Colin Timaxian

Isabelle Raymond-Letron

Céline Bouclier

Linda Gulliver

Ludovic Le Corre

Karim Chebli

Anne Duvoid-Guillou

Patrice Mollard

Karl Balabanian

Dr Gwendal Lazennec
email: gwendal.lazennec@sys2diag.cnrs.fr

Karim Chébli

Anne Guillou

The health status alters the pituitary function and reproduction of mice in a Cxcr2 -dependent manner

Keywords: chemokine receptors, cytokines, microbiota, Cxcr2, pituitary

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Bystander chronic infections are common in rodent animal conventional facilities with a high prevalence of viruses such as mouse norovirus, parvovirus, mouse hepatitis virus, rotavirus, and bacteria such as helicobacter [START_REF] Pritchett-Corning | Contemporary prevalence of infectious agents in laboratory mice and rats[END_REF]. Due to the possible deleterious effects of such infections, this has led to a recent trend of rethinking of the health status of animal facilities and the development of Specific and Opportunistic Pathogen Free (SOPF) -or of specific pathogen free (SPF) animal facilities to limit the influence of the environment on the phenotype of mice, especially in the case of immune or inflammatory studies. In SOPF conditions, mice are devoid of both pathogens and opportunistic infections; whereas in SPF conditions, they are devoid of pathogens only. However, there is quite a debate about using pathogen-free mice as animal models, since several reports have shown that mice exposed to bystander infections better recapitulate the human immune situation than mice housed in pathogen-free conditions [START_REF] Beura | Normalizing the environment recapitulates adult human immune traits in laboratory mice[END_REF] and that infections can affect the response to vaccination [START_REF] Reese | Sequential Infection with Common Pathogens Promotes Human-like Immune Gene Expression and Altered Vaccine Response[END_REF].

Moreover, it has also been shown that transplanting C57BL/6 embryos into domestic wild-type mice trapped in horse stables better recapitulate human immune response than laboratory animals, reinforcing the importance of microbiota [START_REF] Rosshart | Laboratory mice born to wild mice have natural microbiota and model human immune responses[END_REF]. Such pathogen-free influences could also account for some of the difficulties in translating animal studies into treatments for patients. It remains that genetic alterations produced in mouse models frequently do not lead to the same phenotypes as those observed in humans with similar alterations. One relevant example is the response to infection [START_REF] Cypowyj | Immunity to infection in IL-17-deficient mice and humans[END_REF].

Infections are transmitted through different generations of animals in particular during birth, but also during co-housing and breast feeding [START_REF] Mccafferty | Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model[END_REF].

The role of microbiota is not only important in the context of immune studies (Hooper et al, 2012, Honda and[START_REF] Honda | The microbiota in adaptive immune homeostasis and disease[END_REF], but can also affect the outcome of different pathologies such as inflammatory bowel disease [START_REF] Bloom | Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease[END_REF]), Crohn's disease [START_REF] Cadwell | Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine[END_REF], atherosclerosis [START_REF] Wang | Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis[END_REF], arthritis [START_REF] Scher | Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis[END_REF], asthma [START_REF] Thorburn | Evidence that asthma is a developmental origin disease influenced by maternal diet and bacterial metabolites[END_REF] or cancer [START_REF] Roy | Microbiota: a key orchestrator of cancer therapy[END_REF]. Importantly, the genotype of mouse models does not contribute to the totality of phenotype observed and can be largely influenced by the various types of microbiota, with in some cases, a greater impact of the microbiota than the genotype on the phenotype. This has led to the concept of "host gene plus microbe" or metagenome [START_REF] Stappenbeck | Accounting for reciprocal host-microbiome interactions in experimental science[END_REF]. For these reasons, the use of SPF or SOPF husbandry can be viewed as an excellent way to normalize experiments and to limit the inter-individual or inter-housing variability and to improve the reproducibility of the results. However, factors other than microbiota can also deeply affect the phenotype of mouse models including husbandry conditions, such as temperature, light-dark cycles, diet, water, noise, hygrometry and handling of animals by care takers. Never-the-less, the microbiota is sometimes necessary to generate the phenotype. Indeed, a mouse model of Crohn's disease with mice harboring a mutation in Atg16/1 gene, showed the expected phenotype in conventional conditions but not in SPF housing [START_REF] Cadwell | Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine[END_REF][START_REF] Cadwell | A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells[END_REF]. On the other hand, bystander infections can lead to a loss of a particular phenotype, such as in some models of diabetes [START_REF] Okada | The 'hygiene hypothesis' for autoimmune and allergic diseases: an update[END_REF][START_REF] Bach | The effect of infections on susceptibility to autoimmune and allergic diseases[END_REF]. This can be complicated further by the fact that the nature of microbiota can lead to different phenotypes, as exemplified in another model of Crohn's disease with mice deficient for Nod2 [START_REF] Ramanan | Bacterial sensor Nod2 prevents inflammation of the small intestine by restricting the expansion of the commensal Bacteroides vulgatus[END_REF][START_REF] Ramanan | Helminth infection promotes colonization resistance via type 2 immunity[END_REF].

Since one of the primary effects of bystander infections will be alterations to the immune system and the inflammation process [START_REF] Tao | Making Mouse Models That Reflect Human Immune Responses[END_REF], particular attention should be paid to pro-inflammatory cytokines and in particular chemokines. Chemokines are chemotactic cytokines of 60-100 amino acids that can be divided in four subtypes (CXC, CC, C or CX3C), based on the location of cysteines in N-terminus of the protein [START_REF] Zlotnik | Chemokines: a new classification system and their role in immunity[END_REF]. Chemokines are ligands of seven trans-membrane Gi protein-coupled receptors (GPCR), signaling in particular though the phosphatidylinositol-3 kinase (PI3K)/Akt, phospholipase C (PLC)/protein kinase C (PKC) and mitogen-activated protein kinase (MAPK)/p38, Ras/Erk and Janus kinase (JAK2)/signal transducer and activator of transcription (STAT3) pathways [START_REF] Wang | Chemokine signaling in development and disease[END_REF].

Chemokines and their receptors play a major role in the trafficking of immune cells, notably during immune reaction or inflammatory events (McCully et al, 2018), but their role is not restricted to immune processes, as they have been reported to be important in a number of other physiologic or pathologic events. These include angiogenesis (Strieter et al, 2005), metabolism [START_REF] Chavey | CXC ligand 5 is an adipose-tissue derived factor that links obesity to insulin resistance[END_REF], chronic obstructive pulmonary disease (COPD) [START_REF] Henrot | Chemokines in COPD: From Implication to Therapeutic Use[END_REF], neurodegenerative disease and cancer (Lazennec and[START_REF] Lazennec | Recent discoveries concerning the tumor -mesenchymal stem cell interactions[END_REF][START_REF] Lazennec | Chemokines and chemokine receptors: new insights into cancer-related inflammation[END_REF]). Among chemokine receptors, Cxcr2, which is expressed in neutrophils and endothelial cells, appears essential in the control of angiogenesis, through the binding of ELR-motif containing chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8). ELR-motif chemokines harbor the tripeptide glutamic acid-leucine -arginine motif present in the N-terminal part of the protein [START_REF] Strieter | CXC chemokines in angiogenesis[END_REF]. Cxcr2 regulates wound healing [START_REF] Devalaraja | Delayed wound healing in CXCR2 knockout mice[END_REF], angiogenesis [START_REF] Addison | The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokineinduced angiogenic activity[END_REF], multiple sclerosis [START_REF] Liu | CXCR2-positive neutrophils are essential for cuprizone-induced demyelination: relevance to multiple sclerosis[END_REF]), Alzheimer's disease [START_REF] Tsai | The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration[END_REF], atherosclerosis [START_REF] Boisvert | Interleukin-8 and its receptor CXCR2 in atherosclerosis[END_REF], respiratory diseases (Strieter et al, 2005), resistance to infections, [START_REF] Cummings | Expression and function of the chemokine receptors CXCR1 and CXCR2 in sepsis[END_REF] and is involved in cancer [START_REF] Lazennec | Chemokines and chemokine receptors: new insights into cancer-related inflammation[END_REF][START_REF] Ali | Chemokines: novel targets for breast cancer metastasis[END_REF][START_REF] Freund | IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells[END_REF][START_REF] Bieche | CXC chemokines located in the 4q21 region are up-regulated in breast cancer[END_REF]. Cxcr2 knock-out animals exhibit splenomegaly due to an increased number of metamyelocytes and neutrophils, and impairment in the recruitment of neutrophils during acute inflammatory conditions [START_REF] Cacalano | Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog[END_REF].

Here, we report that the action of microbiota on mouse phenotype is dependent on the absence of Cxcr2 protein. In the absence of Cxcr2, mice are clearly affected by the presence of pathogens. However, in the absence of pathogens, Cxcr2 knock-out (KO) mice display a similar external phenotype to that of Wildtype (WT) mice in terms of their ability to reproduce and their gross appearance [START_REF] Cacalano | Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog[END_REF][START_REF] Broxmeyer | Involvement of Interleukin (IL) 8 receptor in negative regulation of myeloid progenitor cells in vivo: evidence from mice lacking the murine IL-8 receptor homologue[END_REF]. By contrast, in conditions of bystander infections, Cxcr2 null mice exhibit an impaired reproductive ability and reduced development of reproductive organs. Using mammary gland and ovary transplant experiments, we show that reproductive function can be restored to Cxcr2 KO mice in a WT context, despite the presence of pathogens. We also show that the absence of Cxcr2, not only leads to susceptibility to infection, it additionally leads to reproductive defects due to major impairment of pituitary function controlling the production of pituitary hormones. This study therefore reveals a novel role for the chemokine receptor Cxcr2 in pituitary physiology, that has been discovered in the context of microbiota infections. This has never been reported for any chemokine receptor.

RESULTS

We have been working for a long time on Cxcr2 ligands [START_REF] Freund | IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells[END_REF][START_REF] Bieche | CXC chemokines located in the 4q21 region are up-regulated in breast cancer[END_REF][START_REF] Freund | Mechanisms underlying differential expression of interleukin-8 in breast cancer cells[END_REF]) and we wished to use Cxcr2 KO animals to analyze its role in vivo. Our study started with the serendipitous finding that Cxcr2 KO animals had distinct breeding abilities in conventional or SOPF animal facilities. In order to evaluate the possible action of microbiota mouse phenotype in the context of Cxcr2 deficiency, mice were housed either in a SOPF animal facility in sterile conditions or in a conventional animal facility with possible bystander infections. In SOPF conditions, Cxcr2 KO animals displayed the same breeding ability as WT animals, confirming prior work of Cacalano [START_REF] Cacalano | Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog[END_REF]) (Fig. 1A). On the contrary, after transfer of SOPF animals in conventional conditions, we observed that after several generations in conventional conditions, Cxcr2 KO animals lost their breeding ability. Both males and females were infertile (Fig. 1B), even when mated with WT animals. Of particular note, the product of such mating, Cxcr2 heterozygous animals, were much less affected by these conditions and could breed nearly normally (although with a delayed time for successful breeding) and generate Cxcr2 KO animals (data not shown). Moreover, Cxcr2 KO females of conventional conditions exhibited a smaller weight than WT animals (Fig. 1D), which was not the case in SOPF conditions (Fig. 1C). Screening of bystander infections of animals housed in conventional conditions showed the presence of mouse norovirus, helicobacter and Entamoeba SP (supplemental Fig. 1). We hypothesized that these infections were responsible for the observed phenotype as rederivating conventional animals to remove pathogens led to animals with full reproduction ability. Moreover, this combination of pathogens was not specific for the phenotype observed, as housing Cxcr2 KO animals in other conventional facilities with another set of pathogens (including mouse norovirus, mouse hepatitis virus, other strains of helicobacter or pinworms) led to the same results (supplemental Fig. 1).

To understand why Cxcr2 KO animals were infertile in conventional conditions, we decided to focus on females. We first performed vaginal smears of WT and KO animals of conventional conditions (Fig. 2A). This showed that WT animals displayed a classical cycling with proestrus, estrus, metestrus and diestrus. On the other hand, KO animals displayed mixed populations of cells, with no real homology to any steps of estrus cycling, suggesting that the mice were not cycling. In SOPF conditions, WT and KO animals displayed a normal estrus cycle (Supplemental Fig. 2). To assess the functionality of the ovary, we analyzed the ovaries of WT and KO animals from conventional or SOPF conditions. We observed that in SOPF conditions, both WT and KO animals displayed a normal histology of the ovary, with all stages of follicles maturation as well as the presence of multiple corpora lutea, suggesting that the mice were able to ovulate (Fig. 2B, upper panel). On the contrary, in conventional conditions, while the ovary of the WT animals appeared to have completely normal histologic appearance, ovaries from KO animals displayed a large number of follicles in all stages of development including atresia, but did not exhibit any corpus luteum, suggesting that KO Cxcr2 mice could not ovulate (Fig. 2B, lower panel).

We next looked at other reproductive organs including uterus and mammary gland. The uterus of WT and KO animals in SOPF conditions appeared normal and of similar size (Fig. 3A, upper panel). In conventional conditions, the uterus of KO animals was smaller in diameter compared to WT animals and was in a rest status, whereas WT uterus was cycling (Fig. 3A lower panel). Maximum uterine thickness and the external uterine diameter were approximately 4-fold reduced in KO animals compared to WT animals (Supplemental Fig. 3A). Whereas uteri of WT mice had well defined layers, uterine layers were less discernable in KO animals, appearing compressed and very cellular. KO mice also showed a loss of the normal convoluted appearance of the uterine luminal epithelium, assuming a more linear profile. The endometrium thickness, the luminal epithelium thickness as well as the number of glandular lumen were also decreased in KO animals, suggesting a non cycling uterus (Supplemental Fig. 3B).

We also observed an altered morphology of the mammary gland in KO animals in conventional conditions.

Whole mount experiments showed a complete branching in WT animals whereas the mammary gland of KO animals displayed a rudimentary branching (Fig. 3B). Mammary glands of KO appeared to have a significant reduction of the numbers of glandular (ductal) profiles (Supplemental Fig. 3C, lower panel).

Epithelial cells lining ducts in KO mice often appeared haphazardly arranged and the mammary gland than its WT counterpart. The situation appeared different in the mammary gland of SOPF animals with a similar branching in WT and KO animals (Supplemental Fig. 3C, upper panel).

In order to understand the reasons for the reproductive defects in KO animals housed in conventional conditions, we decided to first compare the transcriptomic profiles of the mammary gland of WT and KO animals in SOPF with those in conventional conditions by RNAseq. Principal component analysis (PCA) of RNAseq showed that mammary glands of WT and KO animals were close to each other, whereas the one of WT and KO conventional animals were more widely distributed (Supplemental Fig. 4). We observed that the transcriptome of the mammary gland of KO animals was much more altered in conventional, as compared to SOPF conditions, with approximately 10-fold more genes up-regulated in WT animals versus KO animals (Fig. 4A). Among the genes up-regulated in the mammary gland of WT compared to KO animals, only 12 were common between conventional and SOPF conditions (Fig. 4B), and only 4 were common for down-regulated genes. Gene ontology (GO) analysis showed that the common down-regulated genes were essentially related to leukocyte chemotaxis and migration, and host defense (Fig. 4C). In SOPF conditions, the genes down-regulated in the mammary gland of KO animals were essentially those linked to muscle development and differentiation (Fig. 4D and Table 1), whereas the genes up-regulated in KO mice were related to granulocyte migration and leukocyte aggregation/adhesion (Fig. 4E and Table 2). We next focused on the major alterations of the transcriptome of the mammary gland of KO animals in conventional conditions. Gene ontology analysis showed that most of the genes down-regulated in the mammary gland of KO animals were related to three major biological processes (Table 3): mammary gland development and differentiation (Fig. 5A), epithelial cell proliferation (Fig. 5B) and Wnt signaling (Fig. 5C). On the other hand, genes up-regulated in the mammary gland of KO animals in conventional conditions (Table 4) were involved in leukocyte migration (Fig. 5D) and muscle function (Fig. 5E). The RNAseq data were validated by real time PCR on a subset of representative genes of the different GO identified or known to play a role in mammary gland physiology. We observed a common up-regulation of S100a8, S100a9, Mmp8 and Ngp genes related to chemotaxis in the mammary gland of KO animals in conventional and SOPF conditions (Fig. 6). On the other hand, Areg, lactoferrin, CXCL15, Elf5, Sox10, Ido1, Wnt2, Prlr, Krt15 and Gata3 were down-regulated only in the mammary gland of KO animals in conventional conditions (Fig. 6). Of particular note, Areg, Lactoferrin, Wnt2, Prlr, Elf5, and Gata3 are genes known to be critical for development of the mammary gland according to the studies performed with KO animals for these genes (Kelly et We also analyzed the differences in the ovary of WT and KO animals in conventional conditions, by looking at some key genes known to play a role in ovary function. We report a decrease in the expression of Akrc18, Cyp19, Hsd3b2, Prlr and lactoferrin genes in the ovary of KO animals, whereas AR expression was strongly induced (Fig. 7A). Akr1c18 encodes 20-hydroxysteroid dehydrogenase, a progesterone metabolizing enzyme [START_REF] Piekorz | Regulation of progesterone levels during pregnancy and parturition by signal transducer and activator of transcription 5 and 20alpha-hydroxysteroid dehydrogenase[END_REF]. Cyp19 or estrogen synthase is an aromatase of the P450 family involved in, in particular, the aromatization of androgens to estrogens [START_REF] Rosenfeld | Estrogen receptor-and aromatase-deficient mice provide insight into the roles of estrogen within the ovary and uterus[END_REF]. Hsd3b2 encodes hydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroid delta-isomerase 2, which is involved the conversion of 5-ene-3-hydroxysteroids to 4-ene-3-ketosteroid, an essential step in the biosynthesis of progesterone and estrogens in the ovary [START_REF] Payne | The murine 3 beta-hydroxysteroid dehydrogenase multigene family: structure, function and tissue-specific expression[END_REF]. Interestingly, many of these enzymes are regulated by prolactin, and prolactin receptor (Prlr) is critical (Stocco et al, 2007, Bachelot and[START_REF] Bachelot | Corpus luteum development: lessons from genetic models in mice[END_REF]. Androgen receptor (Ar) also plays a critical role in ovary function, and Ar KO leads to premature ovarian failure [START_REF] Shiina | Premature ovarian failure in androgen receptor-deficient mice[END_REF][START_REF] Walters | Role of androgens in normal and pathological ovarian function[END_REF]. The alteration of these key regulatory genes in the ovary suggested to us, a possible impairment of hormone production. We thus measured progesterone and estradiol serum levels in WT and KO animals in conventional conditions. In agreement with the absence of corpus luteum in KO ovaries, we observed a decrease in progesterone levels relative to WT (Fig. 7B). On the other hand, estradiol levels were increased. This led us to hypothesize that an alteration of the hormonal context could explain the reproductive defects observed in KO animals housed under conventional conditions. To test this, we performed ovary transplantation experiments (Fig. 7C). WT mice were ovariectomized and a WT or KO ovary was reimplanted within the oviduct bursa [START_REF] Behringer | Mouse Ovary Transplantation[END_REF]. We observed that both WT and KO transplanted ovaries were able to display a normal phenotype with the presence of corpora lutea (Fig. 7C). Moreover, when transplanted females were bred, they were able to give birth (Fig. 7C). To confirm the role of the hormonal environment in the KO defects, we also performed mammary gland transplantation (Fig. 7D). The mammary fat pads of young WT female mice were cleared of all epithelial structures and either re-implanted with WT or KO mammary gland fragments or left untreated. Control mammary glands without transplant did not develop any ductal branching, whereas both WT and KO transplants could fully restore a functional mammary gland (Fig. 7D). Together, these data suggest that the hormonal environment of WT animals is sufficient to enable the KO ovary and mammary gland to be functional. As steroid hormone production is controlled by the pituitary, we assessed the serum levels of the pituitary hormones FSH, LH, PRL and GH in WT and KO animals in conventional conditions. We report that the four pituitary hormones tested displayed a clear decrease in KO animals (Fig. 8A), suggesting major defects in the pituitary function of KO animals housed under conventional conditions. We did not measure the hormone levels of transplanted animals (Fig. 7C), as these mice were used for breeding and could not be compared with virgin animals.

To understand at the molecular level, the reasons for the pituitary dysfunction in KO animals, we performed an RNAseq analysis of pituitary glands from WT and KO animals in SOPF and conventional conditions (Fig. 8B). Strikingly, very little difference was observed between the pituitaries of WT and KO animals in SOPF conditions. On the other hand, more than 850 genes were either up-regulated or downregulated in the pituitary of KO animals in conventional conditions (Fig. 8B). Principal component analysis (PCA) of RNAseq showed that pituitary of WT and KO animals were close to each other, whereas the one of WT and KO conventional animals were very different (Supplemental Fig. 4). According to gene ontology analysis, the up-regulated pathways in the KO pituitaries were related to immune cell activation (in particular lymphocyte), leukocyte adhesion, and neutrophil motility and extravasation (Fig. 8C and Table 5).

On the other hand, down-regulated genes involving biological processes included those involved in ion transport and synaptic function (Fig. 8D and Table 6), as well as control of ovarian function (Fig. 8E and Table 6). To validate these data, we analyzed the expression of a set of genes representative of the different GO mentioned above by real-time PCR on a larger number of animals (Fig. 9). Elane, S100a8, S100a9, Mpo, Ngp, MMP8, Ltf and Serpina3an were strongly up-regulated in the pituitary of KO animals in conventional conditions and modestly or not regulated at all, in the pituitary of KO animals in SOPF conditions. Elane, S100a8, S100a9 are involved in migration, adhesion, and immune response. Mpo, Ngp, then, morphometry, using multiple histological sections in the present study, identified alterations to the uteri of Cxcr2 KO animals housed conventionally, with global reductions in uterine size , as well as a decreases in endometrial thickness and the number of glands present, suggesting uterus in arrest not cycling under hormonal stimulation.

The mammary glands of Cxcr2 KO females housed in conventional facilities were also minimally developed, harboring a phenotype close to that of juvenile animals with a rudimentary duct branching. In contrast, in SOPF conditions, mammary gland development appeared similar to that of WT animals. Interestingly, even in aged animals, no further development of the mammary gland could be observed in Cxcr2 KO females housed in conventional facilities (data not shown). At the molecular level, the mammary gland of Cxcr2 KO animals showed a clear down-regulation of the expression of genes involved in mammary gland development and differentiation, epithelial cell proliferation, wound healing and Wnt signaling (including lactoferrin, Gata3, cyclin D1, Erbb2, Elf5, Epcam, Prlr, Krt15, Epcam, Wnt4, Wnt5b). For a number of the down-regulated genes, invalidation studies in mice have shown that these genes ( Prlr, cyclin D1, Erbb2, Elf5 ) are crucial for mammary gland development (Zhou, et al The collection of evidence gathered herein, for possible hormonal perturbations affecting Cxcr2 KO animals housed in conventional conditions; including mammary, ovary and uterus histologic changes and molecular defects, led us to evaluate steroid hormone levels in these animals. We observed in particular, a decrease in serum progesterone levels, that could reflect the absence of corpora lutea in the ovary of these animals, the main site of production of progesterone.

To demonstrate the role of the hormonal environment in the phenotype observed for Cxcr2 KO animals in conventional conditions, we decided to perform ovarian transplantation experiments. By transplanting the ovary of Cxcr2 KO animals into the ovarian bursa of ovariectomized WT animals, we observed that KO ovaries could display a normal histology, close to WT ovaries, with the presence of corpora lutea, a sign of successive ovulations. Moreover, the transplanted animals had their fertility restored and were able to give birth to viable mice. Similarly, transplantation of the mammary glands of Cxcr2 KO animals into a WT context also led to the development of normal gland development with correct ductal branching. Taken together, this confirms that the hormonal context of WT animals is sufficient to restore a correct function and development of the Cxcr2 KO ovary and mammary gland. As the function of the ovary, the uterus and the mammary gland are tightly controlled by steroid hormones and at a higher level, by pituitary hormones, we measured the circulating levels of FSH, LH, GH and PRL. The levels of these four pituitary hormones were markedly decreased in Cxcr2 KO animals housed in conventional conditions compared to WT animals, suggesting an alteration to pituitary function. Treating the KO animals with FSH and LH to restore fertility could be interesting issue, but it is likely that the timing will be critical and difficult to assess due to the lack of cycling of these animals.

Transcriptomic and gene ontology analysis of the pituitary of WT and Cxcr2 KO animals revealed as expected, a down-regulation of genes involved in the control of circadian rhythm, ovulation control, and gonad development; which could account for dysregulation of pituitary hormones. This includes genes such as Esr1 (estrogen receptor alpha), Pgr (Progesterone receptor), RMB4 (required for the translational activation of PER1 mRNA in response to circadian clock) [START_REF] Markus | RBM4: a multifunctional RNA-binding protein[END_REF], Crebbp (CREB-Binding Protein, involved in circadian clock) [START_REF] Rexach | Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory formation[END_REF], Foxo3 (a regulator of circadian clock) [START_REF] Chaves | Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription[END_REF]. Moreover, one could expect behavior alterations of Cxcr2 KO animals based on the down-regulation of a number of genes controlling behavior, including for instance Crhbp (Corticotropin Releasing Hormone Binding Protein) [START_REF] Ketchesin | Corticotropin-releasing hormone-binding protein and stress: from invertebrates to humans[END_REF], Oprk1 (opioid receptor, kappa 1) [START_REF] Loh | Antagonizing the different stages of kappa opioid receptor activation selectively and independently attenuates acquisition and consolidation of associative memories[END_REF] or Vip of Cxcr2 in central nervous system are poorly understood. On the other hand, a few reports have suggested a potential role for Cxcr2 in controlling the effects of microbiota. Indeed, it was shown that microbiota promotes the recruitment of Cxcr2-positive neutrophils that can protect from amebic colitis [START_REF] Watanabe | Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis[END_REF]. Finally, so far, no direct evidence of a role of Cxcr2 in the control of reproduction has been reported. Some reports suggest that Cxcr2 might contribute to the development of preeclampsia [START_REF] Wu | CXCR2 is decreased in preeclamptic placentas and promotes human trophoblast invasion through the Akt signaling pathway[END_REF][START_REF] Chen | Association between polymorphisms in CXCR2 gene and preeclampsia[END_REF] and in the control pregnancy tolerance through neutrophils [START_REF] Kang | CXCR2-Mediated Granulocytic Myeloid-Derived Suppressor Cells' Functional Characterization and Their Role in Maternal Fetal Interface[END_REF].

Additionally, loss of Cxcr2 ligand has been shown to result in premature senescence in placenta-derived mesenchymal stem cells [START_REF] Li | Knockdown of IL-8 Provoked Premature Senescence of Placenta-Derived Mesenchymal Stem Cells[END_REF].

In summary, our results highlight a unique unexplored role for Cxcr2 in pituitary and reproductive function that is dependent on chronic infections (summarized in Fig. 10). These findings reinforce an imperative; that in order to be translationally relevant, the use of animal models to understand disease mechanisms must take in account animal husbandry conditions and disease phenotyping requiring the absence or presence of chronic infections. Gene Ontology analysis of Biological Process of down-regulated genes in KO mammary glands of SOPF are mostly related to muscle function. Black arrows mean "is a". Blue arrows mean is "part of". E. Gene Ontology analysis of Biological Process of up-regulated genes in KO mammary glands of SOPF are mostly related to granulocyte chemotaxis (left panel) and to neutrophil aggregation (right panel).

LEGENDS TO FIGURES

RNA extraction and reverse transcriptase, quantitative PCR

Total RNA was isolated using TRIzol reagent (ThermoFisher), as described by the manufacturer. Reverse transcription was performed with 1µg of total RNA using random primers and with M-MLV enzyme (ThermoFisher). Real time quantitative PCR was realized with SYBR green Master Mix (Roche), on a Light Cycler 480 instrument (Roche) as previously described [START_REF] Wang | Chemokine signaling in development and disease[END_REF]. Ribosomal protein S9 (rS9) and GAPDH were used as an internal control. The sequence of the primers used in this study is indicated in Supplemental Table 1. Results are expressed as N-fold differences in target gene expression relative to the internal control gene and termed "mRNA expression", were determined as mRNA expression = 2 -Ctsample , where the Ct value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the average of the internal control genes. Target genes were considered to be non-detectable when the Ct value was above 35.

RNA-seq data processing

RNA integrity and quality was verified using RNA ScreenTape kit and Tapestation 2200 apparatus from AGILENT. cDNA libraries were synthesized using SureSelect-Strand-Specific mRNA library prep (Agilent).

Library quality was checked on Tapestation 2200 apparatus from AGILENT with DNA 1000 ScreenTape.

Samples were sequenced on NextSeq 500 (Illumina) with an average sequencing depth of 30 millions of paired-end reads. Length of the reads was 75 bp. Each 12 Plex Samples was sequenced on one HighOutput FlowCell (2*400Millions of 75bases reads). Raw sequencing data was quality-controlled with the FastQC program. Low quality reads were trimmed or removed using Trimmomatic (minimum length: 40 bp).

Reads were aligned to the mouse reference genome (mm10 build) with the TopHat2 tool (option for no multihits). Mapping results were quality-checked using RNA-SeQC. Gene counts were obtained by read counting software featureCounts. Normalization and differential analysis were performed with the DESeq2 package with Benjamini-Hochberg FDR multiple testing correction (p corr < 0.05; 1.5-fold or higher change) comparing WT and KO animals. Clustering analysis was performed with Genesis software [START_REF] Sturn | Genesis: cluster analysis of microarray data[END_REF]. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF] and are accessible through GEO Series accession number GSE144231 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144231).

Hormone assessment

Whole sera were used to measure the levels of estradiol and progesterone by Elisa with specific kits from Abnova. The levels of LH, FSH, GH and PRL were assessed by luminex with Mouse Pituitary Magnetic Bead Panel (Merck Millipore).

Ovary and mammary transplantations

For ovary transplantations, WT or KO ovaries were obtained from 10 week-old donor mice. WT recipients were anesthetized with isoflurane and ovariectomized. One donor ovary was implanted in the recipient's ovarian bursa. After peritoneal and skin suture, recipients were allowed to recover for 2 weeks. Ovaries were then analyzed at that stage or recipient mice were mated with WT males to evaluate fertility. For mammary gland transplantation, 12 week-old WT and KO donor mice were used to obtain mammary glands. Small pieces of donor glands were implanted in the cleared mammary fat pad of one gland of 2.7 week old WT mice under general anesthesia. The contra-lateral mammary gland was cleared and kept as a control for clearing. Recipient mice were sacrificed at 12 weeks and the transplanted mammary glands were analyzed by whole mount.

Statistics

  ., 2005, Bole-Feysot et al, 1998, Howlin et al, 2006).The Wnt pathway plays a pivotal role in orchestrating proper mammary gland development and maintenance (Jarde and Dale, 2012), and this pathway was impaired in Cxcr2 KO animals. The Wnt pathway is a major paracrine mediator of hormonal action, through Wnt4 and Areg pathways in particular (Ciarloni et al, 2007, Brisken et al, 2000), both of which are down-regulated in the mammary glands of Cxcr2 KO animals housed conventionally. The general scheme of Wnt action in the mammary gland is based on the activation of estrogen receptor alpha (ER-positive luminal cells, which in turn are stimulated by steroid hormones to release Wnt ligands that will act directly or indirectly on the myoepithelial compartment (Jarde and Dale, 2012).

Fig. 1 :

 1 Fig.1: Husbandry in conventional housing conditions alters the reproduction of Cxcr2 KO animals

Fig. 2 :

 2 Fig. 2: Cxcr2 KO animals in conventional housing conditions exhibit cycle defects and altered ovary

Fig. 3 :

 3 Fig. 3: The uterus and mammary gland of Cxcr2 KO animals show defects in conventional conditions

Fig. 4 :

 4 Fig. 4: Differential gene expression in WT and KO mammary glands is more pronounced in conventional

Fig. 5 :

 5 Fig. 5: Cxcr2 KO affects mammary gland function in conventional conditions

Fig. 6 :

 6 Fig. 6: Cxcr2 KO affects mammary gland transcriptome in conventional conditions

Fig. 7 :

 7 Fig. 7: Cxcr2 KO display an alteration of hormonal function in conventional conditions. A. Measure of

Fig. 8 :

 8 Fig. 8: Circulating pituitary hormones and transcriptome in the pituitary of KO animals are drastically

Fig. 9 :

 9 Fig. 9: Gene expression in the pituitary of KO animals in conventional housing conditions

Fig. 10 .

 10 Fig. 10. General scheme of events occurring in Cxcr2 KO mice under pathogen infection.

  al, 2002, Luetteke et al, 1999, Kouros-Mehr et al, 2006, Zhou et al, 2005, Watson and Khaled, 2008).

Table 1 .

 1 Most enriched pathways for genes down-regulated in mammary gland of KO versus WT SOPF animals

Table 2 .

 2 Most enriched pathways for genes up-regulated in mammary gland of KO versus WT SOPF animals

Table 3 .

 3 Most enriched pathways for genes down-regulated in mammary gland of KO versus WT conventional animals

Table 4 .

 4 Most enriched pathways for genes up-regulated in mammary gland of KO versus WT conventional animals

Table 5 .

 5 Most enriched pathways for genes up-regulated in pituitary gland of Cxcr2-KO versus Cxcr2-WT conventional animals

Table 6 .

 6 Most enriched pathways for genes down-regulated in pituitary of KO versus WT conventional animals Mammary glands were spread on glass slides, fixed in Carnoy's fixative for 4 hours. After rehydration, the glands were stained with carmine alum stain overnight. Glands were then cleared in xylene for several weeks to eliminate the fat.

ACKNOWLEDGEMENTS

This work was supported by ARC Foundation and la Ligue contre le Cancer to GL and by an ANR grant 17-CE14-0019 to K.B. We thank N. Binart, A. Richmond and M. Gary-Bobo for useful discussion and help in the experiments and M/L/ Loyalle and A. Pulido their help in mouse husbandry. We are grateful to J. Stingl for training in mammary gland transplantation. We acknowledge the PCEA, RAM, MRI and Setiph facilities in Montpellier. We are grateful to Institut du cerveau et de la moëlle épinière in Paris for RNAseq experiments. We thank CECEMA-UM animal facility for housing and technical help. We are grateful to all master students who have contributed to this work.

DATA AVAILABILITY

The datasets discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF] and are accessible through GEO Series accession number GSE144231 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144231).

Ltf and Serpina3an are contributing to migration and immune response. In contrast, Prl, Crhbp, Akr1c14, Vip and Rln were all down-regulated in the pituitary of conventionally housed KO animals but not in SOPF conditions (Fig. 9). Prl, Crhbp and Vip are involved in ion transport, behavior and reproduction. Reln and Crhbp play a role in ion transport, synapse function and behavior.

DISCUSSION

In this study, we investigated the effects of bystander infections on the physiologic role of the chemokine receptor Cxcr2 using Cxcr2-null mice. Our results have demonstrated that when exposed to common infections found in animal facilities, Cxcr2 knock-out mice, but not WT mice, exhibit reproductive defects.

Of particular note, CXCR2 ligand levels were not altered in the mammary gland and the pituitary gland of WT animals in conventional conditions compared to SOPF conditions, according to RNAseq analysis.

Homozygous males and females were both sub-fertile and females displayed alterations to their secondary sex organs. The first observation accounting for this sub-fertility was that female Cxcr2 KO mice housed in conventional conditions were not able to cycle normally. None of the characteristic phases of the estrous cycle could be identified in these animals by vaginal smear, with mixed populations of vaginal cells suggesting a defect in ovarian function. Microscopic observation of ovaries from Cxcr2 KO animals housed in conventional conditions showed reductions of the size of the ovary and an absence of corpora lutea, whereas in SOPF conditions, both WT and Cxcr2 KO ovaries showed normal histology with all stages of follicle development and corpora lutea present. Interestingly, a number of genes involved in ovarian function and production of steroid hormones (Akr1c18, Cyp19, Hsd3b2, Prlr, lactoferrin, Ar) were downregulated in the ovary of Cxcr2 KO animals in conventional conditions. Akr1c18 (20alpha-hydroxysteroid dehydrogenase), which catabolizes progesterone into 20alpha-dihydroprogesterone (inactive steroid) is necessary for the maintenance of pregnancy [START_REF] Choi | Involvement of 20alpha-hydroxysteroid dehydrogenase in the maintenance of pregnancy in mice[END_REF] and knock out of this gene leads to longer duration of estrous cycle and a reduced number of pups [START_REF] Ishida | Reproductive phenotypes in mice with targeted disruption of the 20alpha-hydroxysteroid dehydrogenase gene[END_REF]. Knocking down Cyp19 (Aromatase P450) has been shown to lead to mice lacking corpus luteum in ovary, accompanied by total infertility [START_REF] Toda | Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol[END_REF]. Hsd3b2 (hydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroid deltaisomerase 2) plays a crucial role in the biosynthesis of many steroids [START_REF] Chapman | Mitochondrial 3 beta-hydroxysteroid dehydrogenase (HSD) is essential for the synthesis of progesterone by corpora lutea: an hypothesis[END_REF]. Prlr (prolactin receptor) KO animals are completely infertile with irregular estrous cycles [START_REF] Horseman | Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene[END_REF].

Lactotransferrin , present in the reproductive tracts of rodents, can regulate secretory function and plays a role in fertilization [START_REF] Yanaihara | High concentrations of lactoferrin in the follicular fluid correlate with embryo quality during in vitro fertilization cycles[END_REF]. Ar (androgen receptor) KO mice show a marked reduction in follicular maturation at maturity, with fewer corpora lutea in their ovaries [START_REF] Hu | Subfertility and defective folliculogenesis in female mice lacking androgen receptor[END_REF]. Not surprisingly (vasoactive intestinal polypeptide) [START_REF] Hill | Vasoactive intestinal peptide in neurodevelopmental disorders: therapeutic potential[END_REF]. The dysfunction of the Cxcr2 KO pituitary involves presumably defects in synapse function, as well as ion transport. Indeed, we report a down-regulation of a number of genes involved in calcium, sodium and potassium transport or in synaptic transmission such as Vip, Oprk1, Kcnb2 (potassium voltage gated channel, Shab-related subfamily, member 2) or Trpc6 (transient receptor potential cation channel, subfamily C, member 6), Cacna1g (calcium channel, voltage-dependent, T type, alpha 1G subunit). Synaptic alteration includes anterograde trans-synaptic signaling, long-term synaptic potentiation and synaptic plasticity and could have major effects on neuronal connections. In addition to down-regulation of the pathways mentioned above, other pathways appeared up-regulated in the pituitary of Cxcr2 KO animals. This includes in particular aggregation and adhesion of leukocytes as well as up-regulation of chemotaxis, extravasation, tethering or rolling and also leukocyte activation, proliferation and differentiation. When comparing our RNAseq results with signatures for B lymphocytes, T lymphocytes, macrophages and neutrophils from Nirmal et al. [START_REF] Nirmal | Immune Cell Gene Signatures for Profiling the Microenvironment of Solid Tumors[END_REF]), it appears, that there is a T lymphocyte enrichment (21.%) and to a lesser extent of B lymphocytes (13.5%), macrophages (12.8%) and neutrophils (6.4%) (Supplemental table 2). This possible infiltration of T and B lymphocytes in the pituitary could be reminiscent of Autoimmune Hypophysitis (AH), which is an inflammatory disease of the pituitary gland, and particularly in relation to lymphocytic hypophysitis [START_REF] Bellastella | Revisitation of autoimmune hypophysitis: knowledge and uncertainties on pathophysiological and clinical aspects[END_REF]. AH can lead to atrophy of the pituitary and to hypo-pituitrism, which is in agreement with what we observed for Cxcr2 KO animals in conventional conditions. Functional disturbances observed in humans with AH include headaches, visual disturbances, hypothyroidism, hypogonadism and an inability to lactate [START_REF] Caturegli | Autoimmune hypophysitis[END_REF].

Whereas multiple links between gut microbiota and the hypothalamic-pituitary axis have been reported [START_REF] Appleton | The Gut-Brain Axis: Influence of Microbiota on Mood and Mental Health[END_REF], our results constitute the very first evidence for the involvement of a chemokine receptor, Cxcr2, in the control of pituitary function and the reproductive system and that this phenomenon is dependent on microbiota (Fig. 10). Although there have to this time been no reports of any action of Cxcr2 in pituitary function, Cxcr2 has been shown to be important for the positioning of oligodendrocyte precursors in the spinal cord [START_REF] Tsai | The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration[END_REF] and in myelin repair (Liu, et al., 2010), but overall the effects

EXPERIMENTAL PROCEDURES Animal Models and housing

All animal experiments conform to our animal protocols that were reviewed and approved by the Institutional Animal Care and Use Committee. Cxcr2-/-mice [START_REF] Cacalano | Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog[END_REF] obtained from the Jackson Laboratory were in BALB/c genetic background as well as their littermate control (WT) mice. Mice were either housed in a SOPF facility or transferred to a conventional animal facility. To favor the acquisition of microbiota from conventional facility to the SOPF animals which were transferred to conventional conditions, the bedding of conventional animals was added to the one of transferred SOPF animals during the first weeks. The phenotype of the animals housed in the conventional facility was first analyzed following at least one or two generations of breeding to enable the establishment of bystander infections in all animals. Due to the reduced fertility of Cxcr2-/-animals in conventional conditions, homozygous animals were obtained by crossing heterozygous animals.

Pathogen screening

The presence of potential infections was monitored by Charles River Research Animal Diagnostic Services on whole animals or body swabs, oral swabs and feces of the animals according to PRIA instructions. The main bystander infections observed are shown in Supplementary Figure 1.

Analysis of estrus cycle

Vaginal smears of WT and KO mice were performed in NaCl 9/1000, fixed in methanol and then stained with Giemsa dye. Slides were scanned with a nanozoomer (Hamamatsu).

Whole mounts of mammary glands

Statistical analyses were carried out using unpaired Mann-Whitney test or when indicated Fisher exact test or Kruskall-Wallis test followed by Dunn's multiple comparison. To assess biological interpretation of the most differentially expressed genes, Gene ontology (GO) enrichment analysis was performed using the DAVID bioinformatics resources (https://david.ncifcrf.gov, Version 6.8).

COMPETING OF INTERESTS

The authors declare that they have no conflict of interest to disclose.

AUTHORS CONTRIBUTIONS

CT, IRL, CB, LG, LLC, KC, AG performed experiments. KC, PM, GL and KB contributed to the design of the experiments and to the editing of the manuscript. GL supervised the project, designed the experiments, performed experiments, analyzed the data and wrote the paper.


I Lauritzen

R Pardossi-Piquard

A Bourgeois

A Bécot

F Checler
email: checler@ipmc.cnrs.fr

Does intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein trigger early neurotoxicity in Alzheimer's disease?

Keywords: Alzheimer's disease, C99, C83, C-terminal APP fragments, dimerization, endolysosomalautophagic dysfunction, synaptic dysfunction, brain network alterations, memory-related behavior, animal models

Background: Alzheimer's disease (AD) is associated with extracellular accumulation and aggregation of amyloid β (Aβ) peptides ultimately seeding in senile plaques. Recent data show that their direct precursor C99 (βCTF) also accumulates in AD-affected brain as well as in AD-like mouse models. C99 is consistently detected much earlier than Aβ, suggesting that this metabolite could be an early contributor to AD pathology. C99 accumulates principally within endolysosomal and autophagic structures and its accumulation was described as both a consequence and one of the causes of endolysosomal-autophagic pathology, the occurrence of which has been documented as an early defect in AD. C99 was also accompanied with C99-derived C83 (αCTF) accumulation occurring within the same intracellular organelles. Moreover, both α-and β-CTFs can dimerize, thereby leading to the generation of higher molecular weight CTFs, which were immunohistochemically characterized in situ by means of aggregate-specific antibodies. Here, we discuss studies demonstrating a direct link between the accumulation of C99 and C99-derived APP-CTFs and early neurotoxicity. We discuss the role of C99 in endosomal-lysosomal-autophagic dysfunction, neuroinflammation, early brain network alterations and synaptic dysfunction as well as in memory-related behavioral alterations, in triple transgenic mice as well as in newly developed AD animal models. Conclusion: this review summarizes current evidence suggesting a potential role of the β-secretasederived APP C-terminal fragment C99 in Alzheimer's disease etiology.

Introduction

Alzheimer's disease (AD) is the most common form of dementia observed in western populations. A consistent histological feature of AD-affected brains is the extracellular accumulation of abnormal protein deposits mainly composed of aggregated Aβ. When autosomal dominant mutations in APP were shown to trigger early and aggressive forms of AD and when cell biology approaches indicated that these mutations systematically triggered modulations of Aβ production or yielded aggregation-prone Aβ species, the amyloid cascade hypothesis was proposed, in which Aβ is at the center of gravity [START_REF] Hardy | Alzheimer's disease: the amyloid cascade hypothesis[END_REF][START_REF] Checler | Processing of the beta--amyloid precursor protein and its regulation in Alzheimer's disease[END_REF]. Aβ peptides are generated by sequential processing of the transmembrane amyloid precursor protein (APP) by β-and γ-secretases [START_REF] Checler | Processing of the beta--amyloid precursor protein and its regulation in Alzheimer's disease[END_REF]. First, APP is cleaved by β-secretase generating the membrane-bound fragment C99 (also referred to as βCTF), which then undergoes a secondary cleavage by γsecretase leading to the release of soluble Aβ peptides. This sequence of proteolytic events is considered as the amyloidogenic pathway. γsecretase is a membrane protein complex composed of Aph-1, Pen-2, nicastrin and presenilins (1 and 2) [START_REF] Kimberly | Gamma--secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph--1, and Pen--2[END_REF][START_REF] Strooper | Deficiency of presenilin--1 inhibits the normal cleavage of amyloid precursor protein[END_REF][START_REF] Takasugi | The role of presenilin cofactors in the gamma-secretase complex[END_REF] and mutations in both PSEN1 and more rarely PSEN2 genes, encoding presenilin 1 and presenilin 2, respectively, are known to cause familial AD [START_REF] Kelleher | Presenilin--1 mutations and Alzheimer's disease[END_REF][START_REF] Checler | Presenilins: multifunctional proteins involved in Alzheimer's disease pathology[END_REF]. Transgenic mouse models harboring mutated APP and/or mutated presenilins recapitulate present age-dependent extracellular amyloid deposits and other ADrelated anatomical stigmata. Mounting data indicate that not only Aβ, but also other catabolites of APP accumulate in the AD brain, and our previous work, based on the 3xTgAD (APPswe, PS1M106V, TauP301L) mouse model, indicated a particularly strong agedependent intraneuronal accumulation of the C99 fragment, the direct precursor of Aβ [START_REF] Lauritzen | The beta--secretase--derived C--terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple--transgenic mouse hippocampus[END_REF][START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF]. In the 3xTgAD mouse, C99 accumulation occurs in specific AD-related brain areas (hippocampus and cortex) and appears at very early stages much before any detectable Aβ. Importantly, works from other groups later confirmed that early intraneuronal C99 accumulation occurs in several other mouse models, including the APP E693Q (Dutch mutation) expressing mouse [START_REF] Kaur | Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein[END_REF], the TgCRND8 (Swedish and Indiana mutations) mouse [START_REF] Cavanagh | betaCTF--correlated burst of hippocampal TNFalpha occurs at a very early, pre--plaque stage in the TgCRND8 mouse model of Alzheimer's disease[END_REF] and the J20 mouse (Swedish and Indiana mutations) [START_REF] Mondragon--Rodriguez | Alzheimer's Transgenic Model Is Characterized by Very Early Brain Network Alterations and beta--CTF Fragment Accumulation: Reversal by beta--Secretase Inhibition[END_REF]. Thus, C99 accumulation appears as a common anatomical and biochemical denominator observed in AD mice models, could well be envisioned as an etiological trigger in AD pathology.

C99 accumulation is both a consequence and a cause of early lysosomal-autophagic dysfunction

Our work showed that C99 mainly accumulates within endosomal, lysosomal and autophagic structures [START_REF] Lauritzen | The beta--secretase--derived C--terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple--transgenic mouse hippocampus[END_REF], which also correspond to the principal intracellular sites for amyloidogenic APP processing [START_REF] Pasternak | The role of the endosomal/lysosomal system in amyloid--beta production and the pathophysiology of Alzheimer's disease: reexamining the spatial paradox from a lysosomal perspective[END_REF]. We found that C99 accumulation in the 3xTgAD mouse did not result from either increased β-secretase or reduced γ-secretase cleavages, but was rather due to early AD-associated lysosomalautophagic impairment [START_REF] Lauritzen | The beta--secretase--derived C--terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple--transgenic mouse hippocampus[END_REF]. Thus, C99 accumulation was similar in both 3xTgAD and 2xTgAD mouse (APPswe, TauP301L), although the latter mice displayed little if any Aβ as expected from the absence of mutated presenilin-1 [START_REF] Lauritzen | The beta--secretase--derived C--terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple--transgenic mouse hippocampus[END_REF]. In agreement with lysosomal dysfunction in both mice, C99 accumulated within abnormally large cathepsins-and lamppositive structures, which number was also increased in C99-positive neurons [START_REF] Lauritzen | The beta--secretase--derived C--terminal fragment of betaAPP, C99, but not Abeta, is a key contributor to early intraneuronal lesions in triple--transgenic mouse hippocampus[END_REF][START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF]. Importantly, endolysomal-lysosomal dysfunc tion is also a key feature in human AD pathology and is consistently viewed as one of the earliest pathological events taking place in this disease. This dysfunction is reflected by the early appearance of morphological and functional abnormal endosomes and a progressive failure of lysosomal and autophagic degradation leading to the build-up of undigested autophagic vesicles [START_REF] Nixon | Amyloid precursor protein and endosomal--lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease[END_REF]. Our data from mouse models indicated that C99 accumulation itself could contribute to this pathology. Indeed, the pharmacological inhibition of γ-secretase in young animals led to increased C99 levels (and C99-derived APP-CTFs, see below) but also to exacerbated lysosomal impairment [START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF]. Strikingly, the group of Dr Levy drew the same conclusions from a study based on a transgenic mouse model harboring the Dutch mutation, the APP E693Q mouse [START_REF] Kaur | Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein[END_REF]. As in the 3xTgAD mouse, C99 accumulation was observed within enlarged LAMP-1, LAMP-2 and cathepsin D-positive structures and the number of these structures were strongly increased in C99-positive cells [START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF][START_REF] Kaur | Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein[END_REF]. These findings showed that C99 could alter lysosomal function in addition to its earlier reported deleterious effects on endosomal dysfunction [START_REF] Nixon | Amyloid precursor protein and endosomal--lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease[END_REF][START_REF] Jiang | Alzheimer's--related endosome dysfunction in Down syndrome is Abeta--independent but requires APP and is reversed by BACE--1 inhibition[END_REF][START_REF] Kim | Evidence that the rab5 effector APPL1 mediates APP--betaCTF--induced dysfunction of endosomes in Down syndrome and Alzheimer's disease[END_REF]. For instance, in fibroblasts from Down's syndrome, a cause of early-onset AD-like pathology due to an extra copy of APP, C99 was found to accumulate within early endosomes and cause both morphological and functional endosomal abnormalities, including up-regulated expression of endocytic proteins, aberrant endocytosis and impaired endosomal transport [START_REF] Jiang | Alzheimer's--related endosome dysfunction in Down syndrome is Abeta--independent but requires APP and is reversed by BACE--1 inhibition[END_REF]. These deleterious effects were reversed in cells treated with a β-secretase inhibitor, but increased in the presence of a γsecretase inhibitor, demonstrating that C99 but not Aβ should underlie these abnormalities [START_REF] Jiang | Alzheimer's--related endosome dysfunction in Down syndrome is Abeta--independent but requires APP and is reversed by BACE--1 inhibition[END_REF]. We and the group of Dr Levy also found that this intraneuronal accumulation is associated with microglial activation and astrogliosis [START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF][START_REF] Kaur | Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein[END_REF]. These inflammatory responses were particularly important in γsecretase inhibitor-treated mice (displaying little if any Aβ and high APP-CTF levels) demonstrating that inflammation was indeed linked to C99 accumulation [START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF]. Again, the group of Dr Levy, working on the APP E693Q mouse, came to the same conclusion, since neuroinflammation in these mice appeared at very early ages before any Aβ detection [START_REF] Kaur | Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein[END_REF]. These observations are in agreement with more general conclusions linking intraneuronal accumulation of biologically inactive aggregated proteins to neuroinflammatory activation [START_REF] Currais | Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain[END_REF]. C99 accumulation exacerbates C99-derived C83 production and generate high molecular weight APP-CTFs-related species. Although, it was found that the major accumulating intraneuronal immunostaining corresponded to C99, our work also indicated that part of it could correspond to C83. This catabolite mainly arises from nonamyloidogenic processing of APP by αsecretase, which occurs primarily on the plasma membrane. However, this apparently paradoxical observation could be explained by the susceptibility of C99 itself to proteolysis by α-secretase [START_REF] Jager | alpha--secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Abeta generation[END_REF][START_REF] Flammang | Evidence that the Amyloid--beta Protein Precursor Intracellular Domain, AICD, Derives From beta--Secretase--Generated C--Terminal Fragment[END_REF] and suggests that αsecretase activity should also be efficient within organelles of the endolysosomal system. Particularly high C99-derived C83 levels were found in animals treated with γ-secretase inhibitors, indicating that γ-secretase inhibition not only protected endogenous C83 from γsecretase-mediated breakdown, but also somehow favored the cleavage of C99 by αsecretase. Strikingly, this interplay between γand α-secretase activities has already been described [START_REF] Chen | Physical and functional interaction between the alpha-and gamma--secretases: A new model of regulated intramembrane proteolysis[END_REF][START_REF] Siegenthaler | g--secretase regulates the a--secretase cleavage of the Alzheimer's disease, amyloid precursor protein[END_REF]. Beside C99 and C83, expression analysis revealed the presence of high-molecular weight APP-CTFs in cells or brain tissue accumulating high levels of C99. We observed these APP-CTFs in cells expressing APP bearing the Swedish mutation, which favors the β-secretase cleavage of APP but also in cells expressing directly the C99 fragment, indicating that these APP-CTFs derived directly from C99 and did not correspond to other putative APP-derived metabolites of higher molecular weight. In this context, recent work has focused on the ƞsecretase-derived APP cleavage product, APP-CTFƞ [START_REF] Baranger | MT5--MMP is a new pro--amyloidogenic proteinase that promotes amyloid pathology and cognitive decline in a transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Willem | eta--Secretase processing of APP inhibits neuronal activity in the hippocampus[END_REF]. Indeed, this 30 kDa APP-CTF can be recognized by anti-APP C-terminal antibodies and it has a size close to one of the high-molecular weight APP-CTFs observed in our study. We hypothesized that this 30kDa fragment could correspond to homodimers of C99, since several in vitro studies have shown that C99 can form homodimers [START_REF] Kienlen--Campard | Amyloidogenic processing but not amyloid precursor protein (APP) intracellular C-terminal domain production requires a precisely oriented APP dimer assembled by transmembrane GXXXG motifs[END_REF][START_REF] Botev | The amyloid precursor protein C--terminal fragment C100 occurs in monomeric and dimeric stable conformations and binds gamma-secretase modulators[END_REF][START_REF] Song | Competition between homodimerization and cholesterol binding to the C99 domain of the amyloid precursor protein[END_REF][START_REF] Winkler | Homodimerization Protects the Amyloid Precursor Protein C99 Fragment from Cleavage by gamma--Secretase[END_REF]. This homodimerization is favored by three glycinexxx-glycine (GxxxG) motifs in the juxtamembrane and transmembrane regions of C99 [START_REF] Kienlen--Campard | Amyloidogenic processing but not amyloid precursor protein (APP) intracellular C-terminal domain production requires a precisely oriented APP dimer assembled by transmembrane GXXXG motifs[END_REF], but its influence on γ-secretasemediated APP processing remains controversial. It was reported that C99 homodimerisation hampered γ-secretase cleavage thus lowering Aβ production [START_REF] Winkler | Homodimerization Protects the Amyloid Precursor Protein C99 Fragment from Cleavage by gamma--Secretase[END_REF], while others reported on increased cleavage yielding enhanced Aβ production [START_REF] Kienlen--Campard | Amyloidogenic processing but not amyloid precursor protein (APP) intracellular C-terminal domain production requires a precisely oriented APP dimer assembled by transmembrane GXXXG motifs[END_REF]. Interestingly, it was also reported that cholesterol could bind to C99 and compete with it, leading to a reduced homodimerization and increased Aβ production [START_REF] Song | Competition between homodimerization and cholesterol binding to the C99 domain of the amyloid precursor protein[END_REF], in agreement with Winkler's team conclusion [START_REF] Winkler | Homodimerization Protects the Amyloid Precursor Protein C99 Fragment from Cleavage by gamma--Secretase[END_REF]. Beside the 30kDa APP-CTF, we also observed the presence of additional high molecular weight APP-CTFs. Although never reported so far, our findings suggested that these immunoractivities could correspond to homodimers of C99 or C83, heterodimers of C99 and C83, as well as heterodimers of other APP-CTFs and cholesterol bound APP-CTFs. Of note, these APP-CTFs were recognized in situ in our mouse models by the aggregatespecific antibodies NU1 and NU4 [START_REF] Lambert | Monoclonal antibodies that target pathological assemblies of Abeta[END_REF] indicating that these species could well harbor an "aggregate-like" conformation within the endolysosomal-autophagic vesicle membranes.

Further immunological characterization of this intracellular staining revealed that these high molecular weight species were not recognized by APP Cterminal directed antibodies, suggesting that dimers formation could well induce a conformation change precluding their Cterminal epitope accessibility/interaction and antibody recognition. Immunostaining of brains of γ-secretase inhibitor-treated mice with NU1 increased intracellular staining, indicating that the label could not be accounted for by aggregated Aβ [START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF]. Thus, our work not only revealed the early occurrence and accumulation of intraneuronal C99 monomers, but was also the first study to document the accumulation of C99-derived C83 and higher molecular weight APP-CTFS (dimers), in situ within the same intracellular structures. One could speculate that either alone or after physical interaction, these species could cooperate to trigger neurotoxic phenotypes.

Brain network or synaptic alterations can be linked to early C99 accumulation

Several lines of evidence highlighted hippocampal synaptic dysfunction as a cause of memory impairment in AD [START_REF] Selkoe | Alzheimer's disease is a synaptic failure[END_REF]. In animal models, this impaired synaptic function appears as an early event and leads to defects in memory much before the appearance of amyloid plaques and neuronal degeneration [START_REF] Nistico | Targeting synaptic dysfunction in Alzheimer's disease therapy[END_REF]. In particular, AD has been associated with defects in synaptic transmission, long-term potentiation (LTP) and long-term depression (LTD), which are thought to underlie the cellular mechanisms of learning behavior and memory consolidation. Mounting evidence suggest that these synaptic dysfunction defects are triggered by soluble Aβ oligomers and much effort in recent years has been made to identify the receptors and signaling pathways implicated in oligomers-induced toxic effects [START_REF] Tanzi | The synaptic Abeta hypothesis of Alzheimer disease[END_REF][START_REF] Tu | Oligomeric Abeta--induced synaptic dysfunction in Alzheimer's disease[END_REF][START_REF] Smith | Binding Sites for Amyloid--beta Oligomers and Synaptic Toxicity[END_REF]. Thus, Aβ was found to bind to receptors for glutamate, nicotinic acetylcholine, cellular prion protein, neurotrophins (p75NTR) and ephrin among others [START_REF] Smith | Binding Sites for Amyloid--beta Oligomers and Synaptic Toxicity[END_REF]. In order to study the implication of C99 in synaptic alterations, we recently analyzed hippocampal LTP in C99-expressing mice (AAV-mediated C99 expression) [START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF]. Indeed, our results indicated that hippocampal LTP was significantly reduced in young C99expressing mice, as compared to control mice infected with control virus. The inhibition of γsecretase did not rescue LTP alterations indicating that C99 rather than Aβ mediated these effects [START_REF] Lauritzen | Intraneuronal aggregation of the beta--CTF fragment of APP (C99) induces Abeta--independent lysosomal-autophagic pathology[END_REF]. In agreement with these findings, two lines of independent observations also indicated that the hippocampal LTP alterations observed in the 3xTgAD mouse (at least at the early stages of the pathology) should likely be linked to C99. Firstly, the synaptic alterations were temporally and spatially correlated with C99 accumulation [START_REF] Pardossi--Piquard | Influence of genetic background on apathy--like behavior in triple transgenic AD mice[END_REF]. Secondly, these electrophysiological modifications were identical in the 2xTgAD (APPswe, TauP301L), which accumulate similar levels of C99, while Aβ levels remain poorly detectable in this mouse model [START_REF] Bourgeois | Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy--like behavior, and spatial learning deficits in 3xTgAD and 2xTgAD mice[END_REF]. Moreover, it was found that spatial learning (using the Morris water maze test) was altered in both the 2xTgAD and 3xTgAD mice, as compared to control mice, although with a higher impact in the 3xTgAD mice, indicating that both C99 and Aβ could well contribute to these alterations [START_REF] Bourgeois | Intraneuronal accumulation of C99 contributes to synaptic alterations, apathy--like behavior, and spatial learning deficits in 3xTgAD and 2xTgAD mice[END_REF]. Recently, it was proposed that memory impairment is not only explained by LTP/LTD defects, but is also associated with changes in brain circuits and network activities reflected by hyperexcitability and spontaneous epileptiform activity [START_REF] Verret | Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model[END_REF]. These alterations were also triggered by pathological Aβ oligomers [START_REF] Verret | Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model[END_REF][START_REF] Palop | Network abnormalities and interneuron dysfunction in Alzheimer disease[END_REF]. Nevertheless, a number of recent works demonstrated that network alterations appeared at very early stages much before Aβ detection, proposing that other candidates could be responsible for these electrophysiological alterations. For instance, the group of Dr William observed alterations in hippocampal network oscillations and thetagamma coupling in the subiculum of the hippocampus of the TgCRND8 AD mouse brain, at early "Aβ-free" stages [START_REF] Goutagny | Alterations in hippocampal network oscillations and theta--gamma coupling arise before Abeta overproduction in a mouse model of Alzheimer's disease[END_REF]. Later work from the same group demonstrated similar electrophysiological alterations in young J20 mice and these abnormalities were correlated with early C99 accumulation, rather than with Aβ accumulation. Of importance, it was found that the treatment with a β-secretase inhibitor (that fully prevented C99 formation) rescued these alterations thus demonstrating that they should be directly linked to C99 [START_REF] Mondragon--Rodriguez | Alzheimer's Transgenic Model Is Characterized by Very Early Brain Network Alterations and beta--CTF Fragment Accumulation: Reversal by beta--Secretase Inhibition[END_REF].

Conclusion

There is a still growing body of evidence suggesting a critical role of C99 in Alzheimer disease mouse model neuropathology. The possibility that C99 could contribute to AD etiology independently of Aβ is supported by C99-induced endosome and lysosome dysfunction, neuroinflammation and electrophysiological alterations in various mice models at early stages of the pathology, at which little if any Aβ is detectable. Very little is still known about the role of C99 in human pathology, since most researches have so far focused on Aβ and have been performed at the end-stages of the disease, at which initial C99 toxicity may be overtaken by late Aβ accumulation and toxicity. Nevertheless, several papers have reported that C99 levels are indeed significantly elevated in human AD brain tissues, as compared to control brains [START_REF] Kim | Evidence that the rab5 effector APPL1 mediates APP--betaCTF--induced dysfunction of endosomes in Down syndrome and Alzheimer's disease[END_REF][START_REF] Hebert | Loss of microRNA cluster miR--29a/b--1 in sporadic Alzheimer's disease correlates with increased BACE1/beta--secretase expression[END_REF][START_REF] Pera | Distinct patterns of APP processing in the CNS in autosomal--dominant and sporadic Alzheimer disease[END_REF] indicating that C99 accumulation also occurs in human pathology and can be monitored also at late stage of the disease. Overall, one can envision an AD setting initiated by C99, while disease progression and late cognitive alterations should be attributed to C99, Aβ and certainly other APP-derived catabolites [START_REF] Dunys | Are N-and C--terminally truncated Abeta species key pathological triggers in Alzheimer's disease?[END_REF].

Ackowledgements

This work was supported through the LABEX (excellence laboratory, program of investment for the future) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer's disease, the Hospital University Federation (FHU) OncoAge and the Fondation Plan Alzheimer. Alexandre Bourgeois and Anaïs Bécot were supported by the DISTALZ LABEX grants.