PhD Yanyi Sun

PhD Cristina Pinto

PhD Stéphane Camus

MSc Vincent Duval

MSc Paul Alayrac

PhD Ivana Zlatanova

PhD Xavier Loyer

PhD a , Jose Vilar

Mathilde Lemitre Bsc

PhD Angélique Levoye

PhD Meritxell Nus

MD Hafid Ait-Oufella

PhD Ziad Mallat

PhD Jean-Sébastien Silvestre
email: jean-sebastien.silvestre@inserm.fr

Splenic marginal zone B lymphocytes regulate cardiac remodeling after acute myocardial infarction in mice

Keywords: Myocardial Infarction, B Lymphocytes, microRNA 21, Hypoxia-inducible factor MI Recombination Signal Binding Protein For Immunoglobulin Kappa

Background. Mature B lymphocytes alter the recovery of cardiac function after acute myocardial infarction (MI) in mice. Follicular B cells and marginal zone B (MZB) cells are spatially distinct mature B cell populations in the spleen and exert specific functional properties. miR21/Hypoxia-inducible factor (HIF)α-related pathways have been shown to govern B cell functions. Objectives. We aimed to unravel the distinct role of MZB cells and that of endogenous activation of miR21/ HIFα signalling in MZB cells during post-ischemic injury. Methods. Acute MI was induced by permanent ligation of the left anterior descending coronary artery in mice. Cardiac function and remodeling were assessed using echocardiography and immunohistochemistry. To determine the specific role of MZB cells, we used mice with B cell lineage-specific conditional deletion of Notch signaling, which leads to selection deficiency of MZB cells. To evaluate the role of HIF-1α isoform, we generated mice with MZB cell lineage specific conditional deletion of Hif1a. Results. Acute MI prompted miR21-dependent increase of HIF-1α, particularly in splenic MZB cells. MZB cell deficiency and MZB cell-specific deletion of miR21 or Hif1a improved cardiac function after acute MI. miR21/HIF-1α signaling in MZB cells was required for Tolllike receptor dependent expression of the monocyte chemo-attractant protein CCL7, leading to increased mobilization of inflammatory monocytes to the ischemic myocardium and to adverse post-ischemic cardiac remodeling. Conclusions. This work reveals a novel function for miR21/HIF-1α pathway in splenic MZB cells with potential major implications for the modulation of cardiac function after acute MI.

Condensed Abstract: Splenic marginal zone B cells are instrumental in the regulation of cardiac function and remodeling after acute myocardial infarction. miR21/HIF-1α signaling adjusts the ability of marginal zone B cells to modulate CCL7 secretion and to subsequently impact inflammatory monocyte-dependent cardiac remodeling after injury.

Introduction

Numerous biological mechanisms converge to remodel the heart after myocardial infarction (MI). Adverse post-ischemic cardiac remodeling and progression to heart failure may be promoted by macroscopic alterations in left ventricle geometry and function, as well as microscopic alterations in the cellular and molecular landscapes of the ischemic heart. Recent evidence implicates both infiltrated and resident immune cells in post-MI remodeling (1) (2).

Notably, B lymphocytes have been shown to produce the chemokine CCL7 and promote cardiac inflammatory monocyte recruitment after acute MI, leading to enhanced tissue damage and myocardial dysfunction (3). Likewise, rapid elimination of cardiac B cells along with reduced CCL7 expression prevented pathological remodeling and heart failure (4).

Modulation of different subsets of B lymphocytes has also been shown to control tissue remodeling in different experimental models of cardiac disease (5) (6) [START_REF] Cordero-Reyes | Full Expression of Cardiomyopathy Is Partly Dependent on B-Cells: A Pathway That Involves Cytokine Activation, Immunoglobulin Deposition, and Activation of Apoptosis[END_REF]. Large B cell clusters were identified in epicardial adipose tissue of patients with coronary artery disease and in infarcted mice. B cell depletion reduced cardiac neutrophil infiltration as well as fibrosis and preserved left ventricular ejection fraction after acute MI [START_REF] Horckmans | Pericardial Adipose Tissue Regulates Granulopoiesis, Fibrosis, and Cardiac Function After Myocardial Infarction[END_REF]. Conversely, IL-10producing CD5 + B cells were expanded in pericardial adipose tissues and activated the resolution of MI-induced inflammation [START_REF] Wu | IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction[END_REF]. Altogether, these results indicate that B cells control left ventricle remodeling but the cellular mechanisms governing the effects of distinct B lymphocyte subtypes from diverse tissue origin remain to be defined. This is highly important given the ongoing and planned clinical trials aiming at targeting B cells in patients with acute MI [START_REF] Zhao | Rituximab in Patients with Acute STelevation Myocardial Infarction (RITA-MI): an Experimental Medicine Safety Study[END_REF].

MicroRNAs (miR) are fundamental to the development of inflammatory cells and also capable of regulating almost every leukocyte-related activity. Notably, they play prominent roles in B cell maturation, and different stages of normal B cell differentiation are characterized by distinct miRNA expression profiles [START_REF] Koralov | Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage[END_REF] [START_REF] Zhang | Patterns of microRNA expression characterize stages of human B-cell differentiation[END_REF]. Among these miRs, several reports have demonstrated a key role for miR21 in inflammatory cells and increased miR21 expression is coupled with conditions involving altered immune response including cardiovascular diseases [START_REF] Dai | The Cell Type-Specific Functions of miR-21 in Cardiovascular Diseases[END_REF]. In B cells, overexpression of miR21 leads to a pre-B malignant lymphoid-like phenotype, suggesting that miR21 may act as an oncogene in B cells [START_REF] Medina | OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma[END_REF].

In the present study, we reveal, for the first time, the involvement of splenic marginal zone B cells in the regulation of cardiac function and remodeling after acute MI. We also show that endogenous activation of miR21/Hypoxia-inducible factor(HIF)1α-related pathway is an integral component of splenic marginal zone B cell-dependent damaging effect on postischemic cardiac remodeling.

Methods

Experimental procedure

All experiments were conducted according to the ethical committee for animal experimentation (University of Paris, CEEA 34) and the National Charter on the ethics on animal experimentation from the French Minister of Higher Education and Research under the reference MESR: n° 01373.01. Acute MI was induced by permanent ligation of the left anterior descending coronary artery (LAD) in mice. Cardiac function and remodeling were analyzed using echocardiography and immunohistochemistry. For evaluation of the role of HIF-1α and HIF-2α in B cells: Cd79a cre/+ mice were crossed with Hif1a flox/flox and/or Hif2a flox/flox animals to generate Cd79a cre/+ /Hif1a flox/flox (referred to as the Hif1a -/-B cell group); Cd79a cre/+ /Hif2a flox/flox (referred to as the Hif2a -/-B cell group); Cd79a cre/+ /Hif1a-Hif2a flox/flox (referred to as the Hif1a-2a -/-B cell group) mice and their littermates Cd79a +/+ /Hif1a flox/flox , Cd79a +/+ /Hif2a flox/flox or Cd79a +/+ /Hif1a-Hif2a flox/flox (referred to as the WT B cell groups).

For evaluation of the role of MZB cells, Cd79a cre/+ animals were crossed with Rbpjk flox/flox mice to generate Cd79a cre/+ /Rbpjk flox/flox (referred to as the MZB ∆ group lacking MZB cells) and Cd79a +/+ /Rbpjk flox/flox (referred to as the MZB WT group) animals.

Statistical analysis

Kruskal-Wallis one-way analysis of variance was used to compare three or more independent experimental groups. Comparisons between groups were then performed using Dunn's multiple comparisons test when the analysis of variance test was statistically significant.

Mann-Whitney Test was used to compare two groups. A p value <0.05 was considered significant. Results are expressed as means with standard deviations or as boxes and whiskers with maximum and minimum values. P values presented in this report have not been adjusted for multiplicity, and therefore inferences drawn from these statistics may not be reproducible.

A detailed description of all experimental procedures is provided in the supplemental Appendix.

Results

Role of B cell specific miR21 in the response to acute MI

As mature B cells mainly reside in the spleen, we first assessed miR21 expression in the spleen of wild-type (WT) mice, which were sham-operated or subjected to a permanent LAD ligation to induce acute MI, at different time-points after the onset of ischemia. We found that miR21 was upregulated at day 1 after the ischemic insult in the spleen, when compared to sham-operated animals (Figure 1A). Using FACS analysis, we then determined miR21 levels in distinct splenic inflammatory cells at day 1 after MI and showed that miR21 was strongly expressed in B cells when compared to other types of splenic inflammatory cells, including neutrophils, T lymphocytes, monocytes and macrophages (Figure 1A). Splenic B cells are known to emerge from B cell progenitors within the bone marrow (BM) and subsequently to undergo final maturation in the spleen [START_REF] Pillai | The follicular versus marginal zone B lymphocyte cell fate decision[END_REF]. To assess the role of miR21 in mediating B cell dependent effects on post-MI cardiac remodeling, we generated WT chimeras through lethal irradiation and reconstitution with BM cells isolated from WT or miR21-deficient animals.

Transplantation of miR21 deficient BM did not impact the number of splenic B cells (Figure 1B) but reduced miR21 levels in splenic B cells when compared to mice transplanted with WT BM (Figure 1C). Left ventricular ejection fraction was similar in both un-operated and sham-operated chimeric animals (Supplemental Figure 1A). After induction of acute MI, cardiac function was higher in WT mice reconstituted with miR21 -/-BM cells when compared to WT chimeras (Figure 1D). Interstitial fibrosis was 1.7-fold lower in WT mice reconstituted with miR21 -/-BM compared to those receiving WT BM (Figure 1E). Infarct size and capillary density were unaffected (Figure 1E). We also substantiated the role of B cell specific miR21 in re-supplementation experiments using B cell transplantation in immunodeficient animals (Supplemental results and Supplemental Figure 1B-D). Altogether, these results indicate that miR21 controls B cell-dependent impact on post-ischemic cardiac remodeling.

B cell specific miR21 impacts the inflammatory response post-MI

Toll-like receptor (TLR) activation has been shown to trigger CCL7 secretion by B cells and governs bone marrow derived monocyte mobilization and infiltration in the ischemic milieu (3) (4). Increased miR21 expression is also associated with TLR activation [START_REF] Sheedy | Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21[END_REF]. Treatment with TLR agonist, CpG, for 24 hours led to a 1.8-fold increase of miR21 expression in cultured splenic B cells (Figure 1F). CpG stimulation was also associated with activation of CCL7 production by cultured WT B cells, and this effect was blunted in cultured B cells isolated from miR21 -/-mice (Figure 1G). CCL7 circulating levels were reduced in chimeric miR21 -/-animals at day 3 after MI (Figure 1H). We then evaluated the impact of CCL7 alteration on the number of monocytes (3) [START_REF] Nahrendorf | Monocyte and macrophage heterogeneity in the heart[END_REF]. Within the population of CD45 + CD11b + Ly6G -cells, classical inflammatory monocytes were characterized as Ly6C High cells (Supplemental Figure 2). miR21 deficiency was associated with a reduction in both circulating and cardiac Ly6C High monocytes (Figure 1I). Re-supplementation experiments using WT and miR21-deficient B cells also revealed variation of CCL7 and inflammatory monocyte levels (Supplemental results and Supplemental Figure1E-F). Thus, miR21 expression fosters CCL7 production by activated B cells, which promotes mobilization and infiltration of Ly6C High monocytes leading to adverse ventricular remodeling and cardiac dysfunction.

miR21 controls HIF-1α levels in B cells

MiR21 has been shown to enhance hypoxia inducible factor-1 (HIF-1)α signaling (18) (19) [START_REF] Richart | MicroRNA-21 coordinates human multipotent cardiovascular progenitors therapeutic potential[END_REF]. HIF-1α is a master transcription factor that controls B cell function [START_REF] Kojima | Differentiation stage-specific requirement in hypoxia-inducible factor-1alpha-regulated glycolytic pathway during murine B cell development in bone marrow[END_REF] [START_REF] Shin | HIF-1alpha-mediated upregulation of TASK-2 K(+) channels augments Ca(2)(+) signaling in mouse B cells under hypoxia[END_REF] [START_REF] Lall | Low-dose radiation exposure induces a HIF-1mediated adaptive and protective metabolic response[END_REF].

Interestingly, aberrant regulation of HIF-1α by miRNAs is a hallmark of chronic lymphocytic leukemia B cells [START_REF] Ghosh | Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells[END_REF]. HIF-1α also drives B cell-related effects in exacerbated collageninduced arthritis and experimental autoimmune encephalomyelitis [START_REF] Meng | Hypoxia-inducible factor-1alpha is a critical transcription factor for IL-10-producing B cells in autoimmune disease[END_REF]. We then speculated that miR21-induced regulation of HIF-1α controlled B cell related effects on post-ischemic cardiac homeostasis. For this purpose, HIF-1α levels were examined in splenic B cell isolated from WT and miR21 -/-mice and stimulated with CpG or anti-IgM. Upregulation of HIF-1α protein content was blunted in activated miR21 -/-B cells when compared to activated WT B cells (Figure 2A).

HIF-1α mediates B cell-related effects in mice with acute MI

To evaluate, the role of HIF-1α in B cells, we generated mice with B cell lineage specific conditional deletion of HIF1α (Hif1a -/-B cells). HIF1α deficiency did not affect cardiac function in un-operated and sham-operated animals (Supplemental Figure 3A). After MI, mice with Hif1a -/-B cells showed improved cardiac function when compared to WT animals (Figure 2B). Cardiac function recovery was associated with reduction in both infarct size and interstitial fibrosis whereas capillary density was unaffected (Figure 2B). HIF-1α invalidation did not reduce total B cell number in the spleen after MI (Figure 3A). CCL7 levels were decreased in blood of mice with Hif1a -/-B cells (Figure 3B). CpG treatment increased HIF-1 α transcriptional activity (Figure 3C) as well as CCL7 mRNA and proteins levels (Figure 3D) in WT cultured B cells. Of note, CCL7 upregulation was abrogated in cultured Hif1a -/-B cells treated with CpG (Figure 3D). As a consequence, the number of Ly6C High monocytes was lowered in the blood and the infarcted heart (Figure 3E). Similarly, cardiac macrophage accumulation (gating strategy is shown in Supplemental Figure 2) as well as the expression of classical pro-inflammatory cytokines such as IL1ß and IL6, were reduced in mice with Hif1a - /-B cells when compared to WT animals, at day 7 after MI (Figure 3F).

HIF-2α is not involved in B cell-related effects post-MI

In mammalian cells, HIFα activity is mainly regulated by two α subunits, HIF-1α and HIF-2α.

Although HIF-1α and HIF-2α are paralogs and share extensive sequence homology, these two α subunits have also non-overlapping and sometimes even opposing roles [START_REF] Imtiyaz | Hypoxia-inducible factor 2alpha regulates macrophage function in mouse models of acute and tumor inflammation[END_REF]. Furthermore, HIF-1α deficiency has also been shown to increase levels of HIF-2α and mutual antagonism between HIFα isoforms can shape HIFα activity [START_REF] Yuan | Mutual antagonism between hypoxia-inducible factors 1alpha and 2alpha regulates oxygen sensing and cardio-respiratory homeostasis[END_REF]. We found that Hif2a mRNA levels were enhanced in miR21 -/-and Hif1a -/-splenic B cells, 1 day after the onset of ischemia (Supplemental Figure 3B). Cardiac function and post-MI remodeling were similar in mice with Hif2a -/-B cells and their WT littermates as well as in un-operated and sham-operated animals (Supplemental Figure 3C-D). On the same note, Hif2a deletion did not impact B cell numbers in the spleen, did not modulate blood CCL7 levels and did not alter circulating and cardiac Ly6C High monocyte number (Supplemental Figure 3 E-G). In contrast, whereas cardiac function was similar in un-operated and sham-operated animals (Supplemental Figure 3H), Hif1a and Hif2a deletion improved cardiac function after MI (Figure 4A). Cardiac function recovery was associated with reduction in infarct size whereas interstitial fibrosis and capillary densities were unaffected (Figure 4A). Hif1a and Hif2a deficiency also reduced Ly6C High monocytes and CCL7 levels in the blood along with a reduction of cardiac macrophage number and Il1ß and Il6 mRNA levels (Figure 4B-E). Altogether, these results indicate that HIF-2α does not control B cell related functions and that HIF-1α isoform plays a prominent role in the orchestration of B cell-induced deleterious effects on post-MI cardiac remodeling.

Marginal zone B cells are instrumental in the regulation of cardiac function and remodeling post-MI

Several subsets of B cells exist. They are broadly divided into innate-like B1 cells and B2 cells, which comprise marginal zone (MZB) and follicular B cells (FOB) with specific developmental pathways and functional properties [START_REF] Pillai | The follicular versus marginal zone B lymphocyte cell fate decision[END_REF]. Within the population of IgM + CD19 + B cells, MZB cells were identified as CD23 low/-CD21 high , and FOB cells as CD23 high CD21 low/- (Supplemental Figure 2). MZB cells displayed higher expression of miR21 when compared to FOB cells and miR21 expression was markedly upregulated in MZB cells of infarcted animals when compared to sham-operated mice (Figure 5A). To determine the specific role of MZB cells, we then used mice with MZB cell deficiency (MZB ∆ ) [START_REF] Nus | Marginal zone B cells control the response of follicular helper T cells to a high-cholesterol diet[END_REF]. As expected, MZB cell number was markedly decreased in the spleen of MZB ∆ mice (Figure 5B). Interestingly, MZB cell deficiency curbed MI-induced miR21 upregulation in splenic B cells (Figure 5C).

Cardiac function was similar in un-operated and sham-operated MZB ∆ and MZB WT animals (Supplemental Figure 3I). The selective depletion of MZB cells improved post-MI cardiac function and the remodeling process (Figure 5D-G), suggesting that MZB cells expressing miR21 played a significant role in the regulation of cardiac function and repair. On the same note, CpG stimulation enhanced CCL7 release in cultured MZB WT cells but not in MZB ∆ cells (Figure 6A). MZB cell-deficient mice also displayed altered circulating levels of CCL7 (Figure 6B) and reduced circulating and cardiac Ly6C High monocytes when compared to their wild-type littermates (Figure 6C). As a consequence, at day 7 after MI, the number of cardiac macrophages as well as Il1b and Il6 mRNA levels were decreased in the infarcted hearts of MZB ∆ mice when compared to MZB WT animals (Figure 6D). In additional set of experiments, we challenged WT and MZB cell-deficient animals with a sequence of ischemia-reperfusion for 40 minutes. MZB cell deficiency also improved cardiac function and reduced interstitial fibrosis in this experimental setting (Supplemental Figure 4).

miR21/HIF1α signaling controls marginal zone B cell-related effects in mice with acute MI

We then assessed the role of miR21/HIF1α signaling selectively in MZB cells. CpG-and IgM-induced HIF-1α upregulation was abrogated in B cells isolated from MZB ∆ mice when compared to those isolated from MZB WT animals (Figure 7A). To substantiate the role of miR21 in HIF-1α regulation within MZB cells, we cultured FOB and MZB cells from WT and miR21 deficient mice and analyzed HIF1α protein levels (Figure 7B). We showed that miR21 deficiency hampered CpG and IgM-induced upregulation of HIF-1α protein levels and CCL7 release in MZB but not in FOB cultured cells (Figure 7B). Finally, we generated chimeric mice in which MZB deficient mice have been partially irradiated and reconstituted with BM cells isolated from MZB ∆ , MZB WT , miR21 -/-or Hif1a -/-animals. Reconstitution restored the number of MZB cells in chimeric animals (Figure 7C) without affecting cardiac function in un-operated and sham-operated animals (Supplemental Figure 5A). Chimeric mice transplanted with MZB miR21 -/-cells displayed reduction of miR21 content in MZB cells (Supplemental Figure 5B) whereas chimeric mice transplanted with MZB Hif1a -/-cells showed decreased Hif1a mRNA levels in MZB cells (Supplemental Figure 5B-C). miR21 and Hif1a mRNA levels were unaffected in FOB cells in chimeric mice (Supplemental Figure 5B-C). Of great interest, transplantation with WT, miR21-deficient MZB or Hif1a-deficient MZB cells reduced circulating CCL7 levels and improved cardiac function when compared to chimeric MZB ∆ animals (Figure 7C-D). These effects were associated with reduction in infarct size and (Figure 7D), interstitial fibrosis, cardiac macrophage as well as Il1ß and Il6 mRNA contents (Supplemental Figure 5 D-F).

Thus, miR21 expression controls HIF-1α content in activated MZB cells and MZB cells govern inflammation-dependent ventricular remodeling and cardiac function (Central illustration).

Discussion

In the present work, we demonstrated that deletion of MZB cells or selective miR21 deficiency in MZB cells abrogated B cell-induced adverse ventricular remodeling after MI, indicating a determinant role for miR21-expressing MZB cells in this context. Although previously unsuspected, this new role of MZB cells in the response to acute ischemic injury is consistent with their innate-like properties and their strategic positioning at the bloodlymphoid interface, where they can sense changes in the systemic environment and mount immediate innate immune responses. miR21 up-regulation was correlated with activation of HIF-1α related signaling and conditional deletion of HIF-1α counteracted MZB cell-induced adverse cardiac remodeling and dysfunction. In mature B cells, persistent (re)-induction of HIF transcription factors limits proliferation, isotype switching, and levels of high-affinity antibodies in splenic germinal centers after immunization [START_REF] Cho | Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system[END_REF]. It is unlikely that those adaptive B cell functions were involved in our experimental conditions very shortly after ischemic injury. However, one can speculate that dying cells and matrix fragments within the ischemic infarcted milieu may produce specific signals, including cytokines or damageassociated molecular patterns, and activate innate-like MZB cells. In this line of reasoning, TLR activation is sufficient to activate the miR21/HIF-1α pathway in cultured MZB cells.

Nevertheless, further investigations are needed to elucidate how B cells are able to recognize these specific biological entities originating from the injured myocardium and how the ischemic milieu commands B cell effector functions. Our work also identifies a determinant role for miR21 and HIF-1α in the regulation of CCL7 production by MZB cells. CCL7 directs Ly6C High monocyte mobilization from the bone marrow and recruitment to inflammatory cardiac tissue (3) [START_REF] Tsou | Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites[END_REF]. Ly6C High pro-inflammatory monocytes are responsible for the scavenging of debris and secretion of pro-inflammatory cytokines and matrix-degrading proteases, which contribute to the myocardial injury (3) [START_REF] Nahrendorf | Monocyte and macrophage heterogeneity in the heart[END_REF]. Of note, bioinformatic analysis and chromatin immunoprecipitation assays revealed three functional hypoxia-response elements in the Ccl7 promoter, indicating that Ccl7 is a direct HIF-1α target gene [START_REF] Qi | Hypoxia inducible factor 1alpha in vascular smooth muscle cells promotes angiotensin II-induced vascular remodeling via activation of CCL7mediated macrophage recruitment[END_REF].

However, additional work will be required to deeply address the mechanisms through which miR21 and HIF-1α regulate CCL7 production.

As B cells have emerged as important players in atherogenesis and post-MI cardiac remodeling, our novel results add to a number of experimental and clinical evidence suggesting that B cells represent a promising therapeutic target for cardiovascular diseases.

Several B cell-directed therapies are already available such as rituximab, an anti-CD20 B cell depleting monoclonal antibody. Small-scale human studies have previously reported some beneficial effects on the cardiovascular system such as variable degrees of reduction in carotid intima-media thickness, improved flow-mediated dilation or decreased arterial thickness in rituximab-treated patients [START_REF] Porsch | Impact of B-Cell-Targeted Therapies on Cardiovascular Disease[END_REF]. Recently, we have conducted a phase 1/2 clinical trial investigating the impact of rituximab in patients with acute MI, and showed that infusion of rituximab within 24 hours of MI was safe and efficiently depleted circulating B cells [START_REF] Zhao | Rituximab in Patients with Acute STelevation Myocardial Infarction (RITA-MI): an Experimental Medicine Safety Study[END_REF].

The detrimental role of MZB cells at the acute phase of MI shown in the present study contrasts with their previously reported atheroprotective properties through modulation of T follicular helper cell functions [START_REF] Nus | Marginal zone B cells control the response of follicular helper T cells to a high-cholesterol diet[END_REF]. What are therefore the clinical implications of these opposing properties? We propose that a therapeutic strategy that depletes MZB cells needs to be both selective and limited to the acute phase after MI, allowing for MZB cell recovery after the acute phase and reducing any potential harm, which can result from a loss of their atheroprotective properties. The B cell depleting strategy currently being tested at the acute phase of MI in humans uses one single infusion of rituximab [START_REF] Zhao | Rituximab in Patients with Acute STelevation Myocardial Infarction (RITA-MI): an Experimental Medicine Safety Study[END_REF]. Rituximab depletes both FOB and MZB cells. It is interesting to note, however, that transitional B cells with innatelike immune properties appear to recover after a few months of a single low-dose rituximab [START_REF] Zhao | Rituximab in Patients with Acute STelevation Myocardial Infarction (RITA-MI): an Experimental Medicine Safety Study[END_REF], suggesting that a low dose may limit the loss of potentially atheroprotective B cells.

Besides, selective targeting of miR21 or HIF-1α signaling in MZB cells, which would probably not impact the atheroprotective properties of MZB cells, would be another potential therapeutic strategy in patients with acute MI

Study limitations

This study did not attempt to identify the specific cardiac derived signals that could activate miR21/HIF-1α signaling in MZB cells. It is also likely that miR21 and HIF-1α control additional targets that could modulate splenic, circulating or cardiac B cell related effects.

Conclusion

This work reveals a prominent role for the miR21/HIF-1α axis in guiding MZB cell function in response to MI through activation of CCL7-dependent mobilization and infiltration of proinflammatory monocytes into the ischemic myocardium.

Perspectives

COMPETENCY IN MEDICAL KNOWLEDGE: Acute MI induces activation of miR21/HIF1-α signaling in splenic marginal zone B cells, leading to the release of the chemoattractant cytokine CCL7 that fosters inflammatory monocyte infiltration in the cardiac tissue and precipitates adverse left ventricular remodeling and cardiac dysfunction.

TRANSLATIONAL OUTLOOK: Targeting B lymphocytes could represent a therapeutic opportunity to limit adverse ventricular remodeling after MI. This strategy is currently under investigation in patients with acute MI. Abbreviations: NS indicates non-stimulated; nd not detected; Sham, sham-operated animals; WT:WT or WT:miR21 -/-chimeric mice transplanted with BM derived cells isolated from WT or miR21 -/-animals, respectively. No corrections for multiple testing were applied.

Central illustration: role of marginal zone B cells after acute myocardial infarction.

Splenic marginal zone B cells are instrumental in the regulation of cardiac function and remodeling after acute myocardial infarction. miR21/HIF-1α signaling adjusts the ability of marginal zone B cells to modulate CCL7 secretion and subsequently impact inflammatory monocyte-dependent cardiac remodeling after injury.

were mechanically ventilated, and ischemia was produced by occluding the left coronary artery with a 7/0 monofilament suture (Peters surgical, France) on a tapered BV-1 needle for 40 minutes. Following 40 minutes of occlusion, the ligature was released. All experiments were performed with 10 to 25-week-old male animals and were carried out using age and littermate matched groups without randomization.

Chimeric mice

To evaluate the role of miR21, bone marrow cells were obtained by flushing tibiae and femora of donor mice. Wild-type mice were lethally irradiated with a total dose of 9.5 Gy and were intravenously injected 24 hours later with 1x10 7 total bone marrow cells from miR21 -/-or their wild-type littermates. Mice were then challenged with MI 8 weeks after bone-marrow reconstitution, as previously described (2). To assess the role of B lymphocyte expressing miR21, 7 days before MI induction, Rag1 -/-mice received either 2 x 10 7 wild-type splenocytes, 1.2 x 10 7 B cell-depleted splenocytes recovered from CD20 monoclonal antibody treated mice (200µg/mouse), as previously described (1), B cell-depleted splenocytes re-supplemented with 8 x 10 6 wild-type B lymphocytes or with 8 x 10 6 miR21 -/-B lymphocytes.

For evaluation of the role of miR21 and HIF-1a in MZB cells, Cd79a cre/+ /Rbpjk flox/flox recipient mice deficient for marginal zone B cells were partially irradiated with a total dose of 4.0 Gy and injected with 5x10 6 total bone marrow cells isolated from Cd79a +/+ /Rbpjk flox/flox (referred to as the MZB WT :MZB ∆ group), Cd79a cre/+ /Rbpjk flox/flox (referred to as the MZB ∆ :MZB ∆ group), miR21 -/-(referred to as the MZB miR21-/-:MZB ∆ group) or Cd79a cre/+ /Hif1a flox/flox (referred to as MZB Hif1a-/-:MZB ∆ group) animals, as previously described (3). Mice were then challenged with MI after 6 weeks.

Technologies). Cells were counted and used for FACS sorting or analyser, as previously described (4).

Single-cell suspensions of spleen, blood and heart were stained with fluorophore-conjugated antibodies in phosphate buffered saline at 4°C. Total cells were gated on Alexa Fluor 700conjugated CD45 (30-F11, BD Biosciences) and the following antibodies were used: Pe-Cy7- IgM + CD19 + , MZB cells as CD23 lo/-CD21 hi , FOB cells as CD23 hi CD21 lo/-, monocytes were identified as CD45 + CD11b + F4/80 -Ly6G -and Ly6C High or Low cells, macrophages as CD45 + CD11b + Ly6G -F4/80 + CD64 + cells.

For MZB and FOB cell purification, B cell enriched populations were first separated by an autoMACS Pro Separator (Miltenyi Biotec) using a B cell purification kit (Miltenyi Biotec) according to the manufacturer's instructions. B cells were then stained for 15 min at 4 °C with anti-CD23-PE and anti-CD21-FITC. After washing and staining with 7-AAD to assess cell viability, MZB (CD21 hi CD23 lo ) and FOB (CD23 hi CD21 lo ) cells were sorted using a FACSAria II (BD Biosciences). The purity of both cell populations was higher than 95%.

Quantitative real-time PCR

Quantitative real-time PCR was performed on a Step One Plus (Applied Biosystems). The following primer sequences were used: Relative expression was calculated using the 2-delta-delta CT method and expressed as arbitrary unit, as previously described (5).

Chemokines levels

Plasma levels of CCL7 were measured using Quantikine Elisa Kits (R&D Systems) according to the manufacturer's instructions.

Statistical analysis

Mice dying within 24 hours of coronary artery ligation surgery (<10% in all genotypes studied) were pre-specified as purely technical failures and excluded from subsequent analysis.

Figure 1 :

 1 Figure 1: Acute myocardial infarction upregulates miR21 levels in splenic B

Figure 2 :

 2 Figure 2: Improved cardiac function in mice with Hif1a deletion in B cells.

Figure 3 :

 3 Figure 3: HIF1a expression in B cells controls inflammatory reaction.

Figure 4 :

 4 Figure 4: HIF2a expression in B cells is dispensable for B cell-related effects.

Figure 5 :

 5 Figure 5: MZB cells are instrumental components of post-MI cardiac repair.

Figure 6 :

 6 Figure 6: MZB cells control inflammatory reaction.

Figure 7 :

 7 Figure 7: miR21-HIF1a signaling shapes the deleterious effect of MZB cells.

  Figure1

  Figure2

  conjugated anti-CD11b (M1/70, BD Pharmingen), phycoerythrin-conjugated anti-Ly6G (1A8, BD Pharmingen), FITC-conjugated anti-Ly6C (AL-21, BD Pharmingen), APC-conjugated anti-IgM (II-41, ebiosciences) PE-conjugated anti-CD23 (B3/B4, ebiosciences), FITCconjugated anti-CD21 (eBio4E3, ebiosciences), BV421-Conjugated anti-CD64 (X54-5/7.1, Biolegend), APC-conjugated anti-F4/80 (MCA497, AbD Serotec, Bio-Rad), Pe-Cy7conjugated anti-CD19 (1D3, BD). All antibodies were used at a dilution of 1:100. The total number of cells was then normalized to tissue weight. Events were acquired on an X20 Fortessa flow cytometer (BD Biosciences) and results were analyzed on FlowJo software. Dead cells were excluded based on forward and side scatter profiles. B cells were identified as

  -3′; β-actin: forward 5'-GGT-CCA-CAC-CCG-CCA-CCA-GT-3', reverse 5'-CGG-CCC-ACG-ATG-GAG-GGG-AAT-3'; Hif1a forward 5miR21 levels were quantified using real time qPCR TaqMan assay (Tm 00397, Applied Biosystems) following the manufacturer's instructions. miR21 expression was normalized to U6 snRNA (Tm 001973, Applied Biosystems) and snoRNA234 (Tm001234; Applied Biosystems) as ubiquitous controls.

Supplemental Figure 2

 2

This work was supported by Fondation pour la Recherche Médicale (DEQ20160334910 to J-S.S., FDT20160435312 to I.Z.), Fondation de France (FDF 00066471 to J-S.S. and C.P.), Fédération Française de Cardiologie (to J-S.S and Y.S.) China scholarship council (CSC, No.201708310220 to Y.S.) French National Research Agency (ANR to J-S.S.) and institutional grants from Université de Paris and the French National Institute for Health and Medical Research.

Supplemental methods

Experimental procedures

Male and female mice of 12 week-old were used. C57BL/6J and Rag1 -/-mice were obtained from Janvier (France). For evaluation of the role of HIF-1a and HIF-2a in B cells: Cd79a cre/+ mice were crossed with Hif1a flox/flox and/or Hif2a flox/flox animals to generate Cd79a cre/+ /Hif1a flox/flox (referred to as the Hif1a -/-B cell group); Cd79a cre/+ /Hif2a flox/flox (referred to as the Hif2a - /-B cell group); Cd79a cre/+ /Hif1a-Hif2a flox/flox (referred to as the Hif1a-2a -/-B cell group) mice and their littermates Cd79a +/+ /Hif1a flox/flox , Cd79a +/+ /Hif2a flox/flox or Cd79a +/+ /Hif1a-Hif2a flox/flox (referred to as the WT B cell groups). For evaluation of the role of marginal zone B cells (MZB), Cd79a cre/+ animals were crossed with Rbpjk flox/flox mice to generate Cd79a cre/+ /Rbpjk flox/flox (referred to as the MZB ∆ group lacking MZB cells) and Cd79a +/+ /Rbpjk flox/flox (referred to as the MZB WT group) animals.

Myocardial infarction

Myocardial infarction was induced by left coronary ligation, as previously described (1). Mice were anesthetized using Ketamine (100 mg/kg body weight) and Xylazine (10 mg/kg body weight) via intra-peritoneal injection, intubated and ventilated using a small-animal respirator.

The chest wall was shaved and a thoracotomy was performed in the fourth left intercostal space.

The pericardial sac was removed, and the left anterior descending artery was permanently ligated using a 7/0 monofilament suture (Peters surgical, France) at the site of its emergence close to the left atrium. The thoracotomy was closed with 6/0 monofilament sutures. The exact same procedure was performed for the sham-operated mice except that the ligation was not tied.

The endotracheal tube was removed once spontaneous respiration was resumed, and mice were placed on a warm pad at 37 °C until awakened. In a second set of experiment, anesthetized mice

Echocardiographic measurements

Transthoracic echocardiography was performed 14 days after surgery using an echocardiograph (ACUSON S3000 ultrasound, Siemens AG, Erlangen, Germany) equipped with a 14-MHz linear transducer (1415SP). The investigator was blinded to group assignment. Mice were anesthetized by isoflurane inhalation. Percentages of ejection fraction (%EF) were calculated, as previously described (4).

Immunohistochemistry

For evaluation of cardiac remodeling, hearts were excised, rinsed in PBS and frozen in liquid nitrogen. Hearts were cut by a cryostat (CM 3050S, Leica) into 5-7µm thick sections. Masson's trichrome and Sirius red stains were performed for infarct size and myocardial fibrosis evaluation, respectively. Infarct size was calculated as the ratio of total infarct circumference divided by total left ventricle circumference. The collagen volume fraction was calculated as the ratio of the total area of interstitial fibrosis to the myocyte area in the entire visual field of the section. Endothelial cells within capillaries were visualized after Bandeiraea Simplicifolia lectin staining (1:100, FITC-conjugated Griffonia simplicifolia, Sigma-Aldrich), and cardiomyocytes after Wheat Germ Agglutinin staining (1:200, Texas Red-conjugated, ThermoFisher).

Flow cytometry

Left ventricles and spleens were isolated, minced with fine scissors and gently passed through the Bel-Art™ Scienceware™ 12-Well Tissue Disaggregator (Fisher Scientific). Cells were collected, filtered through 40µm nylon mesh and washed with PBS with 1%v/v Fetal Bovine Serum. Red blood cells were removed by incubation in hemolysis buffer (StemCell

B lymphocyte isolation and culture

MZB and FOB cells were isolated as described above and cultured in IMDM medium supplemented with 10% Fetal Bovine Serum, β-mercaptoethanol (Gibco), penicillin (100 U/ml), and streptomycin (100 µg/ml) and BAFF (20 ng/ml, R&D systems). Purified B cells (1×10 6 /500µl) were plated in 24-well cell culture plates and stimulated for 8 or 24 hours under the following conditions: CpG ODN1668 (1 µM, InvivoGen) or AffiniPure F(ab')2 Fragment Goat anti-mouse IgM antibody (10 µg/ml, Jackson ImmunoResearch).

Dimethyloxaloylglycine, (DMOG, 1mM, Sigma-Aldrich) was added in some conditions to inhibit HIFα degradation. HIF-1α transcriptional activity was measured using the HIF-1α transcriptional factor activity assay kit (ab133104, abcam) according to the manufacturer's instructions.

Western Blot

Cultured B cells were washed with ice-cold PBS and spun down. Cells were lyzed and proteins denatured by directly resuspending the pellet in 1X Laemmli Buffer (62,5 mmol/l Tris-HCl pH 6.8, 70mM SDS, 10% Glycerol, 1% 2-Mercaptoethanol). Proteins were separated on a freshly casted 9% Bis-Acrylamide gels and transferred onto nitrocellulose membranes (0.2µm Bio-Rad). After Ponceau red staining to evaluate transfer's efficiency and saturating non-specific binding sites with 3% Blotto (3% skimmed milk in TBS/0.05% Tween-20), HIF-1α (Novus, 100-449, 1:1000) and pan-actin (Chemicon international, MAB 1501, 1:1000) were blotted. In some cases, membranes were stripped with freshly prepared stripping buffer (2M Tris-HCL pH 6.8, 10% SDS, 100mM β-Mercaptoethanol) and re-probed. Blots were scanned before reprobing to assess stripping efficiency. ImageJ software (NIH) was used for immunoblots quantification.

Supplemental results

To specifically substantiate the role of B cell expressing miR21, we injected B cell deficient Rag1 -/-mice either with wild-type splenocytes, B cell depleted splenocytes, or B cell depleted splenocytes re-supplemented with wild-type or miR21 -/-B lymphocytes. We first verified that re-supplementation with wild-type or miR21 -/-B lymphocytes restored B cell numbers in spleens of Rag1 -/-mice compared to mice injected with B cell-depleted splenocytes (Supplemental Figure 1B). Of interest, transfer of B cell-depleted splenocytes into Rag1 -/-mice improved left ventricular ejection fraction after MI compared to the transfer of non-depleted splenocytes (Supplemental Figure 1C). This effect was abrogated after re-supplementation of B cell-depleted splenocytes with B lymphocytes isolated from WT mice but not with miR21 -/- B lymphocytes (Supplemental Figure 1C). B cell depletion also reduced infarct size and interstitial fibrosis, which was antagonized by re-supplementation with WT but not miR21 -/-B lymphocytes (Supplemental Figure 1D). Similarly, re-supplementation with WT B lymphocytes was associated with an increase of CCL7 levels compared to mice receiving B cell-depleted splenocytes. In contrast, re-supplementation of B cell-depleted splenocytes with B lymphocytes isolated from miR21 -/-mice failed to increase CCL7 levels (Supplemental Figure 1E). Re-supplementation with WT B lymphocytes increased Ly6C High monocyte numbers in the blood and within the infarcted heart compared to administration of B cell-depleted splenocytes (Supplemental Figure 1F), a phenotype that was abrogated in mice resupplemented with miR21 -/-B lymphocytes (Supplemental Figure 1F).

Supplemental Figures


Lucile Ben

Carole Escartin
email: carole.escartin@cea.fr

Lucile Ben Haim
email: lucile.ben-haim@cea.fr

Astrocytes and neuropsychiatric symptoms in neurodegenerative diseases: exploring the missing links

Keywords: Neuron-astrocyte interactions, depressive-like symptoms, cell-specific targeting, neuronal networks, animal models, behavioral tests

Neurodegenerative diseases (ND) are characterized by primary symptoms such as cognitive or motor deficits. In addition, the presence of neuropsychiatric symptoms (NPS) in ND patients is being increasingly acknowledged as an important disease feature. Yet, their neurobiological basis remains unclear and mostly centered on neurons while overlooking astrocytes, which are crucial regulators of neuronal function underlying complex behaviors. In this opinion article, we briefly review evidence for NPS in ND and discuss their experimental assessment in preclinical models. We then present recent studies showing that astrocyte-specific dysfunctions can lead to NPS. Because many astrocyte alterations are also observed in ND, we suggest that they might underlie NDassociated NPS. We argue that there is a need for dedicated preclinical studies assessing astrocytebased therapeutic strategies targeting NPS in the context of ND. Highlights:  Neuropsychiatric symptoms (NPS) are common features of neurodegenerative diseases  NPS are often well replicated in animal models of neurodegenerative diseases  Astrocytes are crucial regulators of neuronal function  Astrocyte-specific dysfunctions can induce NPS  The role of astrocytes in neurodegenerative disease-associated NPS is overlooked

Introduction

Neurodegenerative disease (ND) main feature is the progressive dysfunction, followed by irreversible loss of neurons in specific brain regions, which determines the nature of each ND's cardinal symptoms. ND primarily affecting the brain include Alzheimer's disease (AD) and other tauopathies, which are mainly characterized by cognitive symptoms, whereas Parkinson's (PD) and Huntington's diseases (HD) classically lead to motor symptoms. In all these diseases, patients also often show neuropsychiatric symptoms (NPS). These deficits are mostly neglected as compared to "typical" cognitive or motor symptoms in ND despite their negative impact on both patient's quality of life and disease progression [START_REF] Lanctot | Neuropsychiatric signs and symptoms of Alzheimer's disease: New treatment paradigms[END_REF][START_REF] Aarsland | Neuropsychiatric Symptoms in Parkinson's Disease[END_REF][START_REF] Paoli | Neuropsychiatric Burden in Huntington's Disease[END_REF]. Dedicated preclinical studies to uncover the neurobiological basis of NPS in ND are sparse. Moreover, current hypotheses have so far largely focused on neurons and overlooked astrocytes, which are key regulators of neuronal function underlying complex behaviors [START_REF] Nagai | Behaviorally consequential astrocytic regulation of neural circuits[END_REF]. In this review, we will quickly summarize clinical evidence for NPS in ND patients. We will then focus on their replication in ND animal models and mention different behavioral tests used for NPS assessment. Then, we will present recent evidence showing astrocyte-dependent regulation of NPS in rodent models of depression. We argue that similar mechanisms of altered astrocyteneuron crosstalk could underlie NPS in the context of ND and be targeted for therapy.

Neuropsychiatric symptoms: a shared feature across ND

NPS include depression, anxiety, apathy, agitation, irritability, sleep disturbances, disinhibition and hallucinations. They have been consistently described in ND patients and are in fact, increasingly included in diagnostic criteria [START_REF] Cummings | The Role of Neuropsychiatric Symptoms in Research Diagnostic Criteria for Neurodegenerative Diseases[END_REF]. NPS can appear at different disease stages but are prevalent in prodromal phases. In this review, we will focus on AD, PD and HD, in which depression, anxiety and apathy typically precede cognitive or motor symptoms. This has been particularly well characterized in PD, where patients frequently show anxiety and depression several years (sometimes decade) before diagnosis [START_REF] Darweesh | Trajectories of prediagnostic functioning in Parkinson's disease[END_REF]. Unsurprisingly, NPS accelerate functional deterioration in ND patients. There is no clear consensus regarding the efficacy of antidepressants in patients with ND. Yet, selective serotonin reuptake inhibitors remain the most commonly prescribed antidepressants, due to their favorable side-effect profile [START_REF] Galts | Depression in neurodegenerative diseases: Common mechanisms and current treatment options[END_REF]. So far, there is no causative evidence showing a direct link between ND pathological mechanisms and NPS. Most studies have focused on "usual suspects" involved in major depressive disorder (MDD) that can also be dysregulated in ND. These include: neurotransmitter (glutamatergic, GABAergic, dopaminergic) and neuromodulatory (serotoninergic, noradrenergic) systems and the hypothalamus-pituitary-adrenal axis [START_REF] Faivre | The hidden side of Parkinson's disease: Studying pain, anxiety and depression in animal models[END_REF][START_REF] Kosel | Behavioural and psychological symptoms of dementia in mouse models of Alzheimer's disease-related pathology[END_REF][START_REF] Gubert | Why Woody got the blues: The neurobiology of depression in Huntington's disease[END_REF]. Interestingly, ND have both critical differences (distinct pathological mechanisms, primarily vulnerable brain regions) and common features (abnormal neuronal networks, neuroinflammation, misfolded proteins). So whether NPS are caused by NDspecific pathology in limbic brain areas or altered connections between ND-affected and limbic regions is not known.

Assessing neuropsychiatric symptoms in ND models

NPS in animal models mainly recapitulate features of depression including anxiety, anhedonia, apathy, decreased social interactions and passive coping. These symptoms can be assessed using multiple well-established tests (Figure 1). We will focus on rodent ND models, which are mostly used in preclinical studies and mention some results obtained in non-human primates.

ND mouse models display a range of NPS, replicating symptoms observed in ND patients (see extensive reviews for AD [START_REF] Kosel | Behavioural and psychological symptoms of dementia in mouse models of Alzheimer's disease-related pathology[END_REF], PD [START_REF] Faivre | The hidden side of Parkinson's disease: Studying pain, anxiety and depression in animal models[END_REF] and HD [START_REF] Gubert | Why Woody got the blues: The neurobiology of depression in Huntington's disease[END_REF], Table 1). Interestingly, transgenic models, neurotoxin models (e.g. MPTP, 6-OHDA for PD) and others such as amyloid-β oligomer infusion [START_REF] Ledo | Amyloid-beta oligomers link depressive-like behavior and cognitive deficits in mice[END_REF], all display NPS. Discrepancies in results shown in Table 1 can stem from several causes. First, ND are progressive and studies performed at different disease stages sometimes gave opposite results.

Second, studies have used males or females, which may produce conflicting results. Traditionally, behavioral testing has mostly been performed on males because of the supposed variability in female performances due to their ovarian cycle or higher basal anxiety but also because some behavioral tests have only been designed for males (e.g. female urine sniffing test) [START_REF] Shansky | Considering sex as a biological variable will require a global shift in science culture[END_REF]. However, considering the higher prevalence of MDD and some ND in women, there is an increasing consensus that both sex should be systematically used in preclinical studies, with appropriate tests and measured outcomes [START_REF] Shansky | Considering sex as a biological variable will require a global shift in science culture[END_REF][START_REF] Gururajan | The future of rodent models in depression Calcium Signaling In Vivo Alters Striatal Microcircuits and Causes Repetitive Behavior[END_REF]. Last, differences in mouse background strains, housing and experimental conditions between laboratories (e.g. light/dark cycle, diet, background music, light exposure) can change behavioral performances, for example by changing anxiety level baseline. Within a disease model, discrepancies can also appear in behavioral test interpretation. For example, some AD mouse models show increased time spent in the center at the open field test (an indication of low anxiety) but with hyperlocomotion, which instead could be interpreted as an anxious behavior. At the splash test, time spent grooming can be interpreted either as apathy (if decreased) or as obsessive-compulsive disorder-like behavior (if increased).

Most studies focused on a given type of NPS, and did not perform a comprehensive behavioral assessment on the same model. The use of test batteries is useful, as some NPS might be disease-specific (e.g. repetitive behavior in HD [14]). Performing multiple and complementary behavioral tests on the same animal also allows z-score calculations to identify potential behavioral patterns and facilitates comparison between cohorts [START_REF] Gururajan | The future of rodent models in depression Calcium Signaling In Vivo Alters Striatal Microcircuits and Causes Repetitive Behavior[END_REF]. The inclusion of positive controls (e.g. stress models), displaying altered performances to most of these tests would also be appropriate.

This can be challenging to implement, especially because laboratories working on ND and psychiatric disorders are usually distinct. Of note, the tail suspension test and the forced swim test, which were originally designed to replicate hopelessness observed in MDD patients, have been instrumental in developing anti-depressant therapy, but are now largely criticized from both an ethical standpoint and scientific relevance [START_REF] Gururajan | The future of rodent models in depression Calcium Signaling In Vivo Alters Striatal Microcircuits and Causes Repetitive Behavior[END_REF].

Developments in behavioral neuroscience converge towards new paradigms to measure "natural" behaviors in rodents, including group-living. Machine-learning tools allowing single animal tracking have recently been developed but their application to group-housed animals is still challenging [START_REF] Mathis | DeepLabCut: markerless pose estimation of user-defined body parts with deep learning[END_REF]. Another example is the Souris city setup, which consists in a compartmentalized social cage where mice live in large groups for prolonged periods of time and are individually tracked through radio-frequency identification chips [START_REF] Torquet | Social interactions impact on the dopaminergic system and drive individuality[END_REF]. This setup allows the detection of specific and stable behavioral traits in individuals. This is of particular relevance for NPS, as inter-individual behavior tends to be highly variable and can even be used as a classifier (i.e. stress susceptible vs resilient mice). SmartCube® is another automated behavioral platform using computer vision and mechanical actuators to detect spontaneous behaviors and reactions to various stimuli in individual mice [START_REF] Brunner | Comprehensive Analysis of the 16p11.2 Deletion and Null Cntnap2 Mouse Models of Autism Spectrum Disorder[END_REF]. The AutonoMouse system allows the simultaneous training and testing of group-housed mice performing discrimination tasks [START_REF] Erskine | AutonoMouse: High throughput operant conditioning reveals progressive impairment with graded olfactory bulb lesions[END_REF]. Last, refined behavioral analysis of facial expressions and body postures, especially during social interactions are being actively developed [START_REF] Ebbesen | Body language signals for rodent social communication[END_REF]. Such tools are being characterized in normal mice with few examples of applications in models of psychiatric diseases. They need to be first characterized in "pure" models of depression/stress before being applied to ND models. Some NPS such as psychosis might not exist or are more challenging to detect in rodents.

Recent studies in mice reported hallucination-like percepts of auditory stimuli [START_REF] Schmack | Striatal dopamine mediates hallucination-like perception in mice[END_REF] and head-twitch response as a specific hallucinogen-induced behavior [START_REF] Dong | Psychedelic-inspired drug discovery using an engineered biosensor[END_REF].

Last, non-human primate models of ND also show a variety of NPS. For example, MPTPlesioned monkeys display apathetic behavior [START_REF] Brown | Dopamine pathway loss in nucleus accumbens and ventral tegmental area predicts apathetic behavior in MPTP-lesioned monkeys[END_REF], anxiety, lack of motivation, excessive grooming [START_REF] Tao | Autologous transplant therapy alleviates motor and depressive behaviors in parkinsonian monkeys[END_REF] and hallucinatory-like responses to non-apparent stimuli [START_REF] Visanji | Pharmacological characterization of psychosis-like behavior in the MPTP-lesioned nonhuman primate model of Parkinson's disease[END_REF]. Monkeys expressing mutant αsynuclein also show anxiety-related stereotypical behavior [START_REF] Niu | Early Parkinson's disease symptoms in α-synuclein transgenic monkeys[END_REF]; while HD transgenic monkeys display increased anxiety and aggression [START_REF] Raper | Increased irritability, anxiety, and immune reactivity in transgenic Huntington's disease monkeys[END_REF].

Astrocytes regulate neuronal function and animal behavior

Research on NPS has so far largely focused on neurons. However, recent studies show that non-neuronal cells such as astrocytes may also play a role in these symptoms. Astrocytes are key partners of neurons and fulfill many vital roles such as trophic and metabolic support; regulation of synaptic transmission; neurotransmitter reuptake or maintenance of K + and water homeostasis [START_REF] Verkhratsky | Physiology of Astroglia[END_REF].

Thanks to the development of new tools allowing precise manipulation of astrocyte Ca 2+ signaling, it is now well established that astrocytes regulate the activity of neurons involved in a myriad of behaviors including memory, motor control, feeding, fear or circadian rhythms [START_REF] Nagai | Behaviorally consequential astrocytic regulation of neural circuits[END_REF]. Furthermore, recent evidence showed that astrocytes are not identical throughout the brain. Instead, they have brain region-specific features allowing tailored interactions with their neuronal neighbors [START_REF] Haim | Functional diversity of astrocytes in neural circuit regulation[END_REF]. This is particularly relevant for NPS that involve distinct, yet highly connected brain circuits [START_REF] Muir | Wiring the depressed brain: optogenetic and chemogenetic circuit interrogation in animal models of depression[END_REF]. It is now well described that abnormal neuron-astrocyte crosstalk contributes to ND pathogenesis. For example in AD, several key astrocyte functions are altered and subsequently impact neuronal networks [START_REF] Escartin | Reactive astrocyte nomenclature, definitions, and future directions[END_REF]. Restoring some of these functions in primarily affected brain regions can have beneficial effects on AD symptoms. However, such approaches have so far not been used to assess the specific impact on NPS in ND. In fact, there is now multiple evidence showing that abnormal local astrocyte functions contribute to NPS in the context of MDD (Figure 2).

Astrocyte dysfunctions in MDD

Post-mortem studies show that astrocyte numbers are decreased in patients with MDD, although the extent depends on the brain region/subregion and method used to detect astrocytes [START_REF] O'leary | Implication of cerebral astrocytes in major depression: A review of fine neuroanatomical evidence in humans[END_REF]. This is associated with decreased expression of astrocyte-specific proteins including GFAP, connexins, glutamate transporters and glutamine synthetase. Yet, some other proteins enriched in astrocytes are upregulated in the brain and cerebrospinal fluid of MDD patients, including the inward rectifying K + channel 4 subunit 1 (KIR4.1) and the secreted protein S100β, respectively [START_REF] Hidese | Cerebrospinal fluid neuroplasticity-associated protein levels in patients with psychiatric disorders: a multiplex immunoassay study[END_REF]. It is important to note that both proteins are also expressed in oligodendrocytes, in the human and mouse brain, so their dysregulation cannot be exclusively assigned to astrocytes. Interestingly, most of these findings are replicated in animal models of depression (see below). In recent years, single nuclei RNA sequencing revealed molecular correlates of many ND and MDD, with unprecedented resolution and showed that not only neurons but also glial cells display profound changes in their expression profile [START_REF] Nagy | Single-nucleus transcriptomics of the prefrontal cortex in major depressive disorder implicates oligodendrocyte precursor cells and excitatory neurons[END_REF][START_REF] Mathys | Single-cell transcriptomic analysis of Alzheimer's disease[END_REF]. Recent studies also integrated data from genome-wide association studies with human brain proteome for both MDD [START_REF] Wingo | Brain proteome-wide association study implicates novel proteins in depression pathogenesis[END_REF] and AD [START_REF] Wingo | Insulin regulates astrocyte gliotransmission and modulates behavior[END_REF]. Comparing such datasets could identify common dysregulated genes and pathways.

Local astrocyte dysfunctions can induce NPS

Recent work revealed that key astrocyte functions and signaling are involved in NPS (Figure 2). Here, we emphasize on innovative studies which used combinatorial, mostly astrocyte-and region-specific approaches. The majority of preclinical studies focused on depressive-like symptoms -hereafter called NPS for clarity -which were mainly assessed using the forced swim test, the tail suspension, the sucrose preference test and the three-chamber test (Figure 1).

Ca 2+ signaling and gliotransmission

Pioneering work showed that decreased vesicular release by astrocytes induces NPS and that ATP supplementation has anti-depressant effects in mouse models of chronic stress. This effect was replicated by activation of Ca 2+ -dependent ATP release by astrocytes and subsequent activation of purinergic receptors P2X2 in the medial prefrontal cortex (mPFC) [37]. More recently, astrocyte release of ATP through the ion channel Calhm2 has also been associated with NPS. Astrocyte-specific knockout of Calhm2 led to decreased ATP concentrations, loss of hippocampal spines and NPS in mice [38]. Moreover, Yu et al. used an original strategy to decrease astrocyte Ca 2+ signaling via a plasma membrane pump, which constitutively extrudes cytosolic Ca 2+ . Attenuation of Ca 2+ signaling in striatal astrocytes was sufficient to trigger repetitive grooming (an obsessive-compulsive disorderlike behavior), associated with altered striatal neuron activity in freely behaving mice [14].

Ionic and metabolic homeostasis

Astrocyte may also impact NPS by regulating synaptic transmission through K + and glutamate Last, another recent study in the mPFC showed that neuron-astrocyte metabolic coupling regulates mouse passive coping response to stress induced by forced swimming. Lactate levels, as measured by in vivo microdialysis, were increased in the mPFC of mice exposed to acute stress. Local virus-mediated knockdown in the mPFC of the astrocytic monocarboxylate transporter 4 (MCT4), which exports lactate in the extracellular space, decreased immobility duration at the forced swim test, interpreted as an abnormal passive coping strategy. On acute slices, MCT4 knockdown was shown to increase pyramidal neuron excitability. Thus, neuron-astrocyte metabolic coupling regulates mPFC neuron excitability and output response to stress [43].

Hormone signaling

Astrocytes express a variety of hormone receptors, which contribute to their interaction with neurons. Using astrocyte-specific, virus-mediated gain-and loss-of-function approaches in rats and mice, Wahis et al. showed that oxytocin receptor-mediated signaling induces Ca 2+ signals in astrocytes and regulates central amygdala neuronal output through NMDA receptors co-activation via D-serine. Activation of oxytocin signaling reduced neuropathic pain-induced anxiety, through the astrocyte receptor. Interestingly, authors showed that only a subset of central amygdala astrocytes with distinct morphological features express oxytocin receptors [44]. Another study showed that astrocyte-mediated insulin signaling modulates NPS in mice. Inducible conditional knockout of insulin receptors in astrocytes induced several types of NPS. Virus-mediated cre-recombination in the NAc but not the mPFC increased passive coping at the forced swim test, without affecting anxiety. The lack of insulin signaling in astrocytes reduced ATP release, and subsequent dopamine-mediated activation of medium sized spiny neurons in the NAc [45].

Inflammation

Under various pathological conditions, astrocyte undergo a range of molecular, morphological and functional changes (i.e. they become reactive), which contributes to neuroinflammation and potentially impacts astrocyte-neuron crosstalk [START_REF] Escartin | Reactive astrocyte nomenclature, definitions, and future directions[END_REF]. Nevertheless, the precise impact of astrocyte reactivity on NPS is still unclear. Leng et al. observed that in mice exposed to chronic unpredictable mild stress or injected with lipopolysaccharide that both trigger NPS, menin levels (encoded by Men1) were decreased [START_REF] Leng | Menin Deficiency Leads to Depressive-like Behaviors in Mice by Modulating Astrocyte-Mediated Neuroinflammation[END_REF]. Surprisingly, astrocyte-but not neuron-specific Men1-depleted mice displayed NPS, whereas memory performances were unchanged.

Mechanistically, menin deficiency in astrocytes was found to activate the NF-κB pathway, which in turn triggered IL1β-mediated microglial activation and neuronal dysfunction.

Shared astrocyte-specific dysfunctions between MDD and ND

Strikingly, many of the astrocyte dysfunctions recently identified as leading to NPS are also involved in the pathophysiology of ND. These include altered Ca 2+ signaling and gliotransmission, abnormal ionic and metabolic homeostasis and inflammation [START_REF] Escartin | Reactive astrocyte nomenclature, definitions, and future directions[END_REF]. These cellular alterations can go in opposite directions in NPS and ND (e.g., Kir4.1 function is increased in depression models whereas it is decreased in HD [START_REF] Tong | Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice[END_REF]) -which could be due to regional differences. Yet, their similarities strongly suggest that circuit-specific alterations of neuron-astrocyte crosstalk could be responsible for NPS in ND. Many studies have shown that restoring these astrocyte functions within primary vulnerable brain regions have beneficial effects on cardinal disease symptoms [START_REF] Escartin | Reactive astrocyte nomenclature, definitions, and future directions[END_REF], but NPS were rarely assessed, except anxiety [START_REF] Guillemaud | Complex roles for reactive astrocytes in the triple transgenic mouse model of Alzheimer disease[END_REF].

Conclusions

Astrocytes are not merely supporting cells for neurons but active and specific regulators of neuronal circuits controlling various behaviors. There is an increasing amount of evidence showing that replicating astrocyte dysfunctions observed in patients with MDD can cause in NPS in rodents.

Because similar dysfunctions are often described in ND models, we suggest that they could underlie NPS associated with ND.

Yet, both fields of ND and neuropsychiatric disorders, have traditionally been neurocentric and for long neglected glial cells, such as astrocytes. The missing link(s) between astrocytes, NPS and ND need to be tackle with cell-and region-specific preclinical studies, on relevant mouse models with advanced behavioral profiling (Figure 3). Ultimately, the challenge will be to translate therapeutic astrocyte targeting to patients, to offer alternative options for NPS treatment.

* This review article discusses current animal models of depression used in preclinical studies. It

argues that new models of various aspects of depression should be developed using a "backtranslation" approach from patients to animal models.

14.

Yu X, Taylor AMW, Nagai J, Golshani P, Evans CJ, Coppola G, Khakh BS: Reducing Astrocyte

  photoactivation of astrocytes in the ventral tegmental area (VTA) promotes avoidance behavior by regulating glutamate-dependent drive on inhibitory GABA neurons, which reduces dopaminergic neuron activity. Interestingly, conditional knockout of the glutamate transporter GLT-1 in VTA astrocytes reduced avoidance but did not affect reward behavior [41]. Xiong et al. showed that epoxyeicosatrienoic acid (EET), an arachidonic acid metabolite, modulates astrocytic ATP release and NPS in a mouse model of chronic stress. Genetic manipulation to either reduce or enhance EET signaling specifically in mPFC astrocytes, impacted NPS in these mice. The astrocyte-specific enzyme involved in EET signaling was shown to regulate ATP-release from lysosomes, which in turn acted on purinergic receptors to modulate synaptic transmission [42].

Figure 1 .

 1 Figure 1. Tests and measured parameters for behavioral assessment of NPS in rodents. This figure depicts the variety of behavioral tests used to assess core NPS including anxiety (cyan), decreased social interactions (orange), anhedonia (green) and passive coping (purple). The dark blue panel summarizes newly-developed automated platforms and machine-learning based setups for highthroughput evaluation of behaviors in group-housed rodents.

Figure 2 .

 2 Figure 2. Key astrocyte functions are dysregulated in depression and their manipulation regulates NPS in rodent models. This figure summarizes recent findings linking astrocyte (dys)functions to NPS in animal models of depression. For each function (colored box), changes are data obtained in MDD patients and animal models of depression as compared with controls. For each function, text in italic below indicates manipulations of local astrocytes and their behavioral consequences on NPS (brain region in brackets). NPS refers to symptoms evaluated by a combination of tests (tail suspension test, forced swim, splash test, three-chamber test, open field elevated plus maze). If a single symptom was affected by the experimental manipulation, it is indicated (e.g. anxiety, passive coping). LOF (loss of function) /GOF (gain of function) refer to direct manipulation of the molecular target by knockdown/knockout or overexpression, respectively. ↓/↑ refer to the activation or inhibition of a cellular process (e.g. Ca 2+ signaling). Abbreviations: AQP: aquaporin, CeM: medial subdivision of the central amygdala central nucleus, Cx: connexin, EET: epoxyeicosatrienoic acid, GFAP: glial fibrillary acidic protein, GLT: glutamate transporter, Gln: glutamine, GS: glutamine synthetase, Glu: glutamate, HPC: hippocampus, IR: insulin receptor, Kir: Inward rectifying K + channel, LHb: lateral habenula, MCT: monocarboxylate transporter, mPFC: medial prefrontal cortex, NAc: nucleus accumbens, OxR: oxytocin receptor, OCD: obsessive compulsive disorder, ST: Striatum, vACC: ventral anterior cingulate cortex, vDG: ventral dentate gyrus, VTA: ventral tegmental area. [51-54].

Figure 3 .

 3 Figure 3. Astrocytes and neuropsychiatric symptoms in neurodegenerative diseases: exploring the missing links. 1. ND patients display NPS at early disease stages. 2. Recent evidence show that astrocyte dysfunctions are observed in MDD patients and in rodent models displaying NPS. 3. Similar astrocyte dysfunctions are also described in ND patients and animal models. Therefore, we suggest that astrocyte dysfunctions could contribute to NPS in ND.

This work was supported by CEA, CNRS and grants from the French National Research Agency (grants # ANR-20-CE16-0012-02, 2011-INBS-0011 (NeurATRIS national infrastructure) and France Alzheimer to C.E.). L.B.H is currently supported by a Fondation pour la Recherche Medicale fellowship (ARF201909009244) and a Fondation Vaincre Alzheimer Pilot award (FR-20007p, 2021).

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