Colin Valet

Aurelie Batut

Alicia Vauclard

Alizee Dortignac

Marie Bellio

Bernard Payrastre

Philippe Valet

Sonia Severin
email: sonia.severin@inserm.fr

Adipocyte Fatty Acid Transfer Supports Megakaryocyte Maturation

Keywords: Bone marrow, Adipocytes, Megakaryocytes, Fatty acids, Obesity

Summary

Megakaryocytes (MKs) come from a complex process of hematopoietic progenitor maturation within the bone marrow that give rise to de novo circulating platelets. Bone marrow microenvironment contains a large number of adipocytes with a still ill-defined role. This study aims to analyze the influence of adipocytes and increased medullar adiposity in megakaryopoiesis. An in vivo increased medullar adiposity in mice caused by high-fat-dietinduced obesity is associated to an enhanced MK maturation and proplatelet formation. In vitro co-culture of adipocytes with bone marrow hematopoietic progenitors shows that delipidation of adipocytes directly support MK maturation by enhancing polyploidization, amplifying demarcation membrane system and accelerating proplatelet formation. This direct cross-talk between adipocytes and MKs occurs through adipocyte fatty acid transfer to MKs involving CD36 to reinforce megakaryocytic maturation. Thus, these findings unveil an influence of adiposity on MK homeostasis based on a dialogue between adipocytes and MKs.

Introduction

Megakaryocytes (MKs) are highly specialized bone marrow (BM) cells releasing up to 10 11 platelets on a daily basis in adult with a rapid turnover to maintain platelet homeostasis. MK maturation is a multi-step process where hematopoietic progenitors (HPs) increase in size, become highly polyploid (by endomitosis without cytokinesis), expand their organelle content and develop a dense membrane demarcation system (DMS), allowing proplatelet projections through medullary sinusoids at the origin of de novo circulating platelets [START_REF] Machlus | The incredible journey: From megakaryocyte development to platelet formation[END_REF][START_REF] Machlus | Interpreting the developmental dance of the megakaryocyte: a review of the cellular and molecular processes mediating platelet formation[END_REF]. This complex and ill-defined process of MK maturation mainly occurs in the BM under the control of thrombopoietin (TPO). Within central cavities of axial and long bones, BM consists of hematopoietic tissue islands and adipose cells surrounded by vascular sinuses [START_REF] Travlos | Normal structure, function, and histology of the bone marrow[END_REF]. BM adipose tissue constitutes 10% of the total fat body mass by filling 50%-70% of the bone cavity in healthy humans [START_REF] Cawthorn | Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction[END_REF][START_REF] Hindorf | EANM Dosimetry Committee guidelines for bone marrow and whole-body dosimetry[END_REF] and increases in mass during metabolic modification conditions as aging, obesity or caloric restriction [START_REF] Cawthorn | Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction[END_REF][START_REF] Doucette | A High Fat Diet Increases Bone Marrow Adipose Tissue (MAT) But Does Not Alter Trabecular or Cortical Bone Mass in C57BL/6J Mice[END_REF][START_REF] Scheller | Region-specific variation in the properties of skeletal adipocytes reveals regulated and constitutive marrow adipose tissues[END_REF]. Considered for a long time as a passive "space fillers", BM adipocytes are specific adipocytes that share some morphological features with peripheral adipocytes but also present unique characteristics and functions [START_REF] Attane | Human Bone Marrow Is Comprised of Adipocytes with Specific Lipid Metabolism[END_REF][START_REF] Cawthorn | Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction[END_REF][START_REF] Craft | Molecular differences between subtypes of bone marrow adipocytes[END_REF][START_REF] Horowitz | Bone marrow adipocytes[END_REF][START_REF] Li | Development, regulation, metabolism and function of bone marrow adipose tissues[END_REF][START_REF] Scheller | Marrow Adipose Tissue: Trimming the Fat[END_REF]. It has been shown that BM adipocytes either in a steady state [START_REF] Ambrosi | Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration[END_REF][START_REF] Naveiras | Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment[END_REF] or under high-fat-diet (HFD)-induced obesity, where medullar adiposity increases, have an impact on general hematopoiesis by regulating hematopoietic stem and progenitor cells, lymphopoiesis and myelopoiesis, thymic aging and memory T cell maintenance (Adler et al., 2014a;[START_REF] Do Carmo | A high-fat diet increases interleukin-3 and granulocyte colony-stimulating factor production by bone marrow cells and triggers bone marrow hyperplasia and neutrophilia in Wistar rats[END_REF][START_REF] Karlsson | Diet-induced obesity in mice reduces the maintenance of influenza-specific CD8+ memory T cells[END_REF][START_REF] Singer | Diet-induced obesity promotes myelopoiesis in hematopoietic stem cells[END_REF][START_REF] Trottier | Enhancement of hematopoiesis and lymphopoiesis in diet-induced obese mice[END_REF][START_REF] Van Den Berg | Diet-induced obesity in mice diminishes hematopoietic stem and progenitor cells in the bone marrow[END_REF]Yang et al., 2009). Considering the critical influence of adipocytes and adipose-rich BM for a normal hematopoiesis and the susceptibility of BM microenvironment to obesity [START_REF] Adler | Obesity-driven disruption of haematopoiesis and the bone marrow niche[END_REF][START_REF] Asada | Complexity of bone marrow hematopoietic stem cell niche[END_REF], we addressed the question whether adipocytes and increased medullar adiposity could influence medullar megakaryopoiesis.

Results

Dysregulated medullar megakaryopoiesis, proplatelet formation and platelet activation in obese mice

To investigate whether a disturbed BM microenvironment with an increased adiposity could influence megakaryopoiesis, MK maturation was analyzed in a context of high-fat-diet (HFD)-induced obesity in mice (Figure S1A) where BM adiposity and triglyceride content increase (Figure 1A) [START_REF] Doucette | A High Fat Diet Increases Bone Marrow Adipose Tissue (MAT) But Does Not Alter Trabecular or Cortical Bone Mass in C57BL/6J Mice[END_REF][START_REF] Singer | Diet-induced obesity promotes myelopoiesis in hematopoietic stem cells[END_REF]. Strikingly, we showed in obese mice that increased medullar adiposity correlates with a significant increase in total and nucleus size of medullar MKs without modifying their number (Figure 1B and Figure S1B).

Polyploidy analysis (Figure S1C) of freshly isolated BM MKs showed an increased percentage of MKs with high ploidy levels in obese mice compared to normal diet (ND) -fed mice (Figure 1C). In contrast, obesity has no impact on extramedullary megakaryopoiesis as spleen size and splenic MK number and size were normal in obese mice (Figure S1D). We also observed that native BM MKs from obese mice displayed a large but poorly invaginated DMS (Figure 1D) and were able to elongate proplatelets faster than MKs from ND -fed mice (Figure 1E and Video S1-S2). Immunostaining of intact BM sections showed an increased proportion of MKs that extended proplatelets in obese mice compared to ND -fed mice. Also, the majority of proplatelet-forming MKs from obese mice extended proplatelets outside BM sinusoidal vessels (Figure 1F) and the proportion of CD41-positive (pro)platelet like particles outside BM sinusoidal vessels was increased in obese mice compared to ND -fed mice (Figure 1G).

Blood count analysis showed that murine obesity resulted in an increased monocyte count as previously described [START_REF] Singer | Diet-induced obesity promotes myelopoiesis in hematopoietic stem cells[END_REF] and in a slightly decreased platelet count and increased platelet volume (Table S1). Following an immune-induced thrombocytopenia, we observed a delayed kinetic in platelet count recovery and a decreased de novo platelet release in the circulation (Figure 1H). Circulating platelet clearance was increased in obese mice (Figure 1I). Transmission electron microscopy analysis of obese mice platelets did not highlight any obvious ultrastructure differences when compared to platelets from ND -fed mice (Figure S1E). Over a collagen matrix, platelets from obese mice display an increased thrombotic capacity compared to platelets from ND -fed mice, by forming bigger thrombi at a physiological arterial shear stress (1500 s-1) and even more at a stenotic arterial shear stress (3000 s-1) (Figure S1F). Tail bleeding time was however normal in obese mice (Figure S1G).

Overall, these data indicate that a diet-induced obesity in mice, which increases medullar adiposity, modifies medullar MK homeostasis, circulating platelet release as well as platelet responses and clearance.

Adipocyte directly improves megakaryopoiesis

Since increased medullar adiposity in mice results in a modified megakaryopoiesis, we next analyzed if adipocytes could directly influence MK maturation. As only few MKs were in direct contact with adipocytes in the BM even with an increased adipose mass (Figure S2A), we set up an in vitro no-contact co-culture assay between adipocytes and freshly isolated mice BM hematopoietic progenitors (HPs) by using a transwell system in presence of TPO to commit HPs towards megakaryocytic maturation for 4 days (Figure S2B). Adipocytes were differentiated from mouse 3T3-F442A preadipocytes cell line or OP9 BM mesenchymal stem cells as shown by morphology changes with the appearance of lipid vacuoles and the increased expression of a specific adipocyte marker, adiponectin (Figure S2C andS2D). TPO itself had no impact on adipocyte differentiation (Figure S2E) and co-culture with HPs/MKs did not induce any adipocyte detachment or death (Figure S2F). We showed that adipocytes increased HPs/MKs survival with a higher proportion of cells negative for Annexin V in coculture (Figure S2G) and significantly upregulated the proportion of cells expressing specific fully differentiated MK markers (CD41 and CD42b) at their surface (Figure 2A). Moreover, larger cells were observed in the HP/MK population co-cultured with adipocytes (Figure S2H).

Polyploidy degree evaluation showed that adipocytes were able to dramatically enhance MK nuclear maturation through a non-physical contact process. Modal ploidy of mature MKs cocultured with adipocytes was significantly higher than non-co-culture MKs, by resulting from a markedly increased proportion of MKs with ploidy levels equal or higher than 64n and a reduced percentage of MKs with lower ploidy levels (Figure 2B). Interestingly, one day of coculture between adipocytes and HPs, either at an early or a late time point of MK differentiation process, was sufficient to significantly influence TPO-induced MK polyploidization (Figure S2I). It is noteworthy that the positive effect of adipocytes on MK nuclear maturation amplification was also observed in absence of TPO (Figure S2J). By transmission electron microscopy, we observed bigger MKs with a fragmented, discontinuous and less structured DMS in MKs co-cultured with adipocytes compared to non-co-cultured MKs where DMS is mainly concentrated in one side of the cell (Figure 2C). This abnormal DMS from MKs in co-culture with adipocytes was associated to an increased proportion of MKs forming proplatelets (Figure 2D). The enhanced CD41/CD42b expression, MK polyploidization and proplatelet formation induced by adipocytes was not observed with nondifferentiated 3T3-F442A or OP9 cells (Figure S3) confirming a specific effect of lipid-filled fully differentiated adipocytes on megakaryopoiesis.

Following TPO binding, c-MPL dimerizes and induces janus kinase 2 (JAK2) autophosphorylation leading to the activation of signal transducers and activators of transcription (STATs), mitogen-activated protein kinases (MAPKs) and phosphoinositide 3kinases (PI3Ks) [START_REF] Geddis | Megakaryopoiesis[END_REF]. In co-culture with adipocytes in presence of TPO, we observed a significantly increased phosphorylation of JAK2, STAT3, AKT and MAPK ERK1/2 (Figure 2E), showing an enhanced activation level of signaling pathways involved in MK differentiation in the presence of adipocytes.

MKs are responsible for adipocyte delipidation

Interestingly, we observed that after 4 days of co-culture with HPs/MKs, adipocytes exhibited a significant reduction in their lipid droplet content (Figure 3A) and triglyceride content (Figure 3B). Surprisingly, MK supernatant was able to induce a significant delipidation of adipocytes (Figure 3C). This adipocyte lipolysis in presence of MK supernatant, occurring at a similar extent than the co-culture between adipocytes and MKs, showed that MK constitutive releasate influences adipocyte homeostasis. Mechanistically, co-cultured adipocytes displayed an increased phosphorylation level of the hormone-sensitive lipase (HSL) on its serine 660 residue (Figure 3D) that promotes its activation for adipocyte delipidation [START_REF] Fruhbeck | Regulation of adipocyte lipolysis[END_REF][START_REF] Nielsen | Dissecting adipose tissue lipolysis: molecular regulation and implications for metabolic disease[END_REF] and a decreased expression of adipocyte specific markers such as PPARγ , aP2 and adiponectin (Figure 3E). The increased CFU-F capacities of co-culture 3T3-F442A and OP9 -cells (Figure 3F) and the fact that these cells were able to re-commit in adipocytes (Figure 3G) provided further evidence that MKs induce a delipidation of 3T3-F442A and OP9 -differentiated adipocytes. Thus, co-culture of adipocytes with HPs/MKs induce adipocyte lipolysis through HSL signaling pathway activation.

MKs uptake fatty acid released by adipocytes via the CD36 scavenger receptor to improve their maturation

We further addressed how adipocytes could influence MK maturation. For that, we analyzed MK maturation after incubating HPs with adipocyte supernatant or with co-culture supernatant in presence of TPO. We observed that adipocyte supernatant increased the polyploidy levels of MKs (Figure 4A and Figure S4A). This enhancement was further increased when MKs were differentiated into the co-culture supernatant (Figure 4A and Figure S4A), showing that HPs/MKs-induced adipocyte release enhances MK maturation.

Adipocytes have been shown to secrete TPO, the main cytokine for megakaryopoiesis [START_REF] Ge | Adipokines identified as new downstream targets for adiponectin: lessons from adiponectin-overexpressing ordeficient mice[END_REF]. We observed that co-culture with MKs decreased TPO mRNA level in adipocytes, (Figure S4B). The decreased TPO mRNA expression in adipocytes co-cultured with MKs (Figure S4B) and the lack of effect of the blocking anti-TPO antibody on adipocytes enhanced MK polyploidization (Figure S4C) led to conclude that adipocyte-secreted TPO was not the causative factor for the increased megakaryopoiesis. Interestingly, when HPs/MKs were cocultured with 14 C palmitate-labeled adipocytes, we detected 14 C palmitate in mature MKs (Figure 4B) associated to a decreased proportion of 14 C palmitate in co-cultured adipocytes and a higher concentration of the radiolabeled fatty acid in the co-culture supernatant (Figure 4B). The 14 C palmitate transferred from adipocytes to MKs was detected in MK neutral lipids and phospholipids (Figure 4B). Altogether, our data demonstrate that HPs/MKs stimulate adipocyte release of fatty acids that are directly uptaken by MKs.

We further investigated the mechanisms by which fatty acid uptake by MKs influences their maturation. HPs were co-cultured with adipocytes in presence of the sulfo-N-succinimidyl oleate (SSO) that has been widely used to irreversibly inhibit the plasma membrane fatty acid translocase CD36 [START_REF] Coort | Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake[END_REF]. Whereas SSO treatment had no effect on non-cocultured MK nuclear maturation (Figure S5A), we observed that inhibiting CD36 significantly reduced MK maturation enhancement induced by adipocytes to a non-co-cultured MK maturation level (Figure 4C and Figure S5B). The abnormal MK DMS ultrastructure induced by adipocyte co-culture was also counteracted by SSO treatment during MK differentiation with the DMS localized in one side of MKs (Figure 4D). Moreover, whereas an acute SSO treatment of MKs after co-culture with adipocytes had no effect on MK proplatelet formation (Figure S5C), SSO treatment along MK differentiation process restored the percentage of MKs forming proplatelets to a level comparable to non-co-cultured MK level (Figure 4E).

These findings demonstrate that the CD36-dependent fatty acid uptake by MKs directly improves MK maturation and proplatelet formation. Nevertheless, inhibiting the entry of fatty acids into mitochondria for β-oxydation with etomoxir had no consequences on MK polyploidization (Figure S5A and S5D) or on proplatelet formation when MK are co-cultured with adipocytes (Figure S5E). Thus, these data exclude the energetic contribution of the CD36-dependent lipid uptake on MK maturation.

We further observed that adding palmitic acid on HPs during TPO-induced megakaryocytic differentiation increased MK size (Figure S5F) and improved MK nuclear maturation (Figure 4F and Figure S5G). Phosphorylation of proteins involved in MK maturation-dependent signaling was significantly increased in MKs differentiated in presence of palmitic acid (Figure 4G). Furthermore, when fluorescent fatty acid Bodipy TM C12-loaded adipocytes (from 3T3-F442 or OP9 cells) were co-cultured with HPs/MKs, fluorescent fatty acids were found in the DMS and at the plasma membrane of mature MKs as assessed by the colocalization of fluorescent fatty acids with the DIOC6 membrane marker (Figure 4H). The absence of Bodipy TM C12 staining in MKs co-cultured with Bodipy TM C12-loaded adipocytes and SSO or in MKs co-cultured with Bodipy TM C12-loaded non-differentiated 3T3-F442A or OP9 confirmed that fatty acids released by adipocytes were the source of lipids for MKs through uptake by CD36 (Figure 4H and Figure S5H). In line with these results, addition of palmitic acid disturbed DMS structure (Figure 4I) and increased the proportion of MKs extending proplatelets (Figure 4J). Altogether, these results showed that adipocyte-derived fatty acids contribute directly to polyploidization, development of MK DMS and therefore to proplatelet formation.

Discussion

Although the analysis of megakaryopoiesis have been the interest of several laboratories since many years, elucidating the molecular and cellular events involved in this process remains an unfinished task. As adipocytes are a major component of the medullar niche homeostasis, we investigated whether they could influence MK biology. Due to the weak proportion of MKs in direct contact with the BM adipocytes, we investigated the paracrine adipocyte/MK communication independent of cell-cell contact by using an in vitro no-contact co-culture assay. We clearly demonstrate that adipocytes strongly influence MK maturation that involved a direct dialogue between HPs/MKs and adipocytes. In one hand, adipocytes support HP survival and enhance their differentiation into the MK lineage by heightening MK polyploidy, modifying MK membrane structure and accelerating proplatelet formation. In the other hand, HPs/MKs induced adipocyte delipidation. Our results also shed light that the initiating event of the crosstalk between adipocytes and MKs remains the MK ability to release soluble factors to modify adipocyte homeostasis by inducing its delipidation, which in turn favors the proper MK differentiation. In line with these results, MKs have been also

shown to influence the BM matrix reorganization and the homeostasis of endothelial cells, osteoblasts/osteoclasts hematopoietic cells within the BM by releasing soluble factors such as cytokines, extracellular matrix (ECM) components and metalloproteinases [START_REF] Bonner | Regulation of PDGF and its receptors in fibrotic diseases[END_REF][START_REF] Chagraoui | Prominent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice[END_REF][START_REF] Guerrero | Gray platelet syndrome: proinflammatory megakaryocytes and alpha-granule loss cause myelofibrosis and confer metastasis resistance in mice[END_REF][START_REF] Malara | The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control[END_REF][START_REF] Malara | Megakaryocyte Contribution to Bone Marrow Fibrosis: many Arrows in the Quiver[END_REF][START_REF] Malara | Megakaryocytes Contribute to the Bone Marrow-Matrix Environment by Expressing Fibronectin, Type IV Collagen, and Laminin[END_REF][START_REF] Pohlers | TGF-beta and fibrosis in different organsmolecular pathway imprints[END_REF]Wickenhauser et al., 1995).

Adipocyte canonical function is to store energy as triglycerides for releasing fatty acids to metabolic tissue utilization. Our study shows a crosstalk between adipocytes and MKs through a direct fatty acid transfer from adipocytes to MKs that is dependent of the fatty acid translocase CD36 known to be expressed at the surface of MKs [START_REF] Coort | Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake[END_REF][START_REF] Pepino | Structure-function of CD36 and importance of fatty acid signal transduction in fat metabolism[END_REF]. In line with previous studies [START_REF] Dhenge | Arachidonic acid and Docosahexanoic acid enhance platelet formation from human apheresis-derived CD34(+) cells[END_REF][START_REF] Murphy | Cholesterol efflux in megakaryocyte progenitors suppresses platelet production and thrombocytosis[END_REF][START_REF] Siddiqui | Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium[END_REF], we clearly point out that fatty acids can directly influence megakaryopoiesis by enhancing ploidy levels of MKs through the regulation of MK maturation related-signaling pathways. We also show that fatty acids released by adipocytes are a source of structural lipids for DMS amplification. As SSO has an impact on proplatelet formation only when it is present during MK differentiation process (i.e when MK differentiation is enhanced by adipocytes), we conclude that the increased proplatelet formation of MKs co-cultured with adipocytes is a direct consequence of the increased nuclear and cytoplasmic/membrane differentiation induced by adipocyte fatty acid uptake by MKs. A close communication between adipocytes and cancer cells via a fatty acid release by adipocytes has also been previously reported [START_REF] Laurent | Periprostatic Adipose Tissue Favors Prostate Cancer Cell Invasion in an Obesity-Dependent Manner: Role of Oxidative Stress[END_REF][START_REF] Nieman | Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth[END_REF][START_REF] Wang | Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells[END_REF]. However, whereas the fatty acid transfer from adipocytes to cancer cells occurs in an energetic way through mitochondrial fatty acid oxydation, it is not the case for the adipocyte lipid transfer in MKs.

In the settlement of obesity, BM environment undergoes considerable changes such as hyperplasia with a ~20-30% increased marrow cellularity, increased adiposity and modifications of growth factor/cytokine secretion, conditions that disrupt chemical, physical and cellular connections of the BM. Literature also shed the light on the role of adipocytes and obesity on the regulation of long term hematopoietic stem and progenitors cells, lymphopoiesis and myelopoiesis (Adler et al., 2014a;[START_REF] Do Carmo | A high-fat diet increases interleukin-3 and granulocyte colony-stimulating factor production by bone marrow cells and triggers bone marrow hyperplasia and neutrophilia in Wistar rats[END_REF][START_REF] Karlsson | Diet-induced obesity in mice reduces the maintenance of influenza-specific CD8+ memory T cells[END_REF][START_REF] Singer | Diet-induced obesity promotes myelopoiesis in hematopoietic stem cells[END_REF][START_REF] Trottier | Enhancement of hematopoiesis and lymphopoiesis in diet-induced obese mice[END_REF][START_REF] Van Den Berg | Diet-induced obesity in mice diminishes hematopoietic stem and progenitor cells in the bone marrow[END_REF]Yang et al., 2009). In addition to these data, our in vitro co-culture assay clearly showed an important influence of adipocytes on megakaryopoiesis and that obesity with an increased medullar adiposity modified the MK phenotype as observed in vitro. Systemic changes such as global metabolism dysregulation and chronic low-grade inflammation changes could influence megakaryopoiesis in addition to the increased medullar adiposity. Nevertheless, the lack of influence of obesity on splenic megakaryopoiesis where adipocytes are absent, strongly argues for a determinant role of increased medullar adiposity on the modified medullar megakaryopoiesis.

The functional importance of the crosstalk between MKs and adipocytes might contribute to a better understanding of the altered thrombosis and hemostasis status observed in the obese population. The non-stretchable compartment of the BM, in which adiposity and MK mass increased, might prevent large MKs from reaching the sinusoids and is likely responsible for platelet presence outside the BM vasculature and decreased circulating platelet count. Obesity also enhanced the clearance of platelets and their thrombotic reactivity. Even if the low-grade inflammation of the obesity might directly impact on platelets, the influence of adipocytes and obesity on MK homeostasis could directly modify the intrinsic characteristics of platelets that impact on their reactivity. This is illustrated by a recent study from Di Paola laboratory showing that aging, which induces metabolic changes similarly to obesity, are responsible for MK gene reprogramming and metabolic profile changes that determine platelet hyperactivity in old mice [START_REF] Davizon-Castillo | TNF-alphadriven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging[END_REF].

Therefore, our study provides insights on how the cellular medullary microenvironment (E) MKs isolated from BSA gradient were lysed and protein samples were immunoblotted using specific antibodies: anti-pJAK2 (Tyr1007/1008), anti-pSTAT3 (Tyr705), anti-pAKT (Ser473), anti-pERK (Thr202/Tyr204). Total JAK2, STAT3, AKT, ERK1/2 and GAPDH were used as control loading. Representative western blots are shown and the ratio phosphorylated/total of each protein was quantified using ImageLab software. Graphs (Sigma-Aldrich) for 7 days at 37°C under 5% CO2 [START_REF] Boucher | Apelin, a newly identified adipokine upregulated by insulin and obesity[END_REF]. Adherent OP9 cells were growth in MEMα Μedium Glutamax TM (Invitrogen) supplemented with 20% fetal bovine serum and 100UI/mL penicillin/100µg/mL streptomycin in Nunclon TM Delta Surface 6

well plates (Thermo Fisher Scientific) and when confluent, were differentiated in ΜΕΜα Μedium Glutamax ΤΜ supplemented with 15% KnockOut TM SR (Invitrogen) for 7 days at 37°C under 5% CO2 (Wolins et al., 2006). Adipogenic differentiation was imaged by using the transmission channel of the ZOE™ Fluorescent Cell Imager (Bio-Rad).

BM hematopoietic progenitor cells (HPs) purification and co-culture with 3T3-F442A or OP9 -differentiated adipocytes: Mature MKs from BM cells were defined as the population of cells generated using the methodology of the Frampton and Shivdasani laboratories [START_REF] Dumon | Differentiation of murine committed megakaryocytic progenitors isolated by a novel strategy reveals the complexity of GATA and Ets factor involvement in megakaryocytopoiesis and an unexpected potential role for GATA-6[END_REF][START_REF] Lecine | Characterization of the hematopoietic transcription factor NF-E2 in primary murine megakaryocytes[END_REF]. BM cells were flushed from femora and tibiae of 10-20 weeks-aged C57BL6/J male mice. The cells were spun down and resuspended in ACK buffer (0.15M NH4Cl; 1mM KHCO3; 0.1mM Na2EDTA pH 7.3) for 5 minutes to lyse mature erythrocytes. After filtration using easy strainer™ 70 µm and centrifugation 300xg for 5 minutes, the pelleted cells were resuspended in DMEM Glutamax TM (Invitrogen)

supplemented with 10% fetal bovine serum (Invitrogen) and 100UI/mL penicillin/100µg/mL streptomycin (Sigma-Aldrich). Lineage cell (Lin + cell) depletion was further performed by incubating cells with antibodies against CD16/CD32 (BD Pharmingen™), Gr1

(eBioscience™), B220 (BD Pharmingen™) and CD11b (eBioscience™) for 30 minutes on ice, followed by incubation with sheep anti-rat IgG immunomagnetic Dynabeads (Invitrogen).

Two cycles of centrifugation (300xg for 5 minutes at 4°C) and resuspension were performed to allow bead-antibody interaction. The use of a magnet enabled collection of non-bound cells in the supernatant. The remaining Lin -cells (called HPs) were then centrifuged and resuspended at 2x10 6 cells/mL in 10% serum-supplemented DMEM Glutamax TM and 100UI/mL penicillin/100µg/mL streptomycin. 1mL of cells was seeded in the upper chamber of the 0.4 µm cell culture insert of 30mm diameter (Transwell chamber, Merck Millipore) over adherent 3T3-F442A or OP9 -differentiated adipocytes (no direct contact) at 37°C under 5% CO2 in the presence of recombinant mouse TPO (50ng/mL, Peprotech) for 4 days to induce HP differentiation into MKs. Isolation of mature MKs was carried out on a bovine serum albumin (BSA) gradient (1.5%/3%) under gravity (1xg) for 40 minutes at room temperature.

Preparation of murine washed platelets: Whole blood was drawn from the inferior vena cava of anesthetized mice with a mixture of Imalgene (25mg/kg) (Merial) and Rompun (10 mg/kg) (Bayer) into a syringe containing acid citrate dextrose (3% trisodium citrate x5.5H20, 1.4% citric acid, 2% glucose) (1 anticoagulant volume / 9 blood volumes). Platelet-rich plasma (PRP) was obtained by mixing blood with 1 volume of modified HEPES-Tyrode's buffer (140mM NaCl, 2mM KCl, 12mM NaHCO3, 0.3mM NaH2PO4, 1M MgCl2, 5.5mM glucose, 5mM HEPES, pH 6.7) containing 0.35% BSA followed by a 300xg centrifugation for 4 minutes. After PGI2 (Sigma-Aldrich) addition at a final concentration of 500nM to the PRP, platelets were pelleted by centrifugation at 1,000xg for 6 minutes and resuspended in modified HEPES-Tyrode's buffer (pH 7.38) in the presence of 0.02IU/mL of the ADP (adenosine-5′-triphosphate diphosphohydrolase) scavenger apyrase (Sigma-Aldrich) and rested for 45 minutes at 37°C.

METHOD DETAILS

Flow cytometry: CD41-CD42b double expression was analyzed on HPs/MKs stained with both FITC-conjugated anti-mouse CD41 (BD Pharmingen™) and PE-conjugated anti-mouse CD42b (Emfret ANALYTICS) antibodies in PBS-1% BSA for 30 minutes at room temperature. Cell survival was analyzed after labeling of HPs/MKs with an anti-annexin V antibody (BD Pharmingen™) in Binding Buffer (10mM Hepes (pH 7.4), 140mM NaCl, 2.5mM CaCl2) for 30 minutes at room temperature. Fresh cell samples were further analyzed on an LSRFortessa TM Cell analyzer flow cytometer (BD Biosciences) and Diva software (BD Biosciences). DNA content analysis was performed in MKs isolated from a BSA gradient.

After fixation with 0.5 % formaldehyde on ice for 30 minutes, MKs were stained with a staining solution containing 2mM MgCl2, 0.05% saponin, 10µg/mL propidium iodide (Invitrogen) and 10U/mL RNAse A (Roche) for 2 hours at 4°C and immediately analyzed by using an LSRFortessa TM Cell analyzaer flow cytometer and Diva software. Gel electrophoresis and immunoblotting: 20µg of protein samples were loaded on 10% polyacrylamide gel. Proteins were separated by SDS-PAGE, transferred on a nitrocellulose membrane (Gelman Sciences) and analyzed using the relevant antibody. The membrane was blocked using 3% BSA in Tris-Buffer-Saline containing 0.1% Tween (TBST) for 60 minutes.

Membranes were probed overnight with the respective primary antibody (see Key Resources Table ). After washing with TBST, the blots were probed with HRP-tagged secondary antibodies and washed again. Signals were detected using Clarity/Clarity Max™ Western ECL substrate (Bio-Rad) and Chemidoc Imaging system (Bio-Rad) The bands were quantified using ImageLab software (Bio-Rad) and normalized to the corresponding total protein loading.

Flow assay on collagen: Biochips microcapillaries (Vena8 Fluoro+, Cellix) were coated with a fibrillar collagen suspension (50μg/mL) for 1 hour and saturated with a solution of 0.5% BSA. Whole blood was drawn from the inferior vena cava of anesthetized mice with a mixture of Imalgene (25mg/kg) (Merial) and Rompun (10mg/kg) (Bayer) into a syringe containing heparin (10IU/mL) (1 volume anticoagulant / 9 volumes blood). Platelets were labeled with DIOC6 (2μM, Invitrogen) in whole blood for 10 minutes at 37°C.

Figure Legends Figure 1 .

 Legends1 Figure Legends

Figure 2 .

 2 Figure 2. Adipocyte directly improves megakaryopoiesis.

  represent fold increase phosphorylation in comparison to non-co-cultured MKs as mean±SD (n=3 and 4 independent experiments for 3T3-F442A and OP9 respectively; *p<0.05, **p<0.01, ***p<0.001 versus MK according to one sample t test). MK: HPs/MKs cultured alone; MK/3T3: HPs/MKs co-cultured with 3T3-F442Adifferentiated adipocytes; MK/OP9: HPs/MKs co-cultured with OP9 -differentiated adipocytes. See also Figure S2, S3 and S6.

Figure 3 .

 3 Figure 3. MKs induced adipocyte delipidation.

Figure 4 .

 4 Figure 4. The CD36-fatty acid uptake by MKs is essential to improve their maturation.

  Using a syringe pump (PHD-2000, Harvard Apparatus) labeled blood was perfused through a microcapillary for 2 minutes at different wall shear rate at 37°C. Platelet adhesion and thrombus formation were visualized with a 40x oil immersion objective for both fluorescent and transmitted light microscopy and recorded in real time (1 frame every 5 seconds) with ORCA Camera (Carl Zeiss). Image sequences of the time lapse recording and analysis of surface coverage were performed offline on single frame by quantification of pixel surface after manual thresholding using ImageJ® software. Thrombi volumes are calculated by thresholding of surface covered by thrombi on slice of Z-stack images and addition of voxel (automatically converted into μm 3 by Zeiss software).Immune-induced thrombocytopenia: Thrombocytopenia was induced by intraperitoneal injection of anti-mouse GPIbα antibody (2 μg/g, Emfret ANALYTICS). Blood samples were collected by tail bleeding at different times and platelet counts were measured using ABX Micros 60 analyser (HORIBA Medical).Platelet lifespan: Circulating platelets were labeled in vivo by mice intravenous injection of Dylight 488 -anti-GPIbβ Ig derivative (0.1 μg/g, Emfret ANALYTICS). 2 hours after antibody injection and every 24 hours for 4 days, the percentage of the Dylight 488 -positive platelet population in whole blood from tail bleeding was determined using an LSRFortessaTM Cell analyser flow cytometer and Diva software (BD Biosciences).Tail bleeding time. 3mm tail-tip transection in mice anesthetized by an intra-peritoneal injection of a mixture of Imalgene (25mg/kg) (Merial) and Rompun (10mg/kg) (Bayer). A stopwatch was started immediately upon transection to determine the time required for the bleeding to stop. Blood drops were removed every 15 seconds with the use of a paper filter. If bleeding did not recur within 30 seconds of cessation, it was considered stopped.QUANTIFICATION AND STATISTICAL ANALYSISData are expressed as mean±SD. Significance of differences, mentioned in the figure legend, was determined by using Prism6 (GraphPad software). p values < 0.05 were considered significant (*p<0.05, **p<0.01, ***p<0.001) using unpaired two-tailed Student, one-sample t test, one-way ANOVA with the Tukey post hoc test or two-way ANOVA with Bonferroni post hoc test.Wickenhauser, C., Hillienhof, A., Jungheim, K., Lorenzen, J., Ruskowski, H., Hansmann, M.L., Thiele, J., and Fischer, R. (1995). Detection and quantification of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) release by normal human megakaryocytes. Leukemia 9, 310-315. Wolins, N.E., Quaynor, B.K., Skinner, J.R.,Tzekov, A., Park, C., Choi, K., and Bickel, P.E. (2006). OP9 mouse stromal cells rapidly differentiate into adipocytes: characterization of a useful new model of adipogenesis. Journal of lipid research 47,450-460. Yang, H., Youm, Y.H., Vandanmagsar, B., Rood, J., Kumar, K.G., Butler, A.A., and Dixit, V.D. (2009). Obesity accelerates thymic aging.Blood 114,[3803][3804][3805][3806][3807][3808][3809][3810][3811][3812]

Acknowledgements

We thank the personnel of Anexplo animal facilities (UMS US006/ Inserm) for animal handling, of experimental histopathology of UMS US006/ Inserm (F. Capilla), of Cytometry core facility of Inserm UMR1048 (E. Riant and A. Zakaroff-Girard), Genotoul imaging core facility (Inserm UMR1048, E. Vega and R. Flores-Flores and CMEAB, I. Fourquaux) and of MetaToul-Lipidomic core facility of Inserm U1048 (J. Bertrand-Michel). The authors thank all members from the B.P. and P.V laboratories and A. Mairal for helpful discussions. C.V. was supported by fellowships from Université Paul Sabatier and Société Francaise d'Hématologie. M.B. was supported by a Fondation pour la Recherche Médicale fellowship (FRM grant number ECO20160736022 [MB]). A.V. was supported by Fondation de France (grant number 00075817 [SS]). Work in B.P. laboratory was supported by Inserm, Fondation pour la Recherche Médicale (FRM grant number DEQ20170336737 [BP]) and Fondation de France (grant number 00075817 [SS]). B.P. is a scholar of the Institut Universitaire de France. Work in P.V. laboratory was supported by Inserm and the Fondation pour la Recherche Médicale (FRM grant number ING20121226373 [PV]).

Authorship contributions

Conceptualization -C.V., P.V., B.P., S.S.; Methodology -C.V., S.S.; Validation -S.S.; Investigation -C.V., A.B., A.V., A.D., M.B., S.S.; Writing -Original Draft, C.V., A.V., S.S.; Writing -Review & Editing -P.V., B.P.; Visualization -C.V., A.B., A.V., M.B., S.S.; Funding Acquisition -S.S.; Supervision and Project Administration -S.S.

Declaration of Interests

The authors declare no competing interests.

STAR Methods

LEAD CONTACT

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr Sonia Severin (sonia.severin@inserm.fr).

MATERIALS AVAILABILITY

This study did not generate new unique reagents.

DATA AND CODE AVAILABILITY

This study did not generate any unique datasets or code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals: All C57BL/6J mice were purchased from Janvier Labs and were backcrossed in house. Mice were housed in conventional cages under specific pathogen-free conditions in a constant temperature (20-22°C) and humidity (50-60%) animal room with a 12/12 h light/dark cycle (lights on at 7:00 AM) and free access to food and water. To isolate HPs, male mice were used at 10-20 weeks old. To induce obesity, male mice were fed at 8 weeks aged (when mice are considered to be adult) with a high-fat-diet (HFD: 20% protein, 20% carbohydrate, 60% fat) (ResearchDiets, NJ, US) for 12 weeks. Normal diet (ND) is composed of 24% protein, 67% carbohydrate, 9% fat (Ssniff, Germany). All procedures were performed in accordance with institutional guidelines for animal research and were approved by the French Ministry of Research in agreement with European Union guidelines.

Eclipse Ti microscope and NIS-Elements AR software, and quantified by using ImageJ® software as the percentage of the field labeled with Red oil.

Proplatelet formation assays:

The analysis of the dynamics of platelet formation from BM explants was performed as previously described [START_REF] Strassel | Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors[END_REF]. Briefly, after flushing, 0.5-1mm sections of marrow were maintained in Tyrode's buffer (0.137M NaCl, 2.68mM KCl, 11.9mM NaHCO3, 0.4mM NaH2PO4, 1mM MgCl2, 2mM CaCl2, 5mM HEPES, 0.35% BSA, 5% mouse serum at pH=7.3) with 15IU/mL heparin in a 5% humidified CO2 atmosphere at 37 °C. Cells were left to migrate out of the explants for 2 hours prior to imaging. Videomicroscopy was performed with an Axio Observer.Z1 microscope operated by the Zen software (Carl Zeiss) in a controlled atmosphere for 15 hours. Images were taken every 5 minutes with an ORCA R2 camera (Hamamatsu) using a 10×, 0.30 EC Plan-Neofluar lens (Carl Zeiss). A motorized multi-position stage (in x, y, z) was used, and an average of 150 stage positions, randomly picked showing 1-2 MKs per field, were followed. Proplatelet formation was quantified manually using Zen software as the percentage of alive MKs that extend proplatelets in a given time point. In vitro proplatet formation analysis was performed by plating for 6 hours on a fibrinogen matrix (100µg/mL) in vitro differentiated MKs and isolated by BSA gradient. The percentage of proplatelet forming MKs was manually quantified by using the transmission channel of the ZOE™ Fluorescent Cell Imager (Bio-Rad).

Real-Time PCR: Total RNAs were isolated by using GeneElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). Total RNAs (500ng) were reverse transcribed using High Capacity cDNA reverse transcription kit (Applied Biosystems) in the presence of random hexamers. Real-time PCR was performed on 6.25ng cDNA and 100-900nmol/L specific oligonucleotide primers (see Key Resources Table) in a final volume of 10μL using the MESA Blue qPCR™ Mastermix for SYBR® Assay (Eurogentec). Fluorescence was


Fabienne Agasse

Indira Mendez-David

Wilhelm Christaller

Rémi Carpentier

Barbara Y Braz

Denis J David

Frédéric Saudou

Sandrine Humbert
email: sandrine.humbert@univ-grenoble-alpes.fr

Chronic corticosterone elevation suppresses adult hippocampal neurogenesis by hyperphosphorylating Huntingtin

Keywords: neurogenesis, corticosterone, Huntingtin, CDK5, depression, BDNF, axonal transport

Chronic exposure to stress is a major risk factor for neuropsychiatric disease, and elevated plasma corticosterone (CORT) correlates with reduced levels of both brainderived neurotrophic factor (BDNF) and hippocampal neurogenesis. Precisely how these phenomena are linked, however, remains unclear. Using a cortico-hippocampal network-on-a-chip, we find that the glucocorticoid receptor agonist dexamethasone (DXM) stimulates the cyclin-dependent kinase 5 (CDK5) to phosphorylate huntingtin (HTT) at serines 1181 and 1201 (S1181/1201), which retards BDNF vesicular transport in cortical axons. Parallel studies in mice show that CORT induces phosphorylation of these same residues, reduces BDNF levels, and suppresses neurogenesis; the adverse effects of CORT are reduced in mice bearing an unphosphorylatable mutant HTT (Hdh S1181A/S1201A ). The protective effect of unphosphorylatable HTT, however, disappears if neurogenesis is blocked. The CDK5-HTT pathway, which regulates BDNF transport in the cortico-hippocampal network, thus provides a missing link between elevated corticosterone levels and suppressed neurogenesis.

INTRODUCTION

In both rodents and humans, the hippocampus generates new neurons throughout the lifespan. This ongoing activity is particularly susceptible to environmental and experiential input such as stress [START_REF] Kempermann | Human Adult Neurogenesis: Evidence and Remaining Questions[END_REF]. Normal adult hippocampal neurogenesis correlates with learning, memory, and mood regulation, and confers resilience to stress-induced depression [START_REF] Anacker | Hippocampal neurogenesis confers stress resilience by inhibiting the ventral dentate gyrus[END_REF])-yet chronic stress in humans and animals, which causes elevated levels of cortisol (or corticosterone), can suppress neurogenesis and reduce hippocampal volume [START_REF] Bremner | Hippocampal volume reduction in major depression[END_REF][START_REF] Schoenfeld | Stress and Loss of Adult Neurogenesis Differentially Reduce Hippocampal Volume[END_REF][START_REF] Vyas | Chronic Stress and Glucocorticoids: From Neuronal Plasticity to Neurodegeneration[END_REF]. What determines which side of this bidirectional relationship carries more weight is a mystery, though the balance can be tipped: antidepressants such as fluoxetine exert their benefits by restoring hippocampal neurogenesis and increasing levels of Brain-derived Neurotrophic Factor (BDNF), which promotes neuronal maturation, dendritic growth and synaptic transmission in the hippocampus [START_REF] Boldrini | Hippocampal angiogenesis and progenitor cell proliferation are increased with antidepressant use in major depression[END_REF][START_REF] Boldrini | Antidepressants increase neural progenitor cells in the human hippocampus[END_REF][START_REF] Chan | Depletion of central BDNF in mice impedes terminal differentiation of new granule neurons in the adult hippocampus[END_REF][START_REF] Chen | Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF]. The precise molecular mechanisms and neural circuits underlying these influences, however, have remained elusive.

Many studies have sought to understand how BDNF functions in the stress response and in the pathophysiology of depression. In depressed patients, both BDNF levels and neurogenesis are diminished [START_REF] Huang | Serum brain-derived neurotrophic factor levels in patients with major depression: effects of antidepressants[END_REF]. BDNF is normally secreted by dentate gyrus neurons to stimulate neurite growth and ramification [START_REF] Wang | Autocrine action of BDNF on dendrite development of adult-born hippocampal neurons[END_REF][START_REF] Waterhouse | BDNF promotes differentiation and maturation of adult-born neurons through GABAergic transmission[END_REF]; it also enters the hippocampus from the entorhinal cortex, which is the primary source of input fibers to the hippocampus via the perforant path [START_REF] Kang | Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus[END_REF]. Interestingly, stimulation of the entorhinal cortico-hippocampal circuit mitigates depression-like symptoms in mice under conditions of chronic stress by increasing hippocampal neurogenesis [START_REF] Bruel-Jungerman | Long-term potentiation enhances neurogenesis in the adult dentate gyrus[END_REF][START_REF] Stone | Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory[END_REF][START_REF] Yun | Stimulation of entorhinal cortex-dentate gyrus circuitry is antidepressive[END_REF], but whether or how BDNF is involved in this antidepressant effect remains unknown. The molecular mechanisms regulating BDNF distribution in the cortico-hippocampal circuit under conditions of stress have yet to be discovered.

Here, we sought to delineate the effects of chronically elevated corticosterone levels on BDNF distribution in the cortico-hippocampal circuit in mice. Since studying the kinetics of BDNF vesicles in projecting axons in vivo is not yet possible, we used microfluidic devices to recreate mature neuronal circuits [START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF] in a close-to-physiological context in order to study axonal transport. We then studied the consequences of corticosterone-influenced BDNF transport on neurogenesis in vivo.

RESULTS

Corticosteroid slows cortical transport of BDNF in a cortico-hippocampal neuronal network

To test in vitro whether stress affects cortical delivery of BDNF to the hippocampus, we reconstructed cortico-hippocampal neuronal networks on silicone-based micro-chips (Figures 1A and 1B). Because the E15.5 embryonic entorhinal cortex on its own provides an insufficient number of cells for culturing, we dissected out whole cortices, which consist of 25% reelin-positive neurons, i.e., cells expressing the chemical type displayed by layer II neurons of the entorhinal cortex that project to the dentate gyrus (DG) (Figure S1A) [START_REF] Witter | Architecture of the Entorhinal Cortex A Review of Entorhinal Anatomy in Rodents with Some Comparative Notes[END_REF]. We plated the cortical and hippocampal neurons in the presynaptic and the postsynaptic compartments, respectively and allowed the cellular network to mature for seven days [START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF]. By this time, most of the connections are oriented, and Tau-positive cortical axons establish synapses with MAP2-positive hippocampal dendrites within the synaptic compartment (Figure 1B).

To mimic the systemic increase of corticosterone that occurs with chronic stress, we applied the glucocorticoid receptor agonist dexamethasone (DXM, 5 µM) to both presynaptic and postsynaptic chambers three hours before imaging with spinning disk confocal microscopy. We recorded vesicular trafficking in the distal part of the microchannels containing the cortical axons, generated kymographs from the videos, and measured the velocity and numbers of BDNF-carrying vesicles moving in anterograde or retrograde directions (Figure 1C). DXM treatment slowed the anterograde transport of BDNF vesicles in WT cortical axons (Mann-Whitney test, **p˂0.01) (Figure 1C) without affecting the overall number of vesicles moving within axons. This indicates that DXM affects the transport of BDNF rather than its production.

To account not only for the velocity of the vesicles but also their number and direction of movement relative to soma/axon terminal orientation, we also calculated the net flux [START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF]. A positive value would indicate more BDNF moving from the soma to the terminals. DXM treatment significantly reduced the net flux of BDNF (Mann-Whitney test, *p˂0.05) (Figure 1C).

Corticosterone augments CDK5 phosphorylation of Huntingtin

To investigate the mechanisms by which DXM reduces BDNF vesicular transport, we considered molecular motors and adaptors, which determine the efficiency and directionality of transport [START_REF] Hirokawa | Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease[END_REF]. Axonal transport of vesicular BDNF relies on huntingtin (HTT), which serves as a scaffold for motor protein complexes [START_REF] Gauthier | Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules[END_REF][START_REF] Saudou | The Biology of Huntingtin[END_REF]. Because stress has been shown to upregulate activity of the serine-threonine kinase cyclin-dependent kinase 5 (CDK5) in the mouse cortex [START_REF] Papadopoulou | Acute and chronic stress differentially regulate cyclin-dependent kinase 5 in mouse brain: implications to glucocorticoid actions and major depression[END_REF], we examined HTT phosphorylation at serines 1181 and 1201, which is accomplished by CDK5 [START_REF] Ben M'barek | Huntingtin mediates anxiety/depressionrelated behaviors and hippocampal neurogenesis[END_REF].

We found that treating cortical neurons with DXM increased phosphorylation of both serine 1181 and 1201 (**p˂0.01, Mann-Whitney test), as measured by antibodies specific to these serines when they are phosphorylated [START_REF] Ben M'barek | Huntingtin mediates anxiety/depressionrelated behaviors and hippocampal neurogenesis[END_REF] (Figures 1D and S1B). DXM also increased cleavage of p35 into p25, the constitutive activator of CDK5 (*p˂0.05). The CDK5 inhibitor roscovitine hindered DXMmediated phosphorylation (Figure 1D), whereas over-activation of CDK5 by transfecting neurons with p25 increased HTT phosphorylation of both serine 1181 and 1201 (*p˂0.05 and **p˂0.01, respectively) (Figures 1E andS1C).

We therefore examined the consequences of CDK5 over-activation on vesicular transport. We found that p25 reduced the anterograde velocity and the net flux of BDNF vesicles by approximately 15% and 65%, respectively (*p˂0.05, Mann-Whitney test) (Figures 2A andS1C). Cortical neurons expressing a kinase-dead form of CDK5 (CDK5-KD) and treated with either DXM or DMSO showed no change in BDNF transport with DXM (Figures 2B andS1C); neither p25 nor CDK5 had any effect on the number or the net flux of moving vesicles. Thus, DXM requires CDK5 activation in order to slow BDNF transport.

To determine whether the effects of DXM and p25 on BDNF transport are mediated by HTT phosphorylation, we reconstituted a cortico-hippocampal network using neurons from Hdh S1181A/S1201A embryos, which lack phosphorylation at S1181/1201 [START_REF] Ben M'barek | Huntingtin mediates anxiety/depressionrelated behaviors and hippocampal neurogenesis[END_REF]. DXM had no effect on the anterograde velocity of BDNF vesicles in Hdh S1181A/S1201A neurons (Figure 2C), nor did hyper-activation of CDK5 by p25 (Figure 2D). Thus, in the absence of HTT phosphorylation at S1181/S1201, neither DXM nor p25 is able to slow the anterograde transport of BDNF. We conclude that DXMmediated stress response alters BDNF transport via the CDK5-HTT pathway.

Corticosterone induces phosphorylation of HTT at CDK5 sites in vivo

We next asked whether chronic administration of low-dose CORT-5 mg/kg/day, administered to WT mice for at least four weeks [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF] to mimic chronic stress-induces HTT phosphorylation by CDK5 in vivo (Figure 3A). Corticosterone treatment increased phosphorylation of HTT at CDK5 sites, as indicated by immunoblotting of protein extracts from the whole cortex (Figures 3B and3C) (*p˂0.05, Mann-Whitney test). CORT treatment also consistently increased the levels of CDK5 and p25 (**p˂0.01). We obtained similar though less marked results in protein extracts from the whole hippocampus.

Mice treated with CORT had lower levels of proBDNF and mature BDNF in cortical extracts than controls (**p<0.001 and *p˂0.05, respectively) (Figure 3D). In the smaller hippocampal-only samples we could measure only mature BDNF: CORT-treated hippocampi had less mature BDNF than controls (****p˂0.0001) (Figure 3E). Together with the fact that the transport assays are done with mCherry-BDNF expressed under a PGK promoter, ensuring that BDNF expression levels are the same between the different conditions, these data confirm that CORT reduces BDNF transport, not maturation. Chronic corticosterone treatment thus raises CDK5 and p25 protein levels, increases HTT phosphorylation at CDK5 sites, and reduces mature BDNF levels in the mouse cortex and hippocampus.

Phospho-ablation of HTT at CDK5 sites preserves neurogenesis under chronic

CORT treatment

Since CORT reduces the number of newborn neurons and alters their morphology [START_REF] Schoenfeld | Differential effects of stress and glucocorticoids on adult neurogenesis[END_REF], we evaluated hippocampal neurogenesis in the dentate gyrus (DG) of WT and Hdh S1181A/S1201A mice in control and CORT conditions (Figure 3A).

We evaluated the dorsal and ventral regions of the DG separately, as they are thought to play different roles in cognitive and emotional processing. Because the results were similar in these two regions (Figure S2), however, we pooled the data (Figures 4A to 4C).

WT and Hdh S1181A/S1201A mice had a similar basal rate of progenitor cell proliferation, according to the number of Ki67 cycling cells (Figures 4A andS2A). CORT treatment reduced proliferation in the WT DG (two-way ANOVA, **p˂0.01) but had no effect on proliferation in Hdh S1181A/S1201A mice (Figures 4A andS2A). To date the birth of the newborn cells, we injected BrdU at the beginning of CORT treatment and counted the surviving BrdU+ cells after four to six weeks of treatment (Figures 4B andS2B). These cells survived longer in Hdh S1181A/S1201A mice than in WT mice (Two-way ANOVA, **p˂0.01), and this effect was maintained under CORT treatment (Two-way ANOVA, **p˂0.01).

To determine whether CORT affects the number of newly generated neurons in the DG, we used DCX, a transient marker of immature neurons. CORT did not affect the number of young neurons in either genotype (Figures 4C and S2C). The absence of an effect on newly generated neurons in conjunction with greater survival and proliferation led us to investigate whether neuronal maturation is accelerated in the mutant DG.

Quantifying the number of BrdU+ cells that differentiated into mature neurons positive for calbindin, a terminal marker for granule neurons, revealed that Hdh S1181A/S1201A mice showed greater neuronal differentiation than WT mice (*p˂0.05) in the basal (vehicle) condition (Figures 4D and4E). Chronic CORT treatment decreased terminal differentiation in WT mice (*p˂0.05) but had no effect on Hdh S1181A/S1201A mice.

We then measured the length and complexity of dendrites in WT and Hdh S1181A/S1201A DCX+ neurons (Figures 4F to 4H). Hdh S1181A/S1201A neurons have longer neurites and greater complexity than WT neurons under control conditions. CORT markedly shortened and simplified dendrites in WT mice compared to vehicle-treated control mice (Mendez- David et al., 2014). CORT also shortened and simplified dendrites in Hdh S1181A/S1201A mice compared to vehicle-treated Hdh S1181A/S1201A mice, but the protective effect of unphosphorylatable HTT was still evident: the dendrites remained significantly longer and the neuritic complexity higher in CORT-treated Hdh S1181A/S1201A mice than CORT-treated WT mice.

Preventing phosphorylation of HTT serines 1181 and 1201 in mice thus allows proliferation, survival and neuronal maturation to continue in the DG even in the presence of chronic CORT administration. We conclude that CORT-mediated effects require HTT phosphorylation at CDK5 sites.

Phospho-ablation of HTT at CDK5 sites decreases depression-related behaviors after chronic CORT treatment

We next examined whether HTT's phosphorylation status affects various stressrelated behaviors in mice (Figure 5A). We first evaluated anxiety-like behaviors. In the open field test, vehicle-treated WT and Hdh S1181A/S1201A mice spent the same amount of time in the center of the field. CORT treatment decreased the amount of time WT mice spent in the center (Two-way ANOVA, ***p˂0.001), as expected [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF], and produced a similar effect in Hdh S1181A/S1201A mice (Two-way ANOVA, **p˂0.01) (Figure S3A). The total distance travelled was similar, so neither genotype nor treatment affected motor performance (Figure S3B). In the elevated plus maze test, vehicletreated WT and Hdh S1181A/S1201A mice spent a similar amount of time in the open arms (Figure S3C). CORT reduced the time both WT (*p˂0.05) and Hdh S1181A/S1201A mice (***p˂0.001) spent in the open arms (Figure S3C). The total entries in open arms were similar for the two genotypes, confirming the absence of motor deficits (Figure S3D). Thus, phospho-ablation of HTT at CDK5 sites does not attenuate the anxiogenic effects of CORT.

To examine neurogenesis-dependent antidepressant effects of the mutation, we used the novelty suppressed feeding test (NSF), in which latency to feed indicates a depressed state [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF]. Mice were starved for 24h and then put in the presence of a food pellet situated at the center of a brightly lit open field. CORT treatment increased the latency to feed in both genotypes (WT vehicle vs CORT: Two-way ANOVA, ****p˂0.0001; Hdh S1181A/S1201A vehicle vs CORT, Two-way ANOVA, **p˂0.01) but with lower efficacy in Hdh S1181A/S1201A mice (WT CORT vs Hdh S1181A/S1201A CORT, Two-way ANOVA, ****p˂0.0001) (Figure 5B).

Unphosphorylatable HTT thus protects against CORT treatment specifically in the NSF paradigm. Since the NSF paradigm is predictive of the behavioral outcome of antidepressants requiring increased hippocampal neurogenesis [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF], our results suggest that the protective effect of phosphomutant HTT requires the maintenance of neurogenesis.

As a negative control, we tested a behavior that is associated with depressive states but is reported in most studies to be independent of neurogenesis: self-neglect (lack of grooming behavior) [START_REF] Culig | Increasing adult hippocampal neurogenesis in mice after exposure to unpredictable chronic mild stress may counteract some of the effects of stress[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF]. We used the splash test (ST), in which a 10% sucrose solution is sprayed on the mouse snout to dirty the coat, and measured the duration of subsequent grooming over a 5-min period. WT and Hdh S1181A/S1201A mice both spent less time grooming under CORT treatment (***p˂0.001 and *p˂0.05, respectively, Two-way ANOVA) (Figure 5C). Phosphoablation does not appear to alter the depressive effects of CORT in this paradigm, which supports the notion that the behavioral differences we did observe between WT and Hdh S1181A/S1201A mice are dependent on hippocampal neurogenesis.

To address this point more directly, we crossed Hdh S1181A/S1201A mice with GFAPthymidine kinase mice (Tk+) and specifically ablated neurogenesis in Tk+ mice by administering ganciclovir, as confirmed by evaluating cells positive for DCX (Figure 5D).

All mice were treated with CORT and behavior was first tested in the NSF paradigm (Figure 5E). As before (Figure 5B), Hdh S1181A/S1201A mice with preserved neurogenesis (Tk-) showed shorter latency to feed than WT (Tk-) mice (One-way ANOVA, ***p˂0.001), confirming the protective effect of the mutation against CORT treatment.

Strikingly, ablation of neurogenesis in Hdh S1181A/S1201A mice resulted in loss of their resistance to depression-like behavior (Tk-vs Tk+: One-way ANOVA, *p˂0.05). There was no difference between WT and Hdh S1181A/S1201A mice with preserved (Tk-) or ablated (Tk+) neurogenesis in grooming duration in the ST (Figure 5F).

DISCUSSION

The molecular mechanisms by which chronic stress conditions reduce BDNF levels in the hippocampus have been poorly understood. Our results dovetail nicely with previous studies showing that CDK5 is activated in several chronic stress paradigms in different brain regions, including the cortex, by increasing either CDK5 levels [START_REF] Papadopoulou | Acute and chronic stress differentially regulate cyclin-dependent kinase 5 in mouse brain: implications to glucocorticoid actions and major depression[END_REF], the number of neurons expressing CDK5 [START_REF] Bignante | Previous stress exposure enhances both anxiety-like behaviour and p35 levels in the basolateral amygdala complex: modulation by midazolam[END_REF], cleavage of the p35 activator of CDK5 into its constitute activated form p25 [START_REF] Adzic | Acute or chronic stress induce cell compartmentspecific phosphorylation of glucocorticoid receptor and alter its transcriptional activity in Wistar rat brain[END_REF][START_REF] Bavley | Cav1.2 channels mediate persistent chronic stress-induced behavioral deficits that are associated with prefrontal cortex activation of the p25/Cdk5-glucocorticoid receptor pathway[END_REF], or the localization of p35 at the cell membrane [START_REF] Zhu | Increased Cdk5/p35 activity in the dentate gyrus mediates depressive-like behaviour in rats[END_REF]. One study showed that CDK5 over-activation by p25 disrupts retrograde transport of various cargos, including signaling endosomes and mitochondria in dorsal root ganglion neurons [START_REF] Klinman | Stress-Induced CDK5 Activation Disrupts Axonal Transport via Lis1/Ndel1/Dynein[END_REF]; the authors found that hyper-activated CDK5 phosphorylates the nuclear distribution protein nudE-like 1 (Ndel1), which, in association with Lis1, increases attachment of the motor protein dynein to microtubules, diminishing organelle motility. Whether this mechanism is involved in the reduced transport of BDNF in response to CORT is unknown. If this is the case, it remains to be established whether and how the CDK5/HTT and CDK5/Ndel1/Lis1 pathways co-ordinate with each other.

Our data support the idea that precise regulation of the CDK5 pathway is required for neuronal homeostasis, as both downregulation and over-activation of CDK5 are detrimental. For instance, genetic inhibition of CDK5 impairs neurogenesis [START_REF] Jessberger | Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus[END_REF][START_REF] Lagace | Cdk5 is essential for adult hippocampal neurogenesis[END_REF], and restoration is beneficial [START_REF] Lee | Impaired neurogenesis and neurite outgrowth in an HIV-gp120 transgenic model is reversed by exercise via BDNF production and Cdk5 regulation[END_REF]. Very few studies have performed both molecular and behavioral studies, however. Zhu et al. reported that mild stress over a three-week period increases CDK5 activity [START_REF] Zhu | Increased Cdk5/p35 activity in the dentate gyrus mediates depressive-like behaviour in rats[END_REF]; the authors used a sucrose preference test to measure anhedonia. Although this report is consistent with our results, we should note that anhedonia is less associated with hippocampal neurogenesis than with impairment of the mesolimbic system [START_REF] Russo | The brain reward circuitry in mood disorders[END_REF]. Bignante et al. studied the rat amygdala in response to acute stress and found that increased CDK5/p35 activity correlated with poor performance in the elevated plus maze, while inhibiting CDK5 reversed these effects [START_REF] Bignante | Previous stress exposure enhances both anxiety-like behaviour and p35 levels in the basolateral amygdala complex: modulation by midazolam[END_REF]. Conversely, non-stressed mice in which CDK5 is depleted in the dopaminergic neurons of the ventral tegmental area responded to the open field test with anxiety-like behavior and showed depressive behaviors in the sucrose preference and novelty suppressed feeding tests [START_REF] Zhong | Cyclin-dependent kinase 5 in the ventral tegmental area regulates depression-related behaviors[END_REF].

It seems, then, that not only must CORT levels be tightly regulated, but that this regulation differs depending on the specific brain regions or neuronal groups involved.

The unphosphorylatable HTT does not interfere with CORT-induced anxiety as assessed in the open field or the elevated plus maze, suggesting that the CDK5-HTT pathway may not affect the functions of the amygdala and the mesolimbic dopaminergic pathway [START_REF] Corral-Frias | Involvement of the ventral tegmental area in a rodent model of posttraumatic stress disorder[END_REF][START_REF] Tye | Amygdala circuitry mediating reversible and bidirectional control of anxiety[END_REF]. Instead, hyper-activation of the CDK5-HTT pathway provides resilience to CORT-induced depressive-like behavior in the novelty suppressed feeding assay, an effect that is lost when neurogenesis is ablated in GFAP-Tk+/Hdh S1181A/S1201A mice. However, abolishing S1181/1201 phosphorylation did not confer complete resistance to stress in the NSF assay (Figure 5B). Also, the mutation did not completely restore decreased DCX neuronal maturation upon CORT treatment (Figures 4F to 4H). Sustained BDNF delivery from the cortical compartment may not fully compensate for the reduction of BDNF levels observed in stressed hippocampi.

Hyper-activated CDK5 may also modulate neurogenesis independently of HTT phosphorylation. For instance, CDK5 phosphorylates TrkB in dendrites of cortical neurons, which is necessary for proper BDNF-stimulated dendritic growth [START_REF] Cheung | Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons[END_REF]. The glucocorticoid receptor is another target of CDK5, and phosphorylation of the nuclear glucocorticoid receptor alters its transcriptional activity [START_REF] Adzic | Acute or chronic stress induce cell compartmentspecific phosphorylation of glucocorticoid receptor and alter its transcriptional activity in Wistar rat brain[END_REF]. Another example is the phosphorylation of the phosphodiesterase-4 by CDK5, which decreases cAMP signaling [START_REF] Plattner | The role of ventral striatal cAMP signaling in stress-induced behaviors[END_REF]: inhibiting this phosphorylation in the ventral striatum improves the performance of mice in the forced swim test [START_REF] Plattner | The role of ventral striatal cAMP signaling in stress-induced behaviors[END_REF]. Although these studies were performed in unstressed conditions, one can imagine that upon CORT treatment of Hdh S1181A/S1201A , CDK5 may still target these pathways and affect anxiety/depressive-like behavior.

Over-activation of CDK5 is also involved in several pathways participating in the neurodegenerative processes in Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [START_REF] Crews | Modulation of aberrant CDK5 signaling rescues impaired neurogenesis in models of Alzheimer's disease[END_REF][START_REF] Kawauchi | Cdk5 regulates multiple cellular events in neural development, function and disease[END_REF][START_REF] Park | Abnormal Mitochondria in a Non-human Primate Model of MPTP-induced Parkinson's Disease: Drp1 and CDK5/p25 Signaling[END_REF].

Historically, HTT has been studied primarily in its polyglutamine-expanded form, which is responsible for Huntington's disease (HD). Strikingly, HD patients suffer from anxiety and depression up to 10 years before the onset of the motor symptoms of the disease [START_REF] Perlis | Prevalence of incompletely penetrant Huntington's disease alleles among individuals with major depressive disorder[END_REF]. In mouse models of HD, anxiety/depressive-like symptoms correlate with a reduction in hippocampal neurogenesis [START_REF] Pla | Mood disorders in Huntington's disease : from behavior to cellular and molecular mechanisms[END_REF], and in humans with HD, neurogenic activity is abnormally low [START_REF] Ernst | Neurogenesis in the striatum of the adult human brain[END_REF]. We previously established that wild-type HTT contributes to several steps of adult hippocampal neurogenesis [START_REF] Ben M'barek | Huntingtin mediates anxiety/depressionrelated behaviors and hippocampal neurogenesis[END_REF][START_REF] Pla | Huntingtin acts non cell-autonomously on hippocampal neurogenesis and controls anxiety-related behaviors in adult mouse[END_REF]. We now show that HTT also participates in the response to chronic stress, suggesting that at least part of the anxiety and depression evident in HD patients may be related to modification of the normal function of HTT. The relationship among HTT, CDK5, and BDNF represents a promising avenue for understanding the regulation of BDNF trafficking, hippocampal neurogenesis and affective mood. (A) WT and Hdh S1181A/S1201A mice were treated for 4-6 weeks with CORT (A-C). (B) Novelty suppressed feeding (NSF) test: latency to feed was expressed as percentage of animals that have not eaten over 10 minutes (left) and mean latency to feed (right) (11 to 27 mice per group).

(C) Splash test: grooming duration was measured after 10% sucrose solution was sprayed on the mouse coat (18 to 29 animals per group).

(D) Ganciclovir was administrated to WT and Hdh S1181A/S1201A mice crossed with GFAP-Tk mice. All mice were treated with CORT (D-E). Middle and right: Cells counts were performed in Hdh S1181A/S1201A /Tk-and in Hdh S1181A/S1201A /Tk+ treated with CORT to quantify DCX+ cells (4 animals per group).

(E) NSF was performed in neurogenesis-ablated (Tk+) and non-ablated (Tk-) mice (6 to 9 mice per group). Latency to feed was expressed as percentage of animals that have not eaten over 10 minutes (left) and mean latency to feed (right) (6 to 9 animals per group).

(F) Splash test: grooming duration was measured after 10% sucrose solution is sprayed on the mouse coat (6 to 9 animals per group).

Error bars, SEM. *p˂0.05, **p˂0.01, ***p˂0.001, ****p˂0.0001. See also Figure S3.

STAR ★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sandrine Humbert (sandrine.humbert@univgrenoble-alpes.fr).

Materials availability

This study did not generated new unique reagents.

Data and Code Availability

This study did not generate/analyze datasets/code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

In vivo experiments were performed with 6-to 8-week-old male C57BL/6J mice, either wild-type (WT) or homozygous for the Hdh S1181A/S1201A mutation (Ben M 'barek et al., 2013), and all derived from heterozygous crosses.

Genetic suppression of hippocampal neurogenesis in WT and homozygous

Hdh S1181A/S1201A male mice was performed by crossing heterozygous Hdh S1181A/S1201A male mice with GFAP-TK heterozygous female mice that express the herpes simplex virus (HSV) TK under the control of the glial fibrillary acidic protein (GFAP) promoter (GFAP-TK mice) [START_REF] Bush | Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice[END_REF]. Mice were generated as previously described [START_REF] Mendez-David | S 47445 Produces Antidepressant-and Anxiolytic-Like Effects through Neurogenesis Dependent and Independent Mechanisms[END_REF]). An agreement (license L-O 15-2015/0) between the NIH and the Université Paris-Sud provides CESP/UMRS 1178 laboratory with the use of transgenic GFAP-TK mice (Mendez- [START_REF] Mendez-David | S 47445 Produces Antidepressant-and Anxiolytic-Like Effects through Neurogenesis Dependent and Independent Mechanisms[END_REF][START_REF] Snyder | Adult hippocampal neurogenesis buffers stress responses and depressive behaviour[END_REF]

METHOD DETAILS

Primary neuronal culture in microfluidic devices

Whole cortices and hippocampi were dissected from E15.5 WT C57BL/6J or HdhS1181A/S1201A mice embryos and re-suspended in growing medium (Neurobasal medium supplemented with 2% B27, 2 mM Glutamax, and 1% penicillin/streptomycin).

We established cortico-hippocampal neuronal networks in microfluidic devices [START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF]) coated overnight at 4°C with poly-D-lysin (0.1 mg/ml) in the upper and synaptic chambers, and with a mix of poly-D-lysin (0.1 mg/ml) and laminin (10 μg/ml) in the lower chamber. This generated a gradient of laminin reducing the number of hippocampal axons reaching the synaptic chamber [START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF].

Microchambers were washed 3 times with growing medium and placed at 37°C before neurons were plated.

First, cortical neurons were plated in the presynaptic compartment so that they can extend axons through the 500 µm-long microgrooves to the synaptic compartment.

Then, hippocampal cells were seeded into the postsynaptic compartment and extended dendrites through 75 µm-long microgrooves to the synaptic compartment. Cells were plated at a density of approximately 7000 cells/mm 2 in the microfluidic devices [START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF]. The day after plating, neurons were infected for 48h with the following lentiviruses (LV): LV.PGK.BDNF-mCh, LV.EF1α-GFP, LV.EF1α.p25-GFP (derived from plasmid #1343, Addgene) and LV.EF1α.CDK5-KD-GFP (derived from plasmid #1344, Addgene). The equivalent of 1x10 7 genomic particles were used to transfect the neurons in their respective chambers and culture dishes. Transduction efficiency has been determined in three different cultures. At DIV7, cortical neurons were fixed in 4% paraformaldehyde, rinsed in PBS, permeabilized in 0.1% Triton and incubated overnight at 4˚C with anti-GFP (1/200, Abcam, ab13970). Preparations were rinsed, incubated with Alexa Fluor® 488 goat anti-chicken (1/500, ThermoFisher, rabbit anti-CDK5 (C8, sc-173) antibody, 1:200 mouse anti-CDK5 (J3, sc-6247) antibody, 1:200 rabbit anti-p35/p25 (C19, sc-820) antibody, 1:500 rabbit anti-BDNF (N20, sc-546) antibody (all from Santa Cruz Biotechnology), 1:1000 rabbit anti-BDNF (ab108319) antibody (Abcam), 1:1000 rabbit anti-huntingtin (D7F7, #5656) antibody (Cell Signalling Technologies) and 1:5000 mouse anti-alpha tubulin (T9026, Sigma-Aldrich). HTTspecific antibodies against phospho-serine 1181 and phospho-serine 1201 were generated following immunization of rabbits with synthetized sequences PSLTNPPSL[pS]PIR (1181) and PGEQASTPM[Sp]PKKV (1201) (Covalab, Lyon, France) and were used at 1:100 in 5% bovine serum albumin-TBST for western blot.

They were validated by immunoblotting of cytoplasmic and nuclei-derived protein fractions obtained from WT and Hdh S1181A/S1201A mouse embryonic fibroblasts (Figure S1B). Membranes were washed with TBST, incubated for 1h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:5000 goat anti-rabbit IgG (H+L), from Jackson Immunoresearch; 1:5000 goat anti-mouse IgG1, SouthernBiotech), and visualized using Pico and ECL western blotting substrate (ThermoFisher Scientific) on a Chemidoc imaging system (Biorad).

Histology and immunolabeling

Mice were sacrificed between 9:00 am and 3:00 pm. Mice were anesthetized with ketamine (250 mg/ml; 100 µl/10 g of body weight, intraperitoneal injections) and intracardiacally perfused with PBS followed by cold paraformaldehyde (PFA) 4%. Brains were harvested, post-fixed for 2h in PFA 4%, cryopreserved in 30% sucrose and embedded in OCT Compound (VWR International). Brains were cryosectioned (35 µm) through the entire hippocampus and slices were kept in free-floating at -20˚C in antifreeze solution (18.75% (w/v) sucrose, 37.5% (v/v) ethylene glycol, 0.05% (w/v) sodium azide in PBS). Proliferation and survival were assessed following immunolabeling of Ki67 (anti-Ki67 antibody: M3062, Spring Biosciences) and BrdU (anti-BrdU antibody:

(#347580, BD Biosciences) positive nuclei. Immature neurons expressing doublecortin (DCX, anti-DCX antibody: sc-8066, Santa Cruz Biotechnology) were immunostained. All staining protocols were already described in previous studies [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF][START_REF] Guilloux | S 38093, a histamine H3 antagonist/inverse agonist, promotes hippocampal neurogenesis and improves context discrimination task in aged mice[END_REF][START_REF] Mendez-David | S 47445 Produces Antidepressant-and Anxiolytic-Like Effects through Neurogenesis Dependent and Independent Mechanisms[END_REF].

The maturation of newly generated neurons in the DG was examined by staining brain slices of WT and Hdh S1181A/S1201A mice to reveal BrdU and calbindin positive neurons. Briefly, brain slices were mounted onto Superfrost Plus GOLD glass slides (Thermo Scientific, K5800AMNZ72), let to dry and adhere overnight, and then rehydrated for 5 min in PBS. Antigen retrieval was achieved by incubating the slices for 10 min at 95˚C in citrate buffer pH 6 (1:10 in ultra-pure water, Zytomed Systems).

Following 3 rinses in PBS, membranes were digested for 7 minutes in 0.1 % trypsin (diluted in 0.1 M Tris pH 7.2 with 0.1% CaCl2), rinses and histones removal was achieved by incubating the slices for 30 min in 2N HCl. Slices were rinsed and blocked in 3% normal donkey serum in PBS containing 0.3% Triton-X100 for 1h at room temperature then incubated overnight at room temperature in 1:100 of mouse anti-BrdU antibody (#347580, BD Biosciences) and 1:500 rabbit anti-calbindin antibody (#13176, Cell Signaling Technologies). Slices were rinsed, incubated in 1:500 647 donkey antirabbit antibody (A31573, ThermoFisher) and 1:250 cy2 donkey anti-mouse antibody Jackson ImmunoResearch) and Hoechst 33342 (2ug/ml) in PBS for 1h.

After 3 rinses in PBS, slices were mounted in Dako mounting medium. Quantifications were performed on 1/6 of the hippocampus for BrdU+ cells; 1/12 for Ki67+ and DCX+ cells and 1/12 for calbindin/BrdU+ cells.

Immunocytochemistry

The proportion of neurons expressing reelin was evaluated in E15.5 whole cortex cultures from WT embryos. Neurons were fixed at 7DIV in 4% paraformaldehyde, rinsed in PBS, permeabilized in 0.1% Triton and incubated overnight at 4˚C with goat antireelin (1/500, AF3820, R&D Systems) and mouse anti-βIII tubulin coupled to Cy3

(1/200, C4585, Sigma-Aldrich). After 1h incubation with Alexa Fluor® 488 donkey antigoat (1/500, ThermoFisher, A11055) and Hoechst33342 (2µg/ml), preparations were rinsed and mounted in Dako mounted medium. The proportion of reelin expressing neurons was evaluated in 2 different cultures, in 2 different wells of culture within each experiment. Images were acquired on the Axio Scan.Z1 slide scanner (Colibri.7; 20X objective; Zeiss) and analyzed using the Zen software (https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html).

Quantification is provided in Supplemental Figure S1.

Sholl analysis

Dendritic complexity and ramification length were assessed using the Neurolucida software as described previously [START_REF] Guilloux | S 38093, a histamine H3 antagonist/inverse agonist, promotes hippocampal neurogenesis and improves context discrimination task in aged mice[END_REF][START_REF] Mendez-David | Rapid anxiolytic effects of a 5-HT(4) receptor agonist are mediated by a neurogenesis-independent mechanism[END_REF][START_REF] Mendez-David | S 47445 Produces Antidepressant-and Anxiolytic-Like Effects through Neurogenesis Dependent and Independent Mechanisms[END_REF].

QUANTIFICATION AND STATISTICAL ANALYSIS

For western blots, densitometry analyses of Pico and ECL signals were performed using NIH Image J software. Data are represented as mean ± SEM of at least 4 animals per condition. Protein levels are displayed as percentage standardized to α tubulin, p35 and HTT. Statistical significance was assessed using a Mann-Whitney test. In histology experiments, cell counts from at least 4 animals per group were expressed as mean ± SEM values. Statistical comparisons were made using the two-way ANOVA followed by the Tukey's multiple comparison post hoc tests. For the NSF test, a Kaplan-Meier survival analysis was applied in which the Mantel-Cox logrank test was used to evaluate differences between experimental groups. Animals that did not eat during the 10 min test period were attributed the value of 600 sec. In the NSF applied on GFAP-tk mice, statistical comparisons were made using the one-way ANOVA followed by the Tukey's

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. CDK5 mediates the decrease of cortico-hippocampal BDNF transport induced by corticosteroid through HTT phosphorylation at S1181/S1201 (A) Representative kymographs show BDNF vesicles trafficking in control and p25 expressing WT cortical axons. Graphs correspond to the corresponding kinetic analyses (at least 44 axons analyzed in 3 different cultures). (B) Representative kymographs show BDNF vesicles trafficking in control and CDK5-KD expressing WT cortical axons treated or not (with DMSO) for 3h with dexamethasone (DXM). Graphs correspond to the corresponding kinetic analyses (at least 45 axons analyzed in 3 different cultures). (C) Representative kymographs show BDNF vesicles trafficking in Hdh S1181A/S1201A cortical axons treated or not (with DMSO) for 3h with dexamethasone (DXM). Graphs correspond to the corresponding kinetic analyses (at least 66 axons analyzed in 3 different cultures).

Figure 3 .

 3 Figure 3. Huntingtin is phosphorylated at CDK5 sites upon chronic CORT treatment. (A) WT mice were treated with CORT or Vehicle for 4 to 6 weeks in their drinking water. (B-E) Whole cortical and hippocampal (Hipp.) extracts were analyzed by immunoblotting for the presence of the phosphorylated forms of HTT at CDK5 sites (P-S1181 and P-S1201), total HTT (D7F7), CDK5, p35, p25, α-tubulin, pro and mature BDNF. Histograms correspond to the quantitative evaluation of the indicated proteins (at least 4 animals). Error bars, SEM. *p˂0.05, **p˂0.01, **** p˂0.0001.

Figure 4 .

 4 Figure 4. Phosphorylation of HTT at S1181/S1201 influences hippocampal neurogenesis. (A-C) Cells counts were performed in the DG of Vehicle-and CORT-treated WT and Hdh S1181A/S1201A mice (at least 4 for animals per condition) to quantify proliferation (Ki67+ cells, A), survival (BrdU+ cells, B) and immature neurons (DCX+ cells, C). *p˂0.05, **p˂0.01, ***p˂0.001. (D) Representative images show BrdU+ surviving cells (cyan) expressing (or not) the terminal marker for granule cell differentiation calbindin (magenta) on DG sections of WT and Hdh S1181A/S1201A mice treated with Vehicle or CORT. (E) The histogram shows the quantification of the number of BrdU+ and calbindin+ cells (3 animals per condition). *p˂0.05, **p˂0.01. (F, G) Dendritic length (F) and complexity (G) were evaluated in both genotypes and treatment conditions (3 animals per condition). (H) Representative drawings of dendrites of DCX-positive neurons. *p˂0.05, **p˂0.01, ***p˂0.001, ****p<0.0001 comparing the Hdh S1181A/S1201A -vehicle group with the WT vehicle group, $ p˂0.05, $$ p˂0.01, $$$ p˂0.001, $$$$ p˂0.0001 comparing the

Figure 5 .

 5 Figure 5. Phosphoablation of HTT at CDK5 sites protects against corticosteroneinduced suppression of hippocampal neurogenesis.

  , which were developed in the laboratory of Dr. Heather Cameron of the National Institute of Mental Health (NIMH). All experimental procedures were performed in authorized establishments (Grenoble Institute of Neurosciences, INSERM U1216, license #B3851610008 and CEE26 authorization 2012-099 at Université Paris-Sud) in strict accordance with the local animal welfare committee (Comité Local Grenoble Institute Neurosciences, C2EA-04), EU guidelines (directive 2010/63/EU) and the French National Committee (2010/63) for care and use of laboratory animals.

  multiple comparison post hoc tests. Statistical significance was set to p˂0.05. All analyses were conducted using Graph pad Prism 7.0. Statistical details of experiments are indicated in the figure legends. Complete statistical analyses related to figures 1 to 5and to supplemental figures S1 to S3 are listed in Supplemental Excel tablesS1 and S2respectively.

ACKNOWLEDGMENTS

We kindly thank Caroline Benstaali, Béatrice Blot, Ayma Galland, Elodie Martin and staff of animal facilities (GIN and SFR-UMS Institut Paris Saclay Innovation Thérapeutique) for technical support; Florence Appaix, Jacques Brocard, Yasmina Saoudi of the GIN imaging facility platform (PIC-GIN) for help with image acquisitions; Vicky Brandt for helpful comments on the manuscript; Heather Cameron (NIMH/NIH) for supplying the GFAP-Tk mice (license agreement L-O 15-2015/0 between the National Institutes of Health and Université Paris-Sud). This work was supported by grants from Agence Nationale pour la Recherche (ANR-15-IDEX-02 NeuroCoG in the framework of the "Investissements d'avenir" program, S.H., F.S.; AXYON: ANR-18-CE16-0009-01, S.H., F.S.), Fondation pour la Recherche Médicale (FRM, équipes labellisées DEQ20170336752, S.H. and DEI20151234418, F.S.), Fondation pour la Recherche sur le Cerveau (FRC, S.H.), AGEMED program from INSERM (S.H., F.S.), ITMO Cancer Aviesan (Alliance Nationale Pour les Sciences de la Vie et de la Santé) in the context of the Plan cancer (slide scanner).

AUTHOR CONTRIBUTIONS

DECLARATION OF INTERESTS

The other authors declare no conflict of interest. A11039) and Hoechst33342 (2µg/ml, ThermoFisher, H3570) to counterstain nuclei, and mounted in Dako mounted medium (Agilent, S302380-2). Confocal images were acquired using an inverted confocal microscopy (LSM710, Zeiss) and analyzed using ImageJ (https://imagej.nih.gov/ij/download.html). Quantification is provided in Supplemental Figure S1.

Live-cell and confocal imaging

Live-cell recordings were acquired using an inverted microscope (Axio Observer, Zeiss) coupled to a spinning-disk confocal system (CSU-W1-T3, Yokogawa) connected to a wide field electron-multiplying CCD camera (ProEM + 1024, Princeton Instrument) and maintained at 37°C and 5% CO2. Images were taken every 200 ms for 30 s to study BDNF-mCh trafficking (×63 oil-immersion objective, 1.46 NA).

Kymograph analysis

Kymographs were generated using KymoToolBox plugin for ImageJ with a length of 100 µm (x-axis) and a total time of 30 s (y-axis). Anterograde and retrograde velocities, vesicle numbers and net flux were analyzed as previously described [START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF][START_REF] Zala | Vesicular glycolysis provides on-board energy for fast axonal transport[END_REF]. Only vesicles with a velocity above 0.12 μm/s were considered motile. For each condition, at least six chambers from three independent cultures were used and a minimum of 40 axons was analyzed.

CORT treatment to mimic stress and behavioral testing

Mice, either wild-type (WT) or homozygous for the Hdh S1181A/S1201A mutation, were treated for four to six weeks with a vehicle (0.45 % hydroxypropyl-β-cyclodextrin, Roquette, Cat#965440) or with CORT (35 μg/ml/day, dissolved in vehicle) added to their drinking water. Mice were injected with 150 mg/kg of BrdU twice per day for 3 days before the CORT treatment session (Figure 2A).

Genetic suppression of hippocampal neurogenesis in WT and homozygote

Hdh S1181A/S1201A male mice was performed by crossing heterozygous Hdh S1181A/S1201A male mice with GFAP-Tk heterozygous female mice that express the herpes simplex virus (HSV) Tk under the control of the glial fibrillary acidic protein (GFAP) promoter 20 (GFAP-Tk mice) [START_REF] Bush | Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice[END_REF]. Male mice from the four genotypes (up to six months old) were given ValGanciclovir (VGCV, Roche), the L-valyl ester of ganciclovir, from Monday to Friday for eight weeks, through their chow, at a concentration of 15 mg/kg/day to kill mitotically active cells. Mice received CORT as previously described during the last four weeks of VGCV feeding.

We evaluated anxiety-and depression-like behavior in mice using the open field (OF), elevated plus maze (EPM), sucrose splash test (ST), and novelty suppressed feeding (NSF) tests as described previously [START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF]Mendez-David et al., 2017a;[START_REF] Mendez-David | Rapid anxiolytic effects of a 5-HT(4) receptor agonist are mediated by a neurogenesis-independent mechanism[END_REF][START_REF] Mendez-David | S 47445 Produces Antidepressant-and Anxiolytic-Like Effects through Neurogenesis Dependent and Independent Mechanisms[END_REF]. Behavioral tests were conducted between 8:00 am and 12:00 pm.

Western Blot

Cortical neurons in culture were treated for 3h with DXM (5 µM), the selective inhibitor of CDK5 roscovitine (10 µM) or both drugs and harvested in protein extraction buffer (Tris-HCl 20 mM, pH7.4, NaCl 137mM, EDTA 2mM, Triton X-100 1%, supplemented with 1/100 protease and phosphatases inhibitors (Sigma-Aldrich, P8340 and P5726) and frozen at -80˚C until protein extraction. Brain samples (whole cortices and hippocampi) were obtained from mice sacrificed by quick cervical dislocation and dissection under a stereomicroscope. Brain tissues were frozen at -80˚C until protein extraction. Protein extraction of neurons was performed by pipetting up and down approximately 10 times with a P1000 Gilson. Cortices and hippocampi were dissociated using 10 strokes of the pestle of a tight-fitting Dounce homogenizer. The supernatants of both samples were collected after centrifugation at 10,000 rpm for 10 min at 4°C. Protein concentration was measured by the BCA method. Proteins (20 µg total) were treated with SDS-PAGE sample buffer [6X concentrated: 350 mM Tris, 10% (w/v) SDS, 30% (v/v) glycerol, 0.6 M DTT, 0.06% (w/v) bromophenol blue], boiled 5 min at 95°C, and loaded on 15% (for CDK5, p35, p25 and BDNF detection) and 6% (for HTT) acrylamide/bisacrylamide gels. Proteins were transferred onto PVDF (polyvinylidine difluoride) membranes, blocked with 5% bovine serum albumin and 0.5% Tween (Euromedex) in TBS (TBST) for 1h at room temperature, and probed overnight at 4˚C with the following primary antibodies diluted in 5% bovine serum albumin-TBST: 1:200