Albert Giralt

Renata Coura

Jean-Antoine Girault
email: jean-antoine.girault@inserm.fr

Pyk2 is essential for astrocytes mobility following brain lesion

Keywords: Gelsolin, wound healing, actin polymerization, lamellipodia, filopodia 2

Pyk2 -/-astrocytes migration is

INTRODUCTION

The central nervous system (CNS) responds to trauma by producing a scar, which is primarily comprised of reactive astroglia [START_REF] Pekny | Astrocyte activation and reactive gliosis[END_REF]. In mammals, the glial scar has beneficial as well as negative properties: it allows prompt repair of the blood-brain barrier, inhibits the entry of inflammatory cells into damaged brain, and limits neuronal death [START_REF] Bush | Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice[END_REF]), but also impairs axonal regeneration [START_REF] Mcgraw | Modulating astrogliosis after neurotrauma[END_REF]) and incorporation of neuronal grafts [START_REF] Kinouchi | Robust neural integration from retinal transplants in mice deficient in GFAP and vimentin[END_REF]. Following focal CNS injury, a subset of astrocytes or astroglial precursors throughout the brain undergo mitosis and morphological changes to yield reactive astroglia, which migrates towards the lesion and organizes into a densely packed glial scar [START_REF] Hampton | The responses of oligodendrocyte precursor cells, astrocytes and microglia to a cortical stab injury, in the brain[END_REF][START_REF] Wang | Vimentin-expressing proximal reactive astrocytes correlate with migration rather than proliferation following focal brain injury[END_REF]). This migration process is dependent on actin dynamics [START_REF] Cowin | Differential expression of F-actin in in utero fetal wounds[END_REF][START_REF] Cowin | Differential effect of wounding on actin and its associated proteins, paxillin and gelsolin, in fetal skin explants[END_REF] placing this cytoskeletal protein at a convergence point of many important intracellular pathways. However, although some mechanisms modulating this phenomenon have been described, the entire molecular framework is not fully understood.

Focal adhesion kinase (FAK) family proteins have been described to play a role in cell migration during different conditions and paradigms [START_REF] Tomar | Focal adhesion kinase: switching between GAPs and GEFs in the regulation of cell motility[END_REF]. Prolinerich tyrosine kinase 2 (Pyk2) is a non-receptor, calcium-dependent protein-tyrosine kinase that belongs to the FAK family [START_REF] Lev | Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions[END_REF][START_REF] Sasaki | Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily[END_REF]. Whereas FAK is expressed in all tissues, Pyk2 is highly enriched in the brain, especially the forebrain where it is regulated by neuronal activity and may be involved in synaptic plasticity [START_REF] Girault | FAK and PYK2/CAKbeta in the nervous system: a link between neuronal activity, plasticity and survival?[END_REF][START_REF] Huang | CAKbeta/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus[END_REF][START_REF] Siciliano | Differential regulation of prolinerich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) and pp125(FAK) by glutamate and depolarization in rat hippocampus[END_REF]. However, although Pyk2 is highly expressed in neurons [START_REF] Menegon | FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern[END_REF] it is also present in astrocytes [START_REF] Cazaubon | Growth factor activity of endothelin-1 in primary astrocytes mediated by adhesion-dependent andindependent pathways[END_REF]. The role of Pyk2 in peripheral tissues has been previously investigated using knockout mice, implicating Pyk2 in macrophages morphology and migration [START_REF] Okigaki | Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration[END_REF], neutrophils degranulation [START_REF] Kamen | Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection[END_REF], bone formation and degradation [START_REF] Buckbinder | Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis[END_REF][START_REF] Gil-Henn | Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2(-/-) mice[END_REF], protection against cardiac arrhythmia [START_REF] Lang | Role of Pyk2 in cardiac arrhythmogenesis[END_REF], and skin wound repair [START_REF] Koppel | Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice[END_REF]. In contrast, the effects of this deletion on the nervous system has not been explored. Despite the fact that the role of Pyk2 in astrocytes is completely elusive, its involvement in several pathways capable to modulate actin dynamics in other cell types [START_REF] Lim | PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility[END_REF][START_REF] Murasawa | Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase[END_REF][START_REF] Ying | PYK2/PDZ-RhoGEF links Ca2+ signaling to RhoA[END_REF]) led us to hypothesize that Pyk2 could regulate astrocytes migration targeted to form the glial scar after physical lesion.

To address directly the function of Pyk2 in the brain we have generated Pyk2 knockout mice in our laboratory. Since we found that many aspects of astrocytes morphology and physiology were unaltered in Pyk2 -/-mice, we focused on astrocytes reactivity and function after a physical lesion into the brain. We observed that astrocytes from Pyk2 -/-mice displayed a slower wound covering as compared to wild type littermate mice after a mechanical stab lesion. We confirmed these data in purified astrocytes in vitro.

We then observed that actin polymerization dynamics was altered in Pyk2 -/-astrocytes.

Gelsolin, an actin-capping protein able to interact with Pyk2, was mislocalized in Pyk2 -/- migrating astrocytes compared to Pyk2 +/+ astrocytes, providing a possible link to the role of Pyk2 in cell migration.

MATERIALS AND METHODS

Pyk2 mutant mice

We have generated a new line of Pyk2 knock-out mice in collaboration with Gen-O-way (Lyon, France). The model was generated by homologous recombination in embryonic stem (ES) cells and the following strategy was used: a targeting vector containing regions homologous to the genomic Pyk2 sequences was constructed for deletion of the Pyk2 exons 15 to 18 (Fig. 1A), corresponding to the kinase domain. Splicing between exons 14 and 19 is out of frame, resulting in a premature STOP codon in exon 19. Mice were genotyped from tail biopsy by Charles River (Erkrath, Germany). The primers used for DNA amplification were: Pyk2 forward: 5'-TGTGCTCAGAGAAAAACGGAGGAACCCT-3', Pyk2 reverse 1: 5'-CATTGATTCCTGCTTCAGCCCTGGTCTAA-3' and Pyk2 reverse 2: 5'-GCCCATCGGGGCGATTTAAATATAATTCG-3'. Breeding strategy generated Pyk2 +/+ , Pyk2

Stab lesions

We performed stab lesions in adult Pyk2 +/+ , Pyk2 +/-and Pyk2 -/-mice as previously described [START_REF] Saadoun | Involvement of aquaporin-4 in astroglial cell migration and glial scar formation[END_REF]. Briefly, mice were anesthetized with a mixture of xylazine (10 mg/ml) and ketamine (25 mg/ml) (Centravet) and mounted in a digitalized stereotactic frame (Stoelting Europe) equipped with a mouse adaptor. After incising the skin, a 27G X 3/4" 0.4x20mm needle was directly introduced through the skull at the following coordinates (in millimeters): AP, +0.6, L, +2.0 from bregma and DV, -2.7 from skull. After 1 min, the needle was removed and the skin was sutured. Mice were maintained in animal house conditions during 72 h and then sacrificed.

Stab lesion characterization

The volume of the stab lesion and the region with reactive astrogliosis was measured using the Cavalieri's method, as previously described [START_REF] Giralt | BDNF regulation under GFAP promoter provides engineered astrocytes as a new approach for long-term protection in Huntington's disease[END_REF], by evaluating coronal sections spaced 80 µm apart. We also calculated the % of orientated cells/field in these coronal sections as described [START_REF] Nagaoka | CPEB-mediated ZO-1 mRNA localization is required for epithelial tight-junction assembly and cell polarity[END_REF]. Briefly, we considered an astrocyte to be orientated when it projected the majority of processes to the wound direction (perpendicular to the wound). Only astrocytes within a 90 µm field surrounding the wound were considered.

Tissue fixation and immunofluorescence

Animals were deeply anesthetized with pentobarbital (60 mg/kg) and intracardially perfused with a 4% (weight/vol) paraformaldehyde solution in 0.12 M sodium phosphate, pH 7.2.

Brains were removed and post-fixed overnight in the same solution, cryoprotected with 300 g/l sucrose in 20 mM sodium phosphate, pH 7.5, 150 mM NaCl (PBS) with 0.2 g/l sodium azide and frozen in dry ice-cooled isopentane. Serial coronal sections (30 µm) obtained with a cryostat were processed for immunohistochemistry as free floating sections. They were washed three times in PBS, permeabilized 15 min by shaking at room temperature with PBS containing (vol/vol) 0.3% Triton X-100 and 3% normal goat serum (Pierce Biotechnology, Rockford, IL, USA). After three washes, brain sections were incubated overnight (o.n.) by shaking at 4°C with antibodies for glial fibrillary acidic protein (GFAP, 1:200, rabbit polyclonal Z033429, DAKO A/S, Glostrup, Germany) or Pyk2 (1:400, mouse monoclonal 610549, BD Transduction Laboratories, San Diego CA, USA) in PBS with 0.2 g/l sodium azide. After incubation with primary antibody, sections were washed three times and then placed 2 h on a shaking incubator at room temperature with the subtype-specific fluorescent secondary 488 anti-rabbit (1:250, Molecular Probes, Sunnyvale, CA). No signal was detected in control sections incubated in the absence of the primary antibody. Nissl staining was performed as described elsewhere [START_REF] Giralt | BDNF regulation under GFAP promoter provides engineered astrocytes as a new approach for long-term protection in Huntington's disease[END_REF].

Astrocytes cultures

Primary cortical astrocyte cultures were obtained from P1 to P3 Pyk2 +/+ , Pyk2 +/-, and Pyk2 Buchs, Switzerland) with L-glutamine (2 mM) and Earle's salts (Gibco-BRL) and placed in an incubator at 37°C with 5% CO 2 . Medium was changed every 3 days. Cells were cultured separately for each pup and a tail biopsy was obtained for genotyping. After two passages, the cultures were purified by agitating in a shaker 16-18 h at 250 r.p.m. Finally, medium with floating cells was replaced with new medium. This protocol provides cultures with a ~95% of astrocytes purity [START_REF] Giralt | BDNF regulation under GFAP promoter provides engineered astrocytes as a new approach for long-term protection in Huntington's disease[END_REF].

Adhesion and proliferation assays

We performed adhesion and proliferation assays as previously described [START_REF] Saadoun | Involvement of aquaporin-4 in astroglial cell migration and glial scar formation[END_REF]. Confluent cultured astroglia were trypsinized and suspended in NM-15. After measurement of cell density using a hemocytometer, 2.8 X 10 4 cells per cm 2 were plated in 24-well plates coated with poly-D-lysine (Sigma). The medium was exchanged 4 hours after plating. Adhesion was defined as the percentage of plated cells remaining immediately after medium exchange. To assess proliferation, the number of cells per well was determined every 2 days by trypsinization and cell counting.

Immunocytochemistry

Cells were fixed 10 min in a 40 g/l paraformaldehyde solution and, after 3 washes with PBS, cells were permeabilized 10 min with triton X-100 in PBS (0.1% vol/vol

Wound healing

As previously described [START_REF] Holtje | Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing[END_REF], confluent astrocytes monolayers growing on glass coverslips were used for scratch assays. Two perpendicular wounds of ~225 µm in width were scratched with sterile P100 tips. Cells were fixed 0, 24 or 48 h after wounding, stained with rhodamine-phalloidin (Sigma) and DAPI (see above), and photomicrographs were taken. Individual images (4 per scratch wound) taken with the 10 x magnification sections were obtained on a DM6000-2 microscope (Leica). The re-population of the lesion site over the whole wound was analyzed by measuring the cell-free area (in µm 2 ) using the ImageJ software.

In vitro actin repolymerization assay

Astrocytes (50-60% confluence) were treated with 1 µM latrunculin B (Calbiochem, San Diego, CA, USA) during 45 min with NM-15 without serum and then medium was removed and substituted by a normal NM-15. Cells were fixed at 0, 15 or 60 min after latrunculin B treatment. Control cells with no treatment were also fixed as control/baseline. Cells were stained for rhodamine-phalloidin (Sigma) and DAPI (see above). Total cellular area was analyzed using the ImageJ software.

Confocal imaging

Fluorescently stained coronal sections (cerebral cortex M1 region) or fixed astrocytes cultures were examined by confocal microscopy, using a Leica SP5 laser scanning confocal spectral microscope with argon and helium-neon lasers. Images were taken from mouse tissue or fixed cells with a 63× numerical aperture lens with 4× digital zoom respectively and standard (one Airy disc) pinhole. For each image obtained in tissue, the entire threedimensional stack of images was obtained by the use of the Z drive present in the Leica SP5 microscope with a separation of 2 μm between each one. For each image obtained in fixed cells, we focused on leading edges of migrating astrocytes, acquiring 0.5-μm separated stacks, which were used for analysis of the gelsolin positive area and mean intensity. Images were analyzed by the freeware NIH ImageJ version 1.33 by Wayne Rasband (National Institutes of Health, Bethesda, MD). The morphological analysis of labeled GFAP-positive astrocytes was performed by using the Sholl approach in the same software.

Immunoblot analysis

Mice were deeply anesthetized in a CO 2 chamber, the brains quickly removed, cortex dissected out, frozen in dry ice and stored at -80°C until use. Briefly, tissue was sonicated in 250 ml of lysis buffer (PBS, 1% Nonidet P40 [vol/vol], 1 g/l SDS, 5 g/l sodium deoxycholate, protease inhibitors cocktail 1:1000 (Sigma), and 2 g/l sodium orthovanadate) and centrifuged at 12 000 r.p.m. for 20 min and the pellet was discarded. For in vitro studies, confluent astrocytes were stimulated during 15 min in serum-free medium containing 10 ng/ml of TNFα (Sigma) or only vehicle and cell homogenates were obtained at 15 or 60 min after TNFα treatment. Cells were lysed in lysis buffer (as above

RESULTS

Brain gross anatomy and astrocytes in Pyk2 knockout mice

We designed and generated a novel line of Pyk2 mutant mice in collaboration with Gen-oway (France). The strategy is summarized in Fig. 1A and detailed under Materials and Methods. Pyk2 +/+ , Pyk2 +/-and Pyk2 -/-mice were born at Mendelian frequencies (Pyk2 +/+ , 24%, Pyk2 +/-, 54 %, and Pyk2 +/-, 22%, from 115 genotyped mice at 4-6 weeks of age) and supervision of their home cages indicated that the gross behavior, general health, body weight (in grams at 12 weeks of age, Pyk2 +/+ , 30.80 ± 1.05, Pyk2 +/-, 30.47 ± 0.84, and Pyk2 -/-, 30.16 ± 1.02; F (2, 25) = 0.09, p = 0.9) and appearance of mice of the three genotypes were normal (Fig. 1B). Immunoblot analysis of hippocampal tissue, the brain region with the highest Pyk2 levels [START_REF] Menegon | FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern[END_REF], showed that total protein levels of Pyk2 were reduced around 50% in Pyk2 +/-mice compared to Pyk2 +/+ mice and undetectable in Pyk2 -/- mice (Fig. 1C andD). Accordingly, phosphorylation levels of Pyk2 Tyr402, its autophosphorylation site leading to its activation [START_REF] Avraham | RAFTK/Pyk2-mediated cellular signalling[END_REF], were reduced around 50% of wild type in Pyk2 +/-mice and undetectable in Pyk2 -/-mice (Fig. 1C andE). In wild type mice Pyk2 immunofluorescence was detected in most forebrain regions, especially in the hippocampus and also the cortex, striatum and amygdala (Fig. 1F). The immunofluorescence was reduced in heterozygous and undetectable in homozygous mutant mice (Fig. 1F). Importantly, the brain gross anatomy of Pyk2 +/-and Pyk2 -/-mice appeared normal (Fig. 1G) indicating that Pyk2 is not essential for the development of the central nervous system.

To assess the shape of astrocytes in Pyk2 knockout mice, we immunostained brain sections for GFAP and compared the appearance of astrocytes in wild type and mutant mice (Fig. 1H). The size and morphology of astrocytes were similar in the two genotypes. To assess more precisely the shape and branching of astrocytes we carried out a Sholl analysis (Fig. 1I), similar to what is usually done to evaluate the dendritic tree of neurons [START_REF] Sholl | Dendritic organization in the neurons of the visual and motor cortices of the cat[END_REF], but can also be applied to evaluate astrocytes complexity [START_REF] Sullivan | Structural remodeling of gray matter astrocytes in the neonatal pig brain after hypoxia/ischemia[END_REF]. There was no difference between wild type and Pyk2 lacking mice. These results showed the lack of gross brain defect in Pyk2 knockout mice as well as the absence of major astrocytic alteration.

Astrocyte migration after cortical stab lesion is slowed in Pyk2 knockout mice

To determine the role of Pyk2 in astroglial activation and cell migration in vivo, we studied reactive gliosis and glial scar formation using a well-established cortical-stab-injury model [START_REF] Hampton | The responses of oligodendrocyte precursor cells, astrocytes and microglia to a cortical stab injury, in the brain[END_REF][START_REF] Wang | Vimentin-expressing proximal reactive astrocytes correlate with migration rather than proliferation following focal brain injury[END_REF]). The stab-injury mouse model, unlike other brain injury models (tumor, infection, trauma), is not associated with significant brain swelling, which could confound interpretation of results [START_REF] Saadoun | Involvement of aquaporin-4 in astroglial cell migration and glial scar formation[END_REF]. Three days after stab lesion, reactive astrocytes were identified by their multiple processes and strong glial fibrillary acidic protein (GFAP) immunoreactivity in the cortical M1 area in all genotypes (Fig.

2).

In wild type mice, although Pyk2 immunoreactivity was much stronger in neurons than in astrocytes, double staining with GFAP revealed that Pyk2 was expressed in astrocytes migrating towards the stab lesion (Fig. 2A). We compared the speed of migration of reactive astroglia in Pyk2 +/+ , Pyk2 +/-, and Pyk2 -/-mice by measuring the volume of the wound, determined by the absence of GFAP labeling, 72 h after stab lesion. Representative GFAPimmunolabeled brain sections and the quantified data (Fig. 2B,C) show that at 72 h the stab lesion area was almost covered by Pyk2 +/+ and Pyk2 +/-astrocytes. In contrast, the stab lesion area was significantly larger in Pyk2 -/-mice as compared to Pyk2 +/+ mice (Fig. 2B,C)

indicating a slower migration rate of Pyk2 -/-astrocytes. To rule out the possibility of defects in astrocytes orientation mechanisms or of decreased astroglial reactivity, we analyzed the orientation of astrocytes towards the wound and the area of the astrogliosis in Pyk2 +/+ , Pyk2 +/-and Pyk2 -/-sections (Fig. 2D). We did not detect any significant differences between genotypes in the percentage of astroglial cells orientated towards the wound (Fig. 2E) or in the astrogliosis area (Fig. 2F). These results indicated that the activation of astrocytes was not modified in the absence of Pyk2, but that their migration was decreased.

Pyk2 knockout does not alter basic properties of astrocytes in culture

To better characterize the function of Pyk2 in astrocytes, we prepared primary cultures of astrocytes. We found that confluent astroglial cells cultured from Pyk2 +/+ and Pyk2 -/-mice had similar appearance by phase-contrast microscopy (Fig. 3A) and similar GFAP expression and immunostaining (Fig. 3B). About 95% of cells were stained for GFAP in wild type cultures, as described elsewhere [START_REF] Giralt | BDNF regulation under GFAP promoter provides engineered astrocytes as a new approach for long-term protection in Huntington's disease[END_REF], as well as in Pyk2 -/-cultures. We then labeled astrocytes for Pyk2 and F-actin. In wild type astrocytes, Pyk2 immunoreactivity had a punctate and diffused appearance throughout the cell body with no specific subcellular enrichment, except for a higher density in the perinuclear region (Fig. 3C). This distribution was similar to that reported in fibroblasts [START_REF] Aoto | Nuclear translocation of cell adhesion kinase beta/prolinerich tyrosine kinase 2[END_REF][START_REF] Sieg | Pyk2 and Src-family proteintyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK-cell migration[END_REF]. In Pyk2 -/- astrocytes, no Pyk2 immunoreactivity was observed, as expected, and the actin filaments looked comparable to those in Pyk2 +/+ astrocytes (Fig. 3C).

To determine whether the absence of Pyk2 altered the basic properties of astrocytes in culture, we measured by immunoblotting the levels of GFAP and S100B, two proteins that are specifically enriched in astrocytes [START_REF] Raponi | S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage[END_REF], and found no significant change in Pyk2 knockout mice (Fig. 3D,E). Similarly TrkB an important receptor in astrocytes, which express both its full length and truncated form (T1) [START_REF] Aroeira | BDNF, via truncated TrkB receptor, modulates GlyT1 and GlyT2 in astrocytes[END_REF][START_REF] Climent | Astrocytes in culture express the full-length Trk-B receptor and respond to brain derived neurotrophic factor by changing intracellular calcium levels: effect of ethanol exposure in rats[END_REF][START_REF] Rose | Truncated TrkB-T1 mediates neurotrophin-evoked calcium signalling in glia cells[END_REF], was unchanged (Fig. 3D,E). We also examined whether cell growth was altered in Pyk2-deficient astrocytes. The cultured Pyk2 +/+ and Pyk2 -/-astroglia had comparable proliferation rates, as assessed by serial cell counting (Fig. 3F). Finally, we examined the adhesion properties of Pyk2-/-astrocytes and found they displayed a similar adhesion to poly-D-lysine substrate (Fig. 3G). Altogether these results indicate that the basic properties of astrocytes in culture are not altered in the absence of Pyk2.

Wound healing migration deficits in Pyk2 -/-astrocytes are cell-autonomous

To better characterize the astrocytes deficit in wound healing in vivo, we investigated whether Pyk2 was also necessary for astrocytes migration in vitro and whether the deficit was a cell-autonomous phenomenon. We studied reactive gliosis and wound repair using in purified astrocytes cultures a well-established in vitro wound healing model [START_REF] Desai | RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells[END_REF][START_REF] Holtje | Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing[END_REF][START_REF] Liang | In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro[END_REF]. Since there was no phenotype in Pyk2 +/-mice in the stab lesion paradigm in vivo, we focused on the comparison between homozygous mutant and wild type astrocytes in culture. Primary astrocytes from newborn Pyk2 +/+ and Pyk2 -/-littermate mice were then cultured to confluence (Fig. 4). The monolayers were scratch-wounded and the abilities of Pyk2 +/+ and Pyk2 -/-astrocytes to heal the wound were compared. As shown in Fig. 4A, at 24 and 48 h post-wounding, Pyk2 -/-astrocytes exhibited a lower capability to fill and close the wound as compared to wild type cells. This was confirmed by the quantification of the wound area (cell-free area) in Pyk2 -/-astrocytes (Fig. 4B). Thus, the ability of Pyk2 -/-astrocytes to heal scratch wound in vitro was severely compromised, indicating that Pyk2 function and/or signaling in astrocytes is essential for scratch wound repair in this setting.

Actin polymerization dynamics are altered in Pyk2 -/-astrocytes

Pyk2 has been reported to be involved in cell migration in macrophages and K562 cell lines [START_REF] Ovcharenko | Enhanced adhesion/migration and induction of Pyk2 expression in K562 cells following imatinib exposure[END_REF]) and regulates actin organization in oocytes [START_REF] Meng | Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte[END_REF]).

Since wound healing-induced migration is dependent on actin dynamics [START_REF] Cowin | Differential expression of F-actin in in utero fetal wounds[END_REF], we examined whether this dynamics was impaired in astrocytes in the absence of Pyk2. Wild type or Pyk2-deficient primary mouse astrocytes were cultured to 50-60% confluence and then treated with latrunculin B (Fig. 5A), a drug which sequesters G-actin and prevents Factin assembly, and previously used to study dynamics in astrocytes in vitro [START_REF] Mitsushima | Abl kinase interacts with and phosphorylates vinexin[END_REF][START_REF] Tomas | Protective effects of lysophosphatidic acid (LPA) on chronic ethanol-induced injuries to the cytoskeleton and on glucose uptake in rat astrocytes[END_REF]. We found that 15 min and 60 min after latrunculin B treatment both Pyk2 +/+ and Pyk2 -/-astrocytes were able to significantly re-polymerize G-actin to F-actin (Fig. 5A). To quantify the speed of repolymerization, we measured the total phalloidinpositive area per cell in the cultures at several time points after latrunculin B treatment. In Pyk2 -/-astrocytes repolymerization was significantly slower than in wild type astrocytes (Fig. 5B). These results suggested that actin dynamics are compromised in Pyk2 -/-astrocytes, potentially accounting for their slower migration than wild type cells in the wound healing paradigm.

Regulation of Pyk2, PKC, and Rac1 by TNF in astrocytes

TNFα is produced by physical lesions in the brain [START_REF] Tchelingerian | Identification and topography of neuronal cell populations expressing TNF alpha and IL-1 alpha in response to hippocampal lesion[END_REF]) and attracts various cell types to the scar [START_REF] Su | Reactive astrocytes in glial scar attract olfactory ensheathing cells migration by secreted TNF-alpha in spinal cord lesion of rat[END_REF]. TNF can be produced by astrocytes [START_REF] Lieberman | Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus[END_REF]) and is active on these cells [START_REF] Benveniste | Tumor necrosis factor-alpha enhances interferongamma-mediated class II antigen expression on astrocytes[END_REF]). Interestingly, Pyk2 was reported to be activated in response to TNFα treatment in cultured H9c2 cells [START_REF] Yang | NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-alpha-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells[END_REF]. To mimic the molecular changes during wound healing and gliosis we therefore treated astrocytes in culture with TNFα (10 ng/ml) or vehicle and measured phosphorylation of Pyk2 at tyrosine 402 (pY402-Pyk2 , Fig. 6A). In Pyk2 +/+ astrocytes pY402-Pyk2 was significantly increased 15 min but not 60 min after TNFα treatment (Fig. 6A,B). As expected, no Pyk2 or pTyr402-Pyk2 signal was detected in Pyk2 -/-astrocytes samples (Fig. 6A). PKC is one of the signaling pathways downstream from TNFα in astrocytes [START_REF] Norris | Signal transduction pathways mediating astrocyte IL-6 induction by IL-1 beta and tumor necrosis factor-alpha[END_REF] and PKC can activate Pyk2 [START_REF] Lev | Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions[END_REF]. We first measured the level of PKC, an isoform prominently expressed in astrocytes [START_REF] Gott | Rat brain glial cells in primary culture and subculture contain the delta, epsilon and zeta subspecies of protein kinase C as well as the conventional subspecies[END_REF]) and reported to be activated by TNF [START_REF] Muller | PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid[END_REF]. We found no significant change in PKC protein levels in astrocytes between genotypes (Fig. 6C, Pyk2 +/+ : 100 ± 12, Pyk2 -/-: 87 ± 24; arbitrary units;

Student's t-test, p = 0.37). We then evaluated PKC activation using an antibody that detects phosphorylated serine/threonine in consensus region for PKC phosphorylation (Fig. 6C). PKCmediated phosphorylation was activated 15 min but not 60 min after TNFα treatment in both Pyk2 +/+ and Pyk2 -/-astrocytes (Fig. 6C,D). Since PKC can activate Pyk2 [START_REF] Lev | Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions[END_REF], this result was compatible with a role of PKC upstream but not downstream from Pyk2 activation in astrocytes in response to TNFα.

Since actin dynamics during cell migration is dependent on small G proteins including Rac1 and RhoA [START_REF] Ridley | Cell migration: integrating signals from front to back[END_REF] we examined whether these proteins were altered in Pyk2-deficient astrocytes. Total protein levels of Rac (Pyk2 +/+ : 100 ± 13, Pyk2 -/-: 113 ± 21;

Student's t-test, p = 0.35) and RhoA (Pyk2 +/+ : 100 ± 29, Pyk2 -/-: 104 ± 15; Student's t-test, p = 0.83) were not altered in Pyk2 -/-as compared to Pyk2 +/+ astrocytes. Then, since Akt, a kinase that can phosphorylate Cdc42/Rac1 at Ser71, can be activated by TNFα [START_REF] Chung | Effects of Tumor Necrosis Factor-alpha on Podocyte Expression of Monocyte Chemoattractant Protein-1 and in Diabetic Nephropathy[END_REF],

we examined the phosphorylation of this residue in astrocytes. PhosphoSer71 was increased at both 15 and 60 min after TNFα treatment, in Pyk2 +/+ as well as in Pyk2 -/-astrocytes, suggesting that the activation of this pathway was not altered by the mutation (Fig. 6E,F).

Our results show that Pyk2 is activated in astrocytes in response to TNFα, one of the factors involved in astrocytes recruitment by a brain injury, but that PKC activation and RhoA and Rac expression are unaltered in Pyk2 knockout cells.

Gelsolin distribution is altered in migrating Pyk2 -/-astrocytes

We next focused on a Pyk2 target that could be involved in the actin polymerization defects.

Pyk2 has been shown to interact with gelsolin and to be involved in actin rings formation in osteoclasts during bones remodeling [START_REF] Wang | Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin[END_REF]. Gelsolin is necessary for general motility of neutrophils, fibroblasts, and macrophages in culture [START_REF] Goncalves | Gelsolin is required for macrophage recruitment during remyelination of the peripheral nervous system[END_REF][START_REF] Witke | Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin[END_REF]. Gelsolin is also enriched in the filopodia of hippocampal neurons growth cone [START_REF] Lu | Delayed retraction of filopodia in gelsolin null mice[END_REF]) and modulates stereocilia length via actin remodeling in cochlea [START_REF] Mburu | Gelsolin plays a role in the actin polymerization complex of hair cell stereocilia[END_REF]. We therefore tested gelsolin localization in migrating astrocytes and whether it could be involved in the impairments we observed in Pyk2 -/-astrocytes in the wound healing paradigm. Although the distribution of Pyk2 punctate immunofluorescence was widespread in migrating astrocytes, it was slightly enriched at the leading edge (Fig. 7A). As expected, 24 h after scratching astrocytes monolayer, gelsolin was specifically localized at the leading edge of migrating wild type astrocytes (Fig. 7B, upper panel). This enrichment appeared less pronounced in Pyk2 -/-astrocytes (Fig. 7B, lower panel). We therefore quantified gelsolin accumulation at the leading edge of Pyk2 +/+ and Pyk2 -/-migrating astrocytes and we found a significantly smaller area (Fig. 7C) and reduced mean labeling intensity (Fig. 7D) in Pyk2 -/- migrating astrocytes as compared to wild type astrocytes. We then evaluated by immunoblot the total protein levels of gelsolin in both Pyk2 +/+ and Pyk2 -/-astrocytes (Fig. 7E, F) and found no significant difference between genotypes. These results indicated that in the absence of Pyk2, gelsolin levels do not change whereas its distribution is impaired during the wound healing-induced migration.

DISCUSSION

Migration of reactive astroglia is an important component of glial scar formation around damaged brain tissue. Our in vitro data and in vivo experiments provide evidence for a specific role of Pyk2 in the migration of reactive astroglia towards the site of injury. In contrast, Pyk2 deletion did not affect other astroglial functions, such as astrogliosis, astrocytes orientation, adhesion, or proliferation. Moreover, our results indicate that actin dynamics are severely compromised in astrocytes lacking Pyk2. We found an altered gelsolin distribution during cell migration towards the site of injury. Thus, our data show that Pyk2 is specifically involved in astrocytes migration towards the site of injury during a physical lesion and that this process involves the recruitment of gelsolin to the cells leading edge.

Pyk2 in astrocytes received little attention so far. An early study showed Pyk2 activation during the formation of stress fibers in response to endothelin-1 [START_REF] Cazaubon | Growth factor activity of endothelin-1 in primary astrocytes mediated by adhesion-dependent andindependent pathways[END_REF]. In this study, Pyk2 activation was associated with ERK activation, but was independent of FAK, Rho, and Src kinases. In a recent study, a broad-spectrum tyrosine kinase inhibitor, quercetin, prevented astrogliosis and astrocyte migration, although the authors did not study Pyk2 [START_REF] Yuan | Quercetin inhibits the migration and proliferation of astrocytes in wound healing[END_REF].

Actin rearrangement is crucial for cell motility and decreased actin turnover could severely affect cell migration during wound repair [START_REF] Goicoechea | The role of palladin in actin organization and cell motility[END_REF][START_REF] Ridley | Cell migration: integrating signals from front to back[END_REF]. In Pyk2-deficient astrocytes, we observed that actin dynamics were compromised, in agreement with the reported role of Pyk2 in actin rearrangement in 3T3-swiss cells [START_REF] Du | Inhibition of PYK2-induced actin cytoskeleton reorganization, PYK2 autophosphorylation and focal adhesion targeting by FAK[END_REF]). Pyk2 could potentially modulate actin dynamics through various mechanisms including regulation of Rac1/RhoA/Cdc42 signaling (reviews in Ben-Ze'ev 1997; [START_REF] Kaibuchi | Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells[END_REF][START_REF] Ridley | Cell migration: integrating signals from front to back[END_REF]. Rac1 and Cdc42 are active and concentrated at the front edge of cells during migration in conditions such as in a wound repair [START_REF] Ridley | Cell migration: integrating signals from front to back[END_REF]. As previously demonstrated, these pathways are directly or indirectly regulated by Pyk2. Pyk2 can activate Rac1 [START_REF] Murasawa | Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase[END_REF][START_REF] Paulino | TROY (TNFRSF19) is overexpressed in advanced glial tumors and promotes glioblastoma cell invasion via Pyk2-Rac1 signaling[END_REF]) and Rac1-deficient mice suffer from deficient wound healing [START_REF] Castilho | Rac1 is required for epithelial stem cell function during dermal and oral mucosal wound healing but not for tissue homeostasis in mice[END_REF]). Pyk2 also activates Cdc42 [START_REF] Ren | Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein[END_REF] and RhoA [START_REF] Lim | PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility[END_REF]. It is possible that a deficient activation of RhoA contributes to the deficient migration of Pyk2-/-astrocytes as suggested in the case of macrophages [START_REF] Okigaki | Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration[END_REF].

We examined signaling response in astrocytes treated with TNFα, a factor released and increased after physical brain injury [START_REF] Tchelingerian | Identification and topography of neuronal cell populations expressing TNF alpha and IL-1 alpha in response to hippocampal lesion[END_REF]. TNFα is known to regulate actin [START_REF] Deli | Exposure of tumor necrosis factor-alpha to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation of actin[END_REF] and calcium mobilization in astrocytes [START_REF] Santello | TNFalpha controls glutamatergic gliotransmission in the hippocampal dentate gyrus[END_REF], as well as astrocytes migration to lesion sites [START_REF] Su | Reactive astrocytes in glial scar attract olfactory ensheathing cells migration by secreted TNF-alpha in spinal cord lesion of rat[END_REF]). Furthermore, TNFα can activate PKC [START_REF] Muller | PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid[END_REF] and in turn PKC can activate Pyk2 [START_REF] Lev | Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions[END_REF]. We found evidence for activation of PKC signaling after TNFα treatment. TNFα also increased Pyk2 in wild type astrocytes, as reported in H9c2 cells [START_REF] Yang | NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-alpha-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells[END_REF]. As expected PKC activation was not prevented in Pyk2 KO astrocytes, as PKC is thought to be upstream from Pyk2. Interestingly, we also found evidence of increased Rac1 phosphorylation on Ser71 in response to TNF. This phosphorylation which is reported to depend on Akt and to alter GTP binding [START_REF] Kwon | Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1[END_REF], was also unaltered in Pyk2-deficient astrocytes.

We evaluated other links between Pyk2 and actin dynamics in astrocytes and focused on the putative role of gelsolin, which is increased in injured neural tissue [START_REF] Zhang | Burn injury induces gelsolin expression and cleavage in the brain of mice[END_REF]). Gelsolin interacts with and is regulated by Pyk2 phosphorylation in osteoclasts, in which it forms actin rings for bone remodeling [START_REF] Wang | Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin[END_REF]. Gelsolin was also observed around the inner edge of actin filaments in moving cells in skin lesions [START_REF] Cowin | Differential effect of wounding on actin and its associated proteins, paxillin and gelsolin, in fetal skin explants[END_REF]. Gelsolin is also necessary for macrophages migration in peripheral nerves injury [START_REF] Goncalves | Gelsolin is required for macrophage recruitment during remyelination of the peripheral nervous system[END_REF]. Importantly, Pyk2 phosphorylates gelsolin on tyrosine residues [START_REF] Wang | Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin[END_REF]. Gelsolin is activated by intracellular calcium increases [START_REF] Kronenberg | Impact of actin filament stabilization on adult hippocampal and olfactory bulb neurogenesis[END_REF]) and can contribute to cell migration [START_REF] Thompson | Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility[END_REF]). In the absence of gelsolin, actin filaments are stabilized and the migration of newborn cells is slowed in the hippocampus [START_REF] Kronenberg | Impact of actin filament stabilization on adult hippocampal and olfactory bulb neurogenesis[END_REF]). In our wound healing experiments we observed increased gelsolin immunoreactivity at the leading edge of migrating astrocytes corresponding to lamellipodia. This enrichment was much less pronounced in Pyk2-deficient migrating astrocytes. This result suggests that Pyk2 activation facilitates astrocytes migration by recruiting gelsolin at the leading edge of migrating cells and that impairment of this recruitment contributes to the defect in migration observed in the absence of Pyk2.

In conclusion, our results indicate that after physical brain injury, astrocytes lacking Pyk2 react normally, orientate themselves properly, and display efficient capacities of cell adhesion and proliferation. However, the ability of astrocytes to migrate towards the wound is compromised since the lack of Pyk2 alters actin dynamics, a deficit that could result, at least in part, from a lack of gelsolin recruitment at the leading edge of migrating astrocytes.

Thus, our findings provide insights into the role of Pyk2 in glial scar formation. Importantly, Pyk2 has been implicated in the migration of glioma cells [START_REF] Lipinski | Differential role of proline-rich tyrosine kinase 2 and focal adhesion kinase in determining glioblastoma migration and proliferation[END_REF][START_REF] Lipinski | The tyrosine kinase pyk2 promotes migration and invasion of glioma cells[END_REF][START_REF] Loftus | A Novel Interaction between Pyk2 and MAP4K4 Is Integrated with Glioma Cell Migration[END_REF], a response that involves the TNF pathway [START_REF] Paulino | TROY (TNFRSF19) is overexpressed in advanced glial tumors and promotes glioblastoma cell invasion via Pyk2-Rac1 signaling[END_REF].

Our results in astrocytes provide further clues about the role of Pyk2 in the phenotype of glial-derived tumors and its interest as a therapeutic target.

FIGURE 1 .

 1 FIGURE 1. Generation and characterization of Pyk2 knockout mice. (A) Strategy used to knock out the Pyk2 gene. Hatched rectangles, Pyk2 coding sequences; grey rectangles, noncoding exon portions; solid lines, chromosome sequences; triangles, LoxP sites; double triangles, FRT sites. (B) Representative mice from each genotype (Pyk2 +/+ , Pyk2 +/-, and Pyk2 -/- ). (C) Immunoblots of hippocampal homogenates probed with antibodies for phosphoTyr402-Pyk2 (pY402-Pyk2), Pyk2, and α-tubulin. (D-E) Quantification by densitometric analysis of immunoblots as in (C) showing the levels of phosphoTyr402-Pyk2 (D) and Pyk2 (E) in mice from the 3 genotypes. Bars are means ± s.e.m. and individual points each corresponding to a mouse are indicated (5-4 per group). Statistical analysis one-way ANOVA (D, F (2, 11) = 26.27, p<0.001; E, F (2, 11) = 169, p<0.001) and post hoc Tukey's test (*** p<0.001). No signal was detected in homozygous mutant mice. (F) Pyk2 immunofluorescence in brain slices from Pyk2 +/+ , Pyk2 +/-, and Pyk2 -/-mice. (G) Nissl stained sections showed no gross anatomy deficiencies in Pyk2 -/-or in Pyk2 +/-compared to Pyk2 +/+ mice. Scale bars, 1 mm. (H) GFAP-immunostained astrocyte in the cortex of a wild type (left panel) and Pyk2-/mouse (right panel). Scale bar, 200 µm. (I) Sholl analysis of astrocytes ramifications in the two genotypes. Data are means + SEM from 30 astrocytes from 3 different mice pergenotype. Two-way ANOVA effect of distance from soma, F (6, 406) = 171.0, p<0.0001, no effect of genotype, F (1, 406) = 0.10, p = 0.75, no interaction, F (6, 406) = 1.54, p = 0.16.

FIGURE 2 .FIGURE 5 .

 25 FIGURE 2. Astrocytes Pyk2 expression and migration after stab injury. (A) Brain sections from a wild type mouse were co-immunolabeled for GFAP (red) and Pyk2 (green). Left panel, low magnification, scale bar, 20 µm. Right panels, higher magnification, scale bar 2 µm. An astrocyte ramification identified by GFAP immunoreactivity and immunoreactive for Pyk2 is indicated by arrowheads. (B) Brain sections were immunolabeled for GFAP 3 days after a cortical stab lesion in Pyk2 +/+ , Pyk2 +/-, and Pyk2 -/-mice. Stab lesion area is indicated with a dashed line. Scale bar, 150 µm. (C) Quantification of the area of the GFAP-negative zone. (D) Higher magnification to show astrocytes orientation. White arrows indicate the direction of the wound. Scale bar, 25 µm. (E) Quantification of astrocytes oriented towards the wound. (F) Quantification of the GFAP-positive area. (C, E, F) Bars represent means ± s.e.m. (4-7 mice

FIGURE 6 .

 6 FIGURE 6. Signaling pathways activated in astrocytes following treatment with TNFα. Confluent Pyk2 +/+ and Pyk2 -/-astrocytes cultures were treated with vehicle (Veh.) for 15 min or TNFα (10 ng/ml) for 15 min or 60 min before lysis. Samples were analyzed by immunoblotting with the indicated antibodies. (A) Immunoblotting for pTyr402-Pyk2, total Pyk2, and α-tubulin as a loading control. Molecular weight marker positions are indicated on the right (kDa). (B) Quantification of results as in (A) for wild type astrocytes. (C) Immunoblotting for PKC phospho-substrates, total PKC, and α-tubulin as a loading control. (D) Quantification of results as in (C). (E) Immunoblot for pSer71-Rac1/Cdc42, total Rac1, and α-tubulin. (F) Quantification of results as in (E). Black dots: Pyk2 +/+ astrocytes. White dots: Pyk2 -/-astrocytes. (B, D, F) Bars represent means ± s.e.m. and individual experimental points are indicted (n = 4-5 culture for each treatment and genotype). Statistical analysis one-way ANOVA: B, F (2,9) = 5.53, p<0.05; D left, Pyk2 +/+ , F (2,9) = 4.76, p<0.05; D right, Pyk2 -/-, F (2,9) = 4.42, p<0.05; F left, Pyk2 +/+ , F (2,9) = 6.66, p<0.05; F right, Pyk2 -/-, F (2,14) = 5.07, p<0.05. Post hoc Tukey's test, *, p<0.05.

FIGURE 7 .

 7 FIGURE 7. Gelsolin recruitment to the leading edge of migrating astrocytes is decreased in the absence of Pyk2. Confluent Pyk2 +/+ and Pyk2 -/-astrocytes cultures were scratched to

  Tubulin (1:1000, T9026, Sigma). All blots were incubated overnight at 4°C with shaking, in the presence of the primary antibody in PBS with 0.2 g/l sodium azide.All results were expressed as the mean ± s.e.m. for each genotype or group. For the statistical analysis of in vivo and in vitro experiments, unpaired Student's t-test, one-way ANOVA or two-way ANOVA were used as specified in the figure legends. P<0.05 was considered significant.

	After several washes in TBS-T, blots were incubated with anti-rabbit IgG IRdye800CW-
	coupled or anti-mouse IgG IRdye700DXcoupled antibodies (1/2000, Rockland
	Immunochemicals, USA) and signal detected by the Odyssey system (Li-Cor) and analyzed
	using ImageJ. A monoclonal α-tubulin antibody (Sigma) was used as a loading control.
	Statistical analysis
	Immunoblots were probed with rabbit polyclonal antibodies for Cdc42 (1:1000, ab14241,
	Abcam, Cambridge, UK), gelsolin (1:1000, ab113229, Abcam), GFAP (1:1000, Z033429, DAKO
	A/S, Glostrup, Germany), PKC  (1:1000, 07-264 Merck-Millipore, Darmstadt, Germany),
	phospho-Ser PKC substrates (1:1000, 2261, Cell Signaling Technology, Beverly MA, USA),
	Pyk2 (1:1000, P3902, Sigma, Saint Louis MI, USA), phospho-Tyr402-Pyk2 (1:1000, 44618G,
	Invitrogen, Carlsbad, CA, USA), phospho-Ser71-Rac1/Cdc42 (1:1000, ab5482, Abcam),
	phospho-Ser188-RhoA (1:1000, RP1361, ECM Biosciences, Versailles KY, USA), S100B

). Proteins (15 mg) from cortex tissue or from astroglial cultures were analyzed by SDS-polyacrylamide gel electrophoresis (7.5% acrylamide, weight/vol) and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). Membranes were blocked in TBS-T (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.05% [vol/vol] Tween 20) with 50 g/l non-fat dry milk and 50 g/l BSA.

(1:1000, ab868, Abcam); or goat polyclonal antibodies for TrkB (1:1000, GT15080, Neuromics, Edina MN, USA); or mouse monoclonal antibodies for Rac1 (1:1000, 240106, Cell Biolabs), RhoA (1:1000, 240302, Cell Biolabs, San Diego CA, USA), Pyk2 (1:1000, 610549, BD Biosciences), or alpha-

ACKNOWLEDGEMENTS

The work was supported by Fondation pour la recherche sur le cerveau (FRC), Fondation pour la recherche médicale (FRM), and an ERC Advanced investigator grant to JAG. JAG's group is affiliated with the Paris School of Neuroscience and the Bio-Psy Laboratory of Excellence.

The authors declare no conflict of interest.

per group). Statistical analysis one-way ANOVA (C, F (2, 16) = 10.33, p = 0.0013; E, F (2, 13) = 0.76, p = 0.48; F, F (2, 14) = 0.055, p = 0.94) and post-hoc Tukey's test, **p<0.01). Quantification of results as in (J) in each sample the amount of protein was normalized to the amount of -tubulin. Individual data points are shown with means ± SEM; Pyk2+/+, n = 5 mice, -/-, n = 7 mice; GFAP, t 10 = 0.012, p = 0.99, S100B, t 10 = 1.57, p = 0.14, TrkB, t 10 = 0.95, p = 0.36. (F) Proliferation of Pyk2 +/+ and Pyk2 -/-astrocytes. Cells were counted (8-9 wells per time point) on alternate days after plating 2.8 x 10 4 astroglia/cm 2 . Statistical analysis, twoway ANOVA, F (1, 10) = 2.174, p = 0.17