Glucocorticoid receptor represses brain-derived neurotrophic factor expression in neuron-like cells
© The Author(s). 2017
Received: 27 October 2016
Accepted: 5 April 2017
Published: 12 April 2017
Brain-derived neurotrophic factor (BDNF) is involved in many functions such as neuronal growth, survival, synaptic plasticity and memorization. Altered expression levels are associated with many pathological situations such as depression, epilepsy, Alzheimer’s, Huntington’s and Parkinson’s diseases. Glucocorticoid receptor (GR) is also crucial for neuron functions, via binding of glucocorticoid hormones (GCs). GR actions largely overlap those of BDNF. It has been proposed that GR could be a regulator of BDNF expression, however the molecular mechanisms involved have not been clearly defined yet. Herein, we analyzed the effect of a GC agonist dexamethasone (DEX) on BDNF expression in mouse neuronal primary cultures and in the newly characterized, mouse hippocampal BZ cell line established by targeted oncogenesis. Mouse Bdnf gene exhibits a complex genomic structure with 8 untranslated exons (I to VIII) splicing onto one common and unique coding exon IX. We found that DEX significantly downregulated total BDNF mRNA expression by around 30%. Expression of the highly expressed exon IV and VI containing transcripts was also reduced by DEX. The GR antagonist RU486 abolished this effect, which is consistent with specific GR-mediated action. Transient transfection assays allowed us to define a short 275 bp region within exon IV promoter responsible for GR-mediated Bdnf repression. Chromatin immunoprecipitation experiments demonstrated GR recruitment onto this fragment, through unidentified transcription factor tethering. Altogether, GR downregulates Bdnf expression through direct binding to Bdnf regulatory sequences. These findings bring new insights into the crosstalk between GR and BDNF signaling pathways both playing a major role in physiology and pathology of the central nervous system.
KeywordsGlucocorticoid receptor Brain-derived neurotrophic factor Glucocorticoids Promoters
The neurotrophin brain-derived neurotrophic factor (BDNF) is a key player in neuronal function. BDNF is highly expressed throughout the brain [1, 2], yet its strongest expression level is found within the hippocampus, a limbic structure of major importance for cognitive functions, such as memorization, learning, behavior, stress, emotions, and mood [3, 4]. In the central nervous system (CNS), BDNF regulates neuronal survival , differentiation and growth . Growing evidence indicates that BDNF is also involved in neuronal homeostasis and brain plasticity-related processes such as memory, learning [7, 8] and drug addiction , as well as in long term potentiation . Alterations in BDNF expression levels within specific neuron subpopulations have been associated with various pathologies, including depression, epilepsy, Alzheimer’s, Huntington’s and Parkinson’s diseases [11–16]. BDNF mainly functions by binding to its high-affinity receptor, tropomyosin-related kinase B (TrkB) activating several pathways such as MAP kinase, PI3 kinase and Phospholipase C . Rodent Bdnf gene exhibits a complex genomic structure comprising of at least 9 exons (I to IX), which are alternatively spliced to generate exon-specific BDNF transcript variants with one common and unique coding exon IX at the 3’ terminal end . Generation of a large set of transcript isoforms is probably of biological significance as in rat hippocampal neuronal cultures, it has been demonstrated that BDNF mRNA variants are differentially distributed in specific dendritic compartments in order to regulate the local availability of BDNF protein . Moreover, BDNF expression was reported to be reduced with aging and associated with a repressed chromatin state on some of its gene regulatory regions . Along this line, epigenetic histone modifications and DNA methylation marks have recently been identified as complex and crucial mechanisms enabling modified expression of various BDNF mRNA isoforms . Altogether, several layers of events driving quantitatively and qualitatively BDNF expression highlight its crucial contribution to CNS function in physiology and pathology [22–24].
Glucocorticoid hormones (GCs) also exert pleiotropic actions on neurons by binding to and activating the glucocorticoid receptor (GR, NR3C1), as well as to the mineralocorticoid receptor (MR, NR3C2) [25, 26]. The latter exhibits a high ligand affinity, and as a consequence it is almost permanently occupied by GCs, while GR is mostly activated under high circulating GC concentrations such as during stress conditions or at the circadian peak of GCs. Both receptors are highly expressed in the hippocampus, acting in balance to regulate various physiological and neurological processes such as stress responses, apoptosis survival and long term potentiation . Interestingly, BDNF activation of TrkB receptors regulates positively GR activity on its target gene expression by phosphorylating two key serine residues on the receptor . Mutating these BDNF-sensitive sites results in the inhibition of the neuroplasticity response to chronic stress , unraveling a crosstalk between GC and neurotrophin signaling pathways. On the other hand, regulation of BDNF expression by stress  has important consequences on the pathophysiology of mood disorders  and in the mechanism of action of antidepressant agents . As exposure to acute or chronic stress triggers a surge of circulating GC concentrations [33, 34], a role of these hormones in modulating BDNF expression has often been suggested [35–41], but most of these reports are based on indirect evidence, and are sometimes contradictory depending on the model and the treatment timeline [42–44]. As a whole, the molecular mechanisms by which GCs regulate BDNF expression are not clearly defined. In the present study, we demonstrated that, upon exposure to the glucocorticoid agonist dexamethasone (DEX), GR directly downregulates Bdnf expression, at least in part, by its binding to a specific DNA region upstream of exon IV. Interestingly, this promoter fragment was already characterized as stimulated by synaptic activity in humans and rats [45, 46]. Along with primary cultures of fetal hippocampal neurons (PCN), we used the newly characterized BZ cell line which was previously generated by targeted oncogenesis strategy  from a mouse hippocampus and which expresses a high level of both BDNF and GR. Altogether, this work unravels new insights about the repression by GR of Bdnf expression, findings that may be of potential physiological importance.
Primary cultures of fetal mouse hippocampal neurons
Pregnant SWISS mice at 18 or 19 days post-fertilization were euthanized by decapitation. Dissection was performed according to a video published in the Journal of Visual Experiments . Hippocampal neurons were isolated and cultured from the embryos using the Pierce Primary Neuron Isolation Kit (Thermo scientific, Courtaboeuf, France) following the manufacturer’s instructions. This kit contained a neuronal media culture supplement (reference: 88286). Cells were typically seeded on culture plates coated with 10 μg/mL poly-D-lysine (Sigma-Aldrich, Lyon, France), at the density of 2.5 × 105 cells/cm2 and grown for 9 days at 37 °C in a 5% CO2 incubator.
Cell culture and reagents
BZ and Neuro-2a (N2A, ATCC number: CCL-131) growth medium was composed of DMEM (PAA, Vélizy-Villacoublay, France) containing 10% fetal bovine serum (FBS) (AbCys SA, Paris, France), 1x nonessential amino acids (PAA), 2 mM glutamine (PAA), 100 U/ml penicillin (PAA), 100 μg/ml streptomycin (PAA), 20 mM HEPES (PAA). For BZ differentiation experiments, serum concentration was lowered to 1% for 2 days. N2A differentiation medium was similar to BZ medium but was supplemented with 5 μg/ml insulin (Sigma-Aldrich), 5 μg/ml transferrin (Sigma-Aldrich), 29 nM sodium selenate (Sigma-Aldrich) and 1 μM retinoic acid (Sigma-Aldrich). For hormonal treatment, cells were grown in Dextran Charcoal Coated-treated (DCC) FBS. Dexamethasone (DEX) (Sigma-Aldrich) and RU 486 (RU) (Sigma-Aldrich) were used at the indicated concentrations diluted in ethanol. For RNA stability analysis, 10 μg/ml of 5, 6-Dichlorobenzimidazole-1-β-D-ribofuranoside (DRB) (Sigma-Aldrich) diluted in DMSO was applied.
BZ cells cultured in x-well tissue culture chambers (Sarstedt, Numbrecht, Germany) were fixed with PBS containing 4% paraformaldehyde (Sigma-Aldrich) solution for 15 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 5 min and blocked by 1% non-fat milk in PBS with 0.1% Tween-20 (PBST) for 1 h at 37 °C. Cells were incubated with anti β-tubulin III antibody TU20 (sc-51670, Santa Cruz, La Jolla, CA, ) or with anti-GR antibody M20 (sc-23476, Santa Cruz, ) overnight at 4 °C and then with secondary antibody Cy3 (Interchim, Thermo scientific) for 45 min in PBST with 1% milk. After washing, BZ cells were incubated with DAPI (4′,6′-diamidino-2-phenylindole), 1: 1000 in PBST for 2 min and observed with a fluorescence Olympus microscope AX70 (Olympus, Hambourg, Germany).
RNA extraction and quantitative real-time PCR
Gene expression was quantified by reverse transcription (RT) followed by quantitative PCR (qPCR). Cells were harvested and total RNAs were extracted with Trizol reagent (Invitrogen, Cergy-Pontoise, France) according to the manufacturer’s instructions and their concentrations were determined using a Nanodrop 2000 spectrophotometer (Thermo scientific). RNAs were reverse-transcribed and processed for real-time PCR on an ABI Step One Plus (Applied Biosystems, Courtaboeuf, France). Briefly, 1 μg of total RNA was treated by DNAse I (Invitrogen), then reverse-transcribed with 50 U MultiScribe reverse transcriptase (Applied Biosystems). After 10-fold dilution, 1/40 of the RT reaction was used for qPCR using the Fast SYBR Green PCR master mix (Applied Biosystems). Final primer concentrations were 300 nM for each primer. Primer sequences for each gene are listed in Additional file 1: Table S1. Reaction parameters were 95 °C for 20 s followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. For plasmids used for standard curves, amplicons were purified from agarose gels and subcloned into pGEMT-easy plasmid (Promega, Charbonnières, France), then sequenced to confirm the identity of each fragment. Standard curves were generated using serial dilutions of standard plasmids, spanning six orders of magnitude and yielding correlation coefficients more than 0.98 and efficiencies of at least 0.95, in all experiments. Standard and sample values were determined in duplicate from each sample. Relative expression within a given sample was calculated as the ratio: attomol of specific transcripts/attomol of 36B4 transcripts.
Protein extraction and Western blotting
Protein expression was assessed by Western blotting. Cells were harvested from the culture plates and total cellular proteins were extracted by lysis buffer containing 1% Triton X-100, 1% proteasome inhibitor cocktail (Sigma-Aldrich). Lysates were cleared by centrifugation at 13,000 g for 20 min and protein concentrations were measured using the BC Assay Protein Quantitation Kit (Uptima, Oakland, CA). Total protein lysates (50 μg) were fractionated on 15% SDS-PAGE then transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Vélizy-Villacoublay, France). Membranes were blocked for 1 h in PBST with 2.5% BSA, and then incubated with primary antibodies overnight at 4 °C, rabbit polyclonal anti-BDNF (sc-546, Santa Cruz ) at 1:200 and mouse monoclonal anti-α-tubulin (Clone DM1A, Sigma-Aldrich) at 1:10,000. Incubation with secondary antibodies conjugated to infrared fluorophores (goat anti-rabbit IgG Dylight 800 and anti-mouse IgG Dylight 680 at 1:10,000 from Thermo Scientific) was performed for 1 h. An Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany) was used to scan membranes at a wavelength of 680 nm (anti-mouse) or 800 nm (anti-rabbit). Data were analyzed with Image Studio 1.1 software (Li-COR).
pCDNA3-GR plasmid was generated by subcloning human GR (NR3C1) into pCDNA3 vector (Invitrogen). Bdnf promoter reporter plasmids were constructed by inserting PCR fragments from Bdnf promoter regions into PGL4-basic luciferase reporter vector (Promega), see Additional file 1: Table S1 for primer sequences. These fragments, named LP6, SP6, LP4, and SP4, are localized according to the distance to exon VI transcription starting site that was set at +1 (see Fig. 4). Specific genomic sequences were amplified by PCR from mouse genomic DNA and inserted into the PGL4 cloning vector in the Xho I and Hind III restriction sites using pGEMT easy vector kit (Promega), then transformed in JM109 E.Coli. Plasmids were sequenced to confirm the identity and orientation. The renilla activities driven by the expression vector PRL-TK (Promega) or total protein concentrations were used for normalizing expression.
On the day prior to transfection, N2A cells were plated in 96-well plates at a density of 2 × 104 cells per well in the N2A differentiation medium. Medium was changed for OptiMEM medium (PAA) on the next day, and cells were transfected using Lipofectamine 2000 reagent (Invitrogen). Plasmid concentrations per well were 50 ng PGL4-Bdnf promoter vectors, 25 ng pCDNA3-GR or empty vector, 13.3 ng PRL-TK renilla expression vector. Six h post-transfection, OptiMEM medium was replaced by N2A DCC medium. Cells were treated with Ethanol (Veh), DEX and/or RU486 24 h after transfection and then were harvested after another 24 h with Passive Lysis Buffer from the dual luciferase reporter assay system kit (Promega). Lysates were transferred to 96-well clearbottom microplates (Fisher), and luminescence intensities were measured with a TRISTAR LB941 automatic luminometer (Berthold, Thoiry, France) with dual injectors. Normalized values were used for statistical analyses. Each experiment was performed with 8 replicates and repeated at least three times.
Chromatin immunoprecipitation (ChIP)
ChIP experiments were performed using the Ideal-ChIP for transcription factor kit (Diagenode, Liège, Belgium) following the manufacturer’s protocol. Specifically, 4 × 106 BZ cells per well of 6-well plates grown in 1% DCC medium were treated by vehicle (ethanol), Dex 10-7 M or Dex 10-7 M together with RU 10-6 M for 1 h. Cells were then fixed by a mix of formaldehyde 16% and fixation buffer from the kit (4: 1 ratio), 1/100 volume final for 8 min. ChiP experiments were performed according to Le Billan et al . Chromatin samples (300 μl) were sheared by 12 cycles 30 s ON and 30 s OFF with the Diagenode Bioruptor Pico system and 2.5 μl samples were kept for input measurements. Two hundred and fifty μl of sheared chromatin samples were incubated overnight at 4 °C with 1 μg IgG from the kit as control or 5 μg anti-GR antibody H300 (Santa Cruz sc-8992-X ). Chromatin shearing was controlled on a 1.5% agarose gel and typically a smear was visualized ranging from 100 to 500 bp. qPCR amplifications of eluted DNA were performed with primers encompassing a short upstream sequence of exon IV, or on Per1 and Ucp1 promoters (Additional file 1: Table S1 for primer sequences). Raw data are expressed as percentage of inputs, according to the Percent of Input Method, ChIP analysis; Thermo Fischer Scientific.
Immunoprecipitations were performed using 30 μl protein A-coated magnetic beads (Diagenode). Beads were washed 3 times with C1 buffer of the High Cell ChIP kit (Diagenode) and incubated for 4 h with anti-GR antibody H300 (Santa Cruz) at 4 °C with protease inhibitors and 0.2% BSA. Then, 350 μg of proteins from BZ cell lysates were incubated overnight with antibody-coated beads. The following day, beads were washed 3 times with buffer C1 and once with buffer W1 (High Cell ChIP kit), and immunoprecipitated proteins were eluted from the beads with 20 μl of Laemmli buffer at 95 °C for 5 min and loaded on a 10% SDS-PAGE gel for Western blotting. Membranes were hybridized with anti-GR antibody M20 (sc-23476, Santa Cruz, .
Targeted mutations of Bdnf promoter construct plasmids were performed using the Quickchange II XL kit (Agilent Technologies, Les Ulis, France) following the manufacturer instructions. Briefly, 10 ng of SP4 luciferase plasmid was amplified by PCR with sense and antisense mutated primers for 18 cycles. PCR reactions were transformed in XL1 blue ultracompetent E. Coli strain (Agilent, les Ulis, France) on LB Agar Ampicillin petri dishes. Bacterial colonies were picked up. Plasmid DNA were extracted and sequenced by Eurofins (Ivry sur Seine, France) to check for the introduction of the mutation and sequence integrity. Primers for mutagenesis are available in Additional file 1: Table S1. Plasmids were transfected in N2A cells as stated for the luciferase assay.
Results are expressed as mean ± SEM of at least six samples for each condition unless stated otherwise. Statistical analyses were performed using nonparametric Mann-Whitney U-tests, unless stated otherwise, using Prism 5 (GraphPad Software, Inc., San Diego, CA).
GR represses Bdnf expression in primary mouse hippocampal cultures
In order to better understand the mechanisms by which GCs decrease neuronal Bdnf expression, it is worth noting that mouse Bdnf gene exhibits a complex genomic structure with 8 untranslated exons (I to VIII) that splice onto the common coding exon IX and two clusters of promoters upstream of I, II, III and IV, V, VI, respectively (Fig. 1c) [18, 55]. We determined the relative expression of the untranslated exon-containing transcripts by RT-qPCR and found that exon IV containing mRNA isoform was by far the most expressed one followed by exon VI and I containing isoforms (Fig. 1d). Expression of all of these 3 exon-containing messengers was repressed by DEX while RU suppressed this effect (Fig. 1e, f and g). Conversely, DEX did not exert any effect on exons VII and VIII containing transcripts (Fig. 1h and I), highlighting an exon-specific regulation of GR on Bdnf gene. Altogether, these data showed that GR was able to repress BDNF mRNA expression in PCN by downregulating specific isoforms even in the presence of noticeable amounts of GCs in the medium.
The BZ cell line is a glucocorticoid responsive model expressing BDNF
GR represses Bdnf expression in BZ cells
GR represses Bdnf expression by acting upstream of exon IV
AP1 and CRE response elements do not mediate GR transrepression
GR binds upstream of exon IV upon DEX treatment
BDNF, the main neurotrophin is a key factor regulating neuronal function in health and diseases, such as survival, growth, synaptic structure and activity. Thus, understanding the molecular mechanisms involved in the regulation of neuronal Bdnf expression is a major issue. However, the effect of glucocorticoids on Bdnf neuronal expression is still a matter of debate . Given the complexity of the central nervous system anatomy and its functional organization, where neurons represent only a small fraction of the total number of cells, we focused our attention in the present study to in vitro neuronal models in order to investigate GC action on Bdnf expression. Besides, GC bind to two receptors in the brain; the high affinity MR, which is occupied even at low concentrations of hormones, and GR with a lower affinity for ligands thus more sensitive to hormonal regulation and variation . In the present work, we decipher the role of GR as a mediator of GC actions and for this purpose we use the glucocorticoid agonist dexamethasone (DEX) that exhibits a low MR activating capacity [61, 62].
We have demonstrated that DEX exposure led to a marked downregulation of BDNF transcript expression in neuronal cells that was mediated by GR. More precisely, total BDNF mRNA levels were reduced after GC treatment in primary hippocampal neuron cultures, but also in the neuron-like cell line BZ. In both models, BDNF transcripts containing exons IV and VI isoforms were highly expressed and specifically repressed by GR activation. Transfection experiments were performed using a long 1.8 kb DNA fragment (LP6) encompassing the closely packed exon IV, V and VI and their 5’ regulatory sequences. LP6-driven activity was significantly repressed by DEX yet prevented by addition of GR antagonist RU. Functional characterization of the transcriptional activity of LP6 construct fragments by luciferase reporter assays led to identify a short 275 pb sequence (SP4) directly upstream of exon IV, and encompassing its transcription start site, which might account for the inhibitory effects of GCs. Analysis by Jaspar online software (URL: http://jaspar.genereg.net/cgi-bin/jaspar_db.pl), revealed that two Jun/Fos response elements, constituting AP1 binding sites, were located on this region as well as two cAMP response elements (CRE), a CREB1 and a closely related CREB3l2 response element, all being located just upstream of the exon IV transcription start site (see Fig. 6a). Chromatin immunoprecipitation assays demonstrated that GR binds to this sequence, GR recruitment being prevented by GR antagonist RU. However, promoter constructs with deletion of AP1 and CREB binding sites were still repressed by DEX excluding their involvement in GR regulation of Bdnf expression. Altogether, we propose that one of the mechanisms responsible for the repression of Bdnf expression by DEX is the binding of GR just upstream of exon IV, through ternary complexes with transcription factors that are still to be determined. Such a repression of gene expression by GR tethered to various transcription factor complexes has been reported in several studies [57, 63]. Nonetheless, no other response elements for proteins susceptible to interact with GR, such as NFκB, were identified on the SP4 sequence using Jaspar software. Another proposed mechanism of direct GR repression was uncovered recently in the form of negative GRE (nGRE) with a specific sequence (CTCCXGGAG) that clearly differed from positive GRE . Anyhow, none of putative nGRE was identified in the SP4 sequence. Eventually, one might speculate that GR binds to a specific GRE outside the long LP6 fragment that lacks any GRE and acts on Bdnf promoter by a folding DNA loop, a mechanism that has been recently described for GR . However, such a mechanism is very unlikely given that GR interacts with the SP4 sequence as demonstrated by transfection and ChIP assays. Nonetheless, we believe these results are of important since they excluded the involvement of specific response elements for transcription factors known to be transrepressed by GR (Jun/fos and CREB) in the SP4 sequence. This raises the hypothesis of a new mechanism involving some partners mediating GR transrepression that remains to be identified.
It is worth noting that this region upstream of exon IV, which displays a high degree of homology between mammalian species emphasizing its biological importance, has been extensively characterized in previous studies by several groups as positively regulated by calcium [45, 66, 67], synaptic activity  and BDNF itself  in humans and rats. Moreover the two CRE elements for CREB (CRE1) and CREB3l2 (CRE2) we identified in the mouse sequence are conserved and involved in this regulation, CRE2 being formerly reported as a BHLHB2 response element [46, 68]. In sum, we unraveled that GR represses the activity of exon IV promoter that was previously shown to be stimulated by synaptic activity . This is consistent with the adverse effects of the overactivation of glucocorticoid pathways on brain physiology .
The BZ cell line was derived few years ago from a mouse hippocampus. Herein, a more detailed characterization of this neuronal cell line was undertaken and revealed that BZ cells constitute a suitable cellular tool to investigate the regulation of BDNF expression. In comparison, the commercially available N2A cell line expresses very low levels of BDNF transcripts, impairing analysis of isoform expression even if total BDNF transcripts could be measured by the sensitive qPCR technique and we found that GC were able to repress Bdnf expression in this model (data not shown). Beyond that, BZ cells appear to be a suitable cell-based system for studying GR signaling pathways in a neuron-like cell context in vitro.
In the present study, quantification of BDNF protein abundance by Western blot in PCN and BZ cells, did not show any significant change with short term (6 h) or long term (24 h) DEX treatments (data not shown). Even if we could not exclude that GCs might reduce BDNF protein expression on a different timeline, recent studies underscored that BDNF mRNA isoforms localization might be a major factor regulating its functions . For instance, transcripts containing exon IV and VI in rat primary hippocampal neuron cultures were found to display a more distal localization relative to the soma than those including exon I, while they were able to stimulate dendritic branching . Based on this fine-tuning spatial code, it could be proposed that the localization of BDNF mRNA isoform translation would be related to distinct BDNF sites of action. Thus, it is very likely that total expression of BDNF or secretion level of the protein might constitute inappropriate indexes to define BDNF biological function.
Altogether, we provide evidence for a functional crosstalk between activated GR and Bdnf expression in mouse neurons that is, at least in part, mediated by GR binding to a restricted sequence upstream of exon IV. In sum, high circulating glucocorticoid levels prevailing under certain circumstances such as stress may alter neuronal Bdnf expression  and specifically its transcripts distribution leading to a remodeling of dendrite and synapse architecture and function. This could be of physiological importance in processes such as memorization and behavior as well as causal in various pathologies associated with modification of GR and BDNF pathways.
Brain-derived neurotrophic factor
GR response element
Primary cultures of hippocampal Neurons
We are indebted to Patrice Penfornis for the initial derivation and subcloning of the BZ cell line (present address: Cancer Institute, University of Mississippi Medical Center, Jackson, MS, USA). We thank Fanny Jung for part of the BZ cell line characterization during her practical training for her advance technology degree (BTS) in biotechnologies. We also thank Simon Travers (Service de Génétique Moléculaire, Pharmacogénomique et Hormonologie, Bicêtre Hospital) for corticosterone measurements by LC/MS-MS.
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Université Paris-Sud and Agence Nationale de la Recherche (Grant 11-BSV1-028-01). HC was funded by a China Scholarship Council (CSC) fellowship.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
ML, HC, and DL designed the study. HC and DL performed the experiments. ML, HC, and DL wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Swiss mice were housed and handled according to the National Institutes of Health Guidelines. The animal work is part of an approved project by the ethical comity CEEA 26 (#2012_021). DL has an agreement to conceive and perform animal procedure (N°R-75UPMC-F1-08) by the French state agency ‘Ministère de l’Agriculture, de l’Agroalimentaire et de la Forêt’.
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