analyzes, the lab will perform a double knockdown of both Lkb1 and p53 in NCC and evaluate if it rescues glial differentiation (Figure

	Technic	Primary antibody	Producing specie	Dilution	References
	IF	LKB1	Rabbit, polyclonal	1/100	Sigma
		Tuj1	Mouse, monoclonal	1/1000	Covance
		S100	Rabbit, Polyclonal	1/1000	Dako
		phospho-p53 (Ser 15)	Rabbit, monoclonal	1/1000	Cell Signaling
		LC3	Mouse, monoclonal	1/100	Novus Biological
		gH2AX	Rabbit, monoclonal	1/100	Abcam
	WB	LKB1 Ley37DG6	Mouse, monoclonal	1/1000	Santa Cruz
		HSP90 (AC88)	Mouse, monoclonal	1/2000	Stressgen
		Actin	Mouse, monoclonal	1/1000	Sigma
		phospho-p53 (Ser 15)	Rabbit, monoclonal	1/1000	Cell Signaling
		Total p53 CM5	Rabbit, polyclonal	1/1000	Leica
		LC3	Mouse, monoclonal	1/1000	Novus Biological
		P62	Mouse, monoclonal	1/1000	Abnova

and

  1.3 ConclusionsSertoli cells is an interesting model of polarized cells sharing functional metabolic similarities with NCC-derived glial cells. Indeed, these cells sustain proliferation, survival and maturation of their neighboring cells, either neurons in case of glial cells or germ, myoid and Leydig cells in case of Sertoli cells. Glial and Sertoli cells both regulate adjacent cell fate through paracrine factors such as amino acids (alanine) or other metabolites (lactate, pyruvate). Therefore, the lab was interested in exploring metabolic regulations exerted by LKB1 in Sertoli cells and I participated to this analysis during my PhD.I emphasized that Lkb1 regulates global tissue architecture, germ cell maturation, neighboring cell proliferation and Sertoli cell metabolism. All these data suggest that Lkb1 is a new regulator of the metabolic dialog between Sertoli cells and germ cells, responsible for spermatozoa formation. However, it is not clear if Lkb1 expressed in Sertoli cells regulates germ cell differentiation directly or if it is a consequence of blood-testis barrier loss. I have shown that in absence of Lkb1 there is the same amount of spermatogonia type B and spermatocytes compared to control. Instead, nothing is known about Lkb1 role in spermatogonia type A, the germ stem cells. Therefore, further studies with the germ stem cell marker, PLZF

  GCN5 and could affect early embryonic development in this model. Hence, we could study this hypothesis in vitro by inactivating GCN5 in NCC and analyze LKB1 acetylation on lysine 48, p53 accumulation, NCC differentiation and migration. If the differentiation and/or migration are

impaired and p53 is accumulated and activated, we could double knockdown GCN5 and p53 to see if it rescues the phenotype. Further, we could analyze if this role of GCN5 as a p53 regulator is linked to its capacity to acetylate LKB1 on lysine 48. If in vitro results are promising, in vivo experiments could be performed on chick embryos in collaboration with Sylvie Dufour (Institut H. Mondor pour la Recherche Biomédicale, Créteil, France).

  . As calcium regulates NCC migration (see Introduction, Chapter IV.3.1), defects of lysosomal Ca 2+ uptake may also lead to neurocristopathies. In 2003, Hanson et al. showed that several lysosomal storage disorders are associated with dermal melanocytosis, a pathology due to perturbed migration of melanocytes from neural crest to the developing epidermis

Acknowledgments

We are grateful to E. Fontaine for helpful comments about the results. We thank S. Michallet, B. Sefrin and P. Vernet from the animal facility of the Institute for Advanced Biosciences and F. Blanquet and C. Colomb from the animal facility PHTA of Grenoble. We are grateful for D. Bouvard for discussions and for giving us the R26R mice and the help of G. Chevallier with mouse breeding and genotyping. We thank V. Blanc-Marquis for her technical help. S. Michallet was very helpful with excellent technical assistance. C. Caron helped us with human sample studies. J. Delaroche and A. Bertrand from the electron microscopy facility of the Neuroscience Institute of Grenoble performed semi-thin sections, staining and electronic microscopy experiments and analyses. We thank N. Gadot and the ANIPATH facility, University Lyon1 Laennec, for tissue sections.

This work was founded by "l'Institut National du Cancer" (Programme recherche translationnelle en cancérologie) and "la Ligue régionale contre le cancer" (comité de l'Isère). Acknowledgments We thank P. Vernet from the animal facility of the Institute for Advanced Biosciences and A. Piras and C. Colomb from the animal facility PHTA of Grenoble. A. Bertrand, from the electron microscopy facility of the Neuroscience Institute of Grenoble performed semi-thin sections, staining and electronic microscopy experiments and analyses supervised by KPG. We thank N. Gadot and the ANIPATH facility, University Lyon1 Laennec, for tissue sections. We thank the photonic microscopy-cell imaging facility "MicroCell" from the Institute for Advanced Biosciences. The confocal microscopy tool was partly funded by the Asscoiation for Research on Cancer,

Supplementary material S2 Fig. Lkb1 silencing impacts CNCC survival, and morphogen expression. (A) When compared to control embryos electroporated with scramble dsRNA, Lysotracker staining of 7ss embryos electroporated with dsLkb1 reveals expanded cell death. (B) HNK1 immunofluorescence with lysotracker revealed that NCC display increased cell death in the absence of Lkb1. (C) Quantification of the average percentage of dying NCC (HNK1 and lysotracker positive cells) in control or dsLkb1 conditions. Increased cell death in NCC in the absence of Lkb1 was statistically significant as evaluated by a Student t-test. (D) Fgf8, which is strongly expressed in ANR, is down-regulated by Lkb1-silencing in CNCC. (E) Quantification of gene expression by qRT-PCR showed that Lkb1 silencing significantly affected the mRNA levels of Fgf8 (Fgf8∆=60%), Wnt1 (Wnt1∆=40%), and Wnt8b (Wnt8b∆=90%). By contrast, Shh expression was up-regulated (1.5 fold). The number of embryos used for qRT-PCR analyses was n=4 in control condition, n=3 in dsLkb1 and n=4 in dsLkb1+AMPK-CA. Statistical analyses were performed using Mann-Whitney U test. AMPKα1 (A-C) and AMPKα2 (D-F) transcripts at 5ss (A, D), 10ss (B, E), 12-13ss (C, F). At neurula stages, at which electroporation experiments are performed, AMPKα1 and AMPKα2 are expressed in the diencephalic and mesencephalic neural crest cells (5ss; A, D). The expression of these two genes is maintained in migratory neural crest cells once they have started to migrate, according to a similar pattern at 10ss (B,E) and 12-13 ss (C-F). (G) Absence of signal after in situ hybridization with AMPK sense probes (ISH Control).

Supplementary material S3 Fig. Gene expression pattern ofAMPKα1andAMPKα2in chick embryos. Accumulation of

Conclusions and perspectives

The present study underlined the existence of a crosstalk between Lkb1 and p53 signaling in NCC and pointed out that this crosstalk is crucial for NCC-derivatives development. Increased p53 signaling has been described in biopsies of patients with neurocristopathies and p53 loss in neurocristopathy mouse models rescues the pathological phenotype. To complete this study, it will be interesting to analyze if combined loss of Lkb1