The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation

The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation

Neurobiology of Disease 98 (2017) 137–148 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locat...

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Neurobiology of Disease 98 (2017) 137–148

Contents lists available at ScienceDirect

Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation Majistor Raj Luxman Maglorius Renkilaraj a,1, Lucas Baudouin b,1, Claire M. Wells c, Mohamed Doulazmi d, Rosine Wehrlé a, Vidjeacoumary Cannaya a, Corinne Bachelin b, Jean-Vianney Barnier e, Zhengping Jia f, Brahim Nait Oumesmar b, Isabelle Dusart a, Lamia Bouslama-Oueghlani a,b,⁎ a

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de Biologie Paris Seine, Neuroscience Paris Seine, F-75005 Paris, France Sorbonne Universités, UPMC Univ Paris 06, INSERM U 1127, CNRS UMR 7225, Institut du Cerveau et de la Moelle épinière, F-75013 Paris, France c Division of Cancer Studies, King's College London, UK d Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de Biologie Paris Seine, Adaptation Biologique et vieillissement, F-75005 Paris, France e Institute of Neuroscience Paris-Saclay, CNRS-Université Paris-Sud, UMR9197, F-91405 Orsay, France f Neurosciences & Mental Health, The Hospital for Sick Children, and Department of Physiology, Faculty of Medicine, University of Toronto, 555 University, Toronto, Ontario M5G 1X8, Canada b

a r t i c l e

i n f o

Article history: Received 26 April 2016 Revised 4 November 2016 Accepted 2 December 2016 Available online 6 December 2016 Keywords: PAK3 Differentiation Oligodendrocyte OPC Intellectual disability Myelin Development

a b s t r a c t Oligodendrocyte and myelin deficits have been reported in mental/psychiatric diseases. The p21-activated kinase 3 (PAK3), a serine/threonine kinase, whose activity is stimulated by the binding of active Rac and Cdc42 GTPases is affected in these pathologies. Indeed, many mutations of Pak3 gene have been described in non-syndromic intellectual disability diseases. Pak3 is expressed mainly in the brain where its role has been investigated in neurons but not in glial cells. Here, we showed that PAK3 is highly expressed in oligodendrocyte precursors (OPCs) and its expression decreases in mature oligodendrocytes. In the developing white matter of the Pak3 knockout mice, we found defects of oligodendrocyte differentiation in the corpus callosum and to a lesser extent in the anterior commissure, which were compensated at the adult stage. In vitro experiments in OPC cultures, derived from Pak3 knockout and wild type brains, support a developmental and cell-autonomous role for PAK3 in regulating OPC differentiation into mature oligodendrocytes. Moreover, we did not detect any obvious alterations of the proliferation or migration of Pak3 null OPCs compared to wild type. Overall, our data highlight PAK3 as a new regulator of OPC differentiation. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Oligodendrocytes (OLs) are the myelin forming cells of the central nervous system (CNS). Myelin plays critical functions, increasing action potential speed (Baumann and Pham-Dinh, 2001), and providing metabolic support for axons. Myelin is thus essential for the neuronal integrity in the CNS (Lee et al., 2012; Morrison et al., 2013). Myelinating OLs are generated through lineage progression of oligodendrocyte precursors (OPCs), which occurs through several developmental stages. In the final stages of differentiation, OPCs lose their mitogenic and migration abilities and finally acquire their myelinating skill (Baumann and Pham-Dinh, 2001; Emery, 2010; Pfeiffer et al., 1993). Aberrations in one or more of these stages of oligodendroglial development lead to

⁎ Corresponding author at: Institut du Cerveau et de la Moelle épinière (ICM), Hôpital Pitié-Salpêtrière Paris, 75013, France. E-mail address: [email protected] (L. Bouslama-Oueghlani). 1 Majistor Raj Luxman Maglorius Renkilaraj and Lucas Baudouin contributed equally to this work. Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2016.12.004 0969-9961/© 2016 Elsevier Inc. All rights reserved.

alterations observed in many pathologies, such as multiple sclerosis and leukodystrophies (Baumann and Pham-Dinh, 2001; El Waly et al., 2014). OLs and myelin abnormalities have also been reported in mental/psychiatric diseases (Edgar and Sibille, 2012; Takahashi et al., 2011). While previous studies focused on neurons in the neuropathogenesis of these diseases, recent reports demonstrated the critical role of myelin plasticity in cognitive and behavioral functions (Gibson et al., 2014; McKenzie et al., 2014; O'Rourke et al., 2014). Furthermore, it has been also reported that Clemastine, a pro-myelinating compound, is able to rescue impaired myelination of the prefrontal cortex and to reverse avoidance behavior in adult mice undergoing prolonged social isolation (Liu et al., 2016). Therefore, oligodendroglial cells could be potential targets for reversing cognitive disorders in neuropsychiatric diseases, such as depression, autism and schizophrenia. Myelin defects have been also reported in patients presenting intellectual disability like Down syndrome (Abraham et al., 2012; Koo et al., 1992; Olmos-Serrano et al., 2016; Vlkolinsky et al., 2001). However, previous studies on X-linked non-syndromic intellectual disability disorders in which Pak3 (p21 activated kinase 3) gene is mutated have been mainly focused on neuronal functions (Allen et al., 1998; Gedeon et al., 2003;

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Kreis and Barnier, 2009; Peippo et al., 2007b; Rejeb et al., 2008). Nevertheless, independent transcriptomic analyses revealed that Pak3 is expressed in OPCs and oligodendrocytes (Cahoy et al., 2008; Dugas et al., 2006; Nielsen et al., 2006). PAK3 is a serine/threonine kinase belonging to group I of the PAK family and acts downstream to the Rho GTPases Rac and Cdc42 (Kreis and Barnier, 2009). PAK3 is implicated in spine morphogenesis and synapse formation (Boda et al., 2004; Dubos et al., 2012; Kreis et al., 2007), in the differentiation of neural progenitors into neurons in Xenopus (Souopgui et al., 2002), and in the differentiation of mouse cortical GABAergic interneurons (Cobos et al., 2007). Pak3 null mice exhibit significant abnormalities in synaptic plasticity, specifically of the hippocampal late-phase LTP and learning and memory deficits (Meng et al., 2005). In the present study, we analyzed the expression of PAK3 in oligodendroglial cells and the impacts of Pak3 loss-of-function on oligodendrocyte development and myelination. We showed that PAK3 is highly expressed at the OPC stage and down-regulated in differentiated OLs. Moreover, Pak3-null mice displayed a transient delay of OPC differentiation and consequently a developmental myelination defect in the corpus callosum and to lesser extent in the anterior commissure. To decipher the cell autonomous effects of Pak3 deletion in oligodendroglial cells, we also examined proliferation, migration and differentiation in OPC cultures derived from Pak3-null and wild type mouse brains. Our data indicate that PAK3 is a new regulator of OPC differentiation. 2. Material and methods 2.1. Animals Swiss mice (Janvier, Le Genest Saint Isle, France), Pak3 knockout mice (Pak3 KO) and their wild type littermates (Meng et al., 2005) were used in this study. All in vivo experiments have been performed using males. The different experiments were performed at least in triplicates. The experimental plan was designed in accordance with the European Union Guidelines for the care and use of experimental animals. 2.2. Brain section preparation Postnatal day 7 (P7), P10, P14 and 2 month-old (P60) mice were anesthetized with sodium pentobarbital (200 mg/kg, i.p.). All animals were perfused through the ascending aorta with 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS), pH 7.4. Brains were removed, post-fixed 4 h and cryoprotected in 30% sucrose for 2 days. The forebrains were cut in the coronal plane (20-μm-thick sections) on a cryostat. The sections were then processed for immunohistochemistry. 2.3. Rodent OPC cultures The rodent OPC culture protocol was adapted from McCarthy and de Vellis (McCarthy and de Vellis, 1980). The primary cultures of glial cells were obtained from forebrain animals at P1–P2. The tissue was dissociated mechanically until homogenization in DMEM GlutaMAX (Invitrogen) containing 10% of bovine serum (Invitrogen), 100 units/ ml penicillin (Life Technologies), 100 μg/ml streptomycin (Invitrogen). Cells were plated on polyornithine (50 μg/ml, Sigma-Aldrich) coated flasks. Cultures were maintained in an incubator (37 °C with 5% CO2). The medium was changed the 4th day of culture and thereafter every two days. The secondary cultures of oligodendrocytes were obtained from primary glial cultures of 8–10 days in vitro. Two steps of shaking (250 rotations per min, 37 °C) lead to the purification of OPCs. The first shaking (1 h) leads to the elimination of microglial cells. The second shaking (18 h) detaches OPCs from the astrocyte layer. The supernatant is submitted to different steps of preferential adhesion (30 min on Petri dishes (Falcon), 37 °C, 5% CO2) in order to eliminate microglial cells and astrocytes, which had not been eliminated in the previous steps of

purification. The obtained OPCs are maintained in culture in a complete medium, which is replaced after approximately 16 h, by a proliferation medium containing DMEM GlutaMax, B-27 (Sigma) and 1% antibiotics (Sigma), FGF (Fibroblast Growth Factor, 25 ng/ml; Sigma) and PDGFBB (Platelet Derived Growth Factor BB, 10 ng/ml; Sigma) or a differentiation medium (DMEM GlutaMax, B-27 and 1% antibiotics and 40 ng/ml of T3 thyroid hormone, T3, from Sigma). 2.4. Antibodies (immunostainings) The following primary antibodies were used in this study: goat antiOlig2 (1/500, R&D Systems Europe) to visualize oligodendroglial cells, rat anti-MBP antibody (MBP: myelin basic protein, 1/500; Millipore), mouse monoclonal anti-APC (CC1; 1/1000, Calbiochem) to detect differentiated oligodendrocytes, rat anti-CD140a that labels PDGFRα + OPCs (1/500, anti-PDGFRα, BD Pharmingen) and rabbit anti-Ki67 (1/200, BD Pharmingen) to stain proliferating cells, O4 (1/500, mouse monoclonal IgM, Millipore) to detect pre- and differentiated oligodendrocytes and NG2 antibody (1/500 rabbit polyclonal, Millipore) to stain OPCs. FITCconjugated Phalloidin (1 μg/ml, Sigma) and Hoechst (1 μg/ml, Sigma) were used, respectively, to stain actin filaments and nuclei. The secondary antibodies used were CY3-conjugated goat antimouse (1:200 dilution; Jackson ImmunoResearch Laboratories), CY3conjugated goat anti-rabbit (1:200 dilution; Jackson ImmunoResearch Laboratories, CY3-conjugated donkey anti-rabbit (1:500 dilution; Jackson ImmunoResearch Laboratories), CY3-conjugated donkey anti -goat (1/200 dilution, Jackson Immunoresearch), CY3-conjugated goat antirat (1/200, Jackson ImmunoResearch Laboratories). Alexa Fluor 488conjugated donkey anti-mouse (1/400, Invitrogen) and Alexa Fluor 647-conjugated donkey anti-mouse (1/200, Invitrogen). 2.5. Immunostainings 2.5.1. Brain sections Antigen retrieval for Ki67 antibody staining was performed by heating sections in Vector solution (Vector, H-330) up to boiling and then cooling down the solution at room temperature for 20 min. Sections were next washed several times with PBS. In all cases, sections were incubated for 1 h in 0.1 M PBS containing 0.2% gelatin, 0.1% sodium azide (PBSGA) and 0.1 M lysine, before applying the primary antibodies, diluted in PBSGA overnight. After several times of washing steps in PBS, the primary antibodies were revealed with the corresponding secondary antibodies. After 2 h incubation in PBSGA containing the secondary antibodies, the sections were washed several times in PBS and mounted in Mowiol (Calbiochem). For tissue sections, images were acquired with Zeiss Apotome.2 and Axiovision software (Carl Zeiss). 2.5.2. Cultures Dissociated cultures of OPCs and oligodendrocytes were immunostained with different primary antibodies (NG2, MBP, O4, PDGFRα). Cells were first permeabilized in PBS containing 0.1% Triton X100 during 5 min. Then, they were incubated with the primary antibody in PBS/BSA 5% solution during 2 h and then 1 h with the secondary antibody. Several washing steps in PBS were performed after the incubation with the primary and secondary antibodies. Immunostainings were analyzed using a DMR microscope equipped with a Coolscan camera (Princeton Instruments). Images were captured using Metaview software (Universal Imaging Corporation). Finally, figure panels were prepared using Adobe Photoshop version 9.0 software (Adobe System, Inc.). 2.6. Electron microscopy Electron microscopy was performed as described previously (Bachelin et al., 2005). Animals (P14 and P60) were perfused intracardially with a mixture of 5% glutaraldehyde (Euromedex) and 4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer saline. Brains were cut

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with a vibratome into 100 μm thick slices. Sections were post-fixed in 2% osmium tetroxide (Euromedex) and dehydrated in graded series of ethanol prior embedding in epon (Euromedex). Ultra-thin sections (80 nm) were analyzed with a Hitachi 120kv HT 7700 electron microscope. The corpus callosum and the anterior commissure were analyzed in this study.

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phalloidin and anti-Olig2 antibody. Images were randomly acquired at × 40 objective. The number of processes was counted and the total length of all processes per cell was measured using ImageJ software. WT and Pak3 KO oligodendrocytes (3 days in a differentiation medium) were stained with O4 antibody and pictures were randomly acquired at the same magnification. The contours of individual oligodendrocytes were drawn using Image J drawing tool and cell surface was quantified.

2.7. Migration assays and video-microscopy OPCs were seeded on μ-Slides (8 wells, IBIDI) coated first with polyornithine (100 μg/ml, Sigma-Aldrich), then with laminin (50 μg/ ml, Sigma-Aldrich). OPCs were cultured in the OPC proliferation medium. Images were acquired each 5 min during 8 h using a camera (Coolsnap HQ) connected to an inverted microscope (Leica DMI 600B). Migrating WT and Pak3 KO OPCs were tracked on pre-selected fields in the same experiment. Three independent experiments were performed. OPCs migration was evaluated using ImageJ software (plug-in Manual Tracking, Fabrice Cordelière, Curie Institute Paris). Cells that migrated N50 μm during 8 h were considered in this study and the means of the migration speed and the distance were quantified using the Image J software. 2.8. Western blot Protein extracts from cell cultures of OPCs and oligodendrocytes were prepared in a Laemmli buffer (NuPage LDS, Invitrogen) completed with 2-mercaptoethanol (2.5%; Sigma-Aldrich). Brain hemispheres were lysed in RIPA buffer (50 mM Hepes, 150 MM NaCl, 5 mM EDTA, 1% NP-40, 0.5% SDS; pH 7.7). Lysates were clarified by centrifugation. The DCA protein assay (Biorad) was used to determine protein concentration. Samples were denatured by heating for 5 min at 95 °C in Laemmli buffer. Then they were submitted to an electrophoretic migration in a separating gel (4–20% of acrylamide precast gels; Biorad) and then transferred to a nitrocellulose membrane (Biorad). TBS supplemented with 0.1% tween-20 and 5% dry milk powder was used for blocking and antibody incubations. Primary rabbit polyclonal antiPAK3 (1/1000, Cell signaling), rabbit polyclonal anti-MBP (1/2000, Millipore), mouse monoclonal anti-GAPDH (1/1000, Millipore) antibodies were used for western-blot experiments. Secondary antibodies were peroxidase-conjugated goat anti-rabbit polyclonal antibody (1/20000 to detect PAK3 and 1/100000 to detect MBP, Jackson) and goat antimouse monoclonal antibody (1/200000, Jackson). Membranes were incubated overnight at 4 °C with the primary antibody and then for 1 h at room temperature with the secondary antibody. They were washed several times in TBS supplemented with 0.1% Tween-20. Chemiluminescent detection was performed using ECL (ClarityTM Western ECL Substrate, Biorad). Relative expressions of PAK3 and MBP (14 kDa, the most abundant isoform of MBP) were measured using densitometric analysis over three separate experiments using ImageJ. 2.9. Quantifications and statistical analysis 2.9.1. In immunohistochemistry experiments Positive cells have been counted manually in the corpus callosum and the anterior commissure in at least 6 adjacent coronal sections of at least 3 different animals. Quantifications were performed on optical sections obtained on the Zeiss Apotome.2. The total area has been measured and the density was calculated. The density of Olig2+, Olig2+/ CC1 + and Olig2 +/Ki67 + cells have been quantified at P7, P10, P14 and P60 mice. We selected sections starting from the first rostral section, where the corpus callosum has been completely formed. 2.9.2. Morphology of OPCs and oligodendrocytes OPCs from WT and Pak3 KO mice obtained after the last step of shaking were maintained 3 days in culture in a proliferation medium containing PDGF and FGF. They were fixed (4% PFA) and stained with

2.9.3. Cell survival For each experiment the same number of WT and Pak3 KO OPCs (50 000 cells) was plated in a differentiation medium. The total number of phalloidin+ cells was counted from 20 fields (randomly acquired at ×20 magnification) at 2 DIV, 3 DIV and 6 DIV. The rate of survival between 2 and 3 days is defined as the following ratio (number of phalloidin cells at 3 days/number of phalloidin + cells at 2 days) ×100. The rate of survival between 3 and 6 days is defined as the following ratio (number of phalloidin cells at 6 days / number of phallodin+ cells at 3 days) × 100. Three independent experiments have been performed. 2.9.4. Quantification of oligodendrocyte differentiation in vitro This rate has been evaluated using two independent approaches. In the first one, we performed MBP immunostaining to label mature oligodendrocytes and phalloidin to stain the total number of cells. In the second approach, O4 has been used as another marker of oligodendrocyte differentiation and whose proportion has been calculated among NG2+ and O4+ cells. Images were acquired at × 20 magnification in at least 20 randomly chosen regions. In the first set of experiments the number of MBP + cells and total number of phalloidin + cells have been counted in each image. The rate of differentiation is defined as (number of MBP + cells / number of phalloidin+ cells) × 100. In the second set of experiments, the number of O4+ cells and the number of NG2+ cells have been counted in at least 20 randomly chosen regions. The rate of differentiation is defined as (number of O4+ cells / number of O4+ cells + NG2+ cells) × 100. 2.9.5. Density of myelinated axons Myelinated axons were counted in 34 μm2 fields (44,000 magnification, randomly sampled by taking 4 electron micrographs at each corner of each grid). At least 3500 axons were analyzed per animal and per structure (3 animals per genotype). 2.9.6. Statistics Data were analyzed with SPSS statistical software version 22.0 (Chicago, Illinois, USA). Normality in the variable distributions was assessed by the Shapiro-Wilk test. Furthermore, the Levene test was performed to probe the homogeneity of variances across groups. Variables that failed the Shapiro-Wilk or the Levene test were analyzed with nonparametric statistics using the Mann–Whitney rank sum tests for pair-wise multiple comparisons. Variables that passed the normality test were analyzed by Student's t-test for comparing two groups. Categorical variables were compared using the Pearson's Chi-squared test. A p value of b 0.05 was used as a cut-off for statistical significance. Data are presented as mean ± SEM. The statistical tests are described in each figure legend. 3. Results 3.1. PAK3 is expressed in OPCs and mature oligodendrocytes Pak3 mRNAs have been previously detected in oligodendroglial cells (Cahoy et al., 2008; Dugas et al., 2006; Nielsen et al., 2006) but expression at the protein level has not been reported. Due to the poor quality of commercial antibodies against PAK3, it was not possible to detect endogenous PAK3 in immunostaining experiments. We therefore analyzed the expression of the PAK3 protein in oligodendroglial cells by

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western blot. PAK3 was detected in cultured primary OPCs and differentiated (OLs) isolated from mouse brain (Fig. 1A). Furthermore, PAK3 is also highly expressed in the mouse brain at P7, as previously reported (Meng et al., 2005). Specific detection of PAK3 was demonstrated by the lack of PAK3 expression in the breast cancer cell line MDA-MB231. Interestingly, we detected a significantly higher level of PAK3 in OPCs compared to differentiated OLs (Fig. 1A, B). The same results were also obtained in OPCs and OLs from rat (data not shown). Thus, our data indicate that PAK3 protein level is down-regulated upon OPC differentiation.

3.2. The density of differentiated OLs is decreased in the developing white matter of Pak3 null mice We first analyzed whether oligodendroglial cells are affected in the Pak3 KO mouse brain (Meng et al., 2005). We compared the densities of the total Olig2 + oligodendroglial cells (OPCs and OLs stained for Olig2 (Ligon et al., 2004)) and differentiated Olig2+CC1+ OLs (Bhat et al., 1996) between WT and Pak3 KO mice at different postnatal stages. Cell quantifications were performed in the corpus callosum (CC) and the anterior commissure (AC) of Pak3 knockout (Pak3 KO) and wild type (WT) mouse littermates at P14. To assess the potential role of PAK3 in oligodendroglial differentiation, we also compared the

densities of Olig2 + CC1 + differentiated OLs in Pak3 KO and WT CC (Fig. 2) and AC (only cell quantification was presented for this structure), at P14, a developmental period where OPC differentiation is high (Emery et al., 2009; Ming et al., 2013). A significant decrease of the Olig2+ cells density in Pak3 KO was observed in the CC (Fig. 2A1, A3, B1, B3, C) but it did not reach significance in the AC (Fig. 2D). However, we found a significant decrease in the density of differentiated OLs in both structures in Pak3 KO (Fig. 2A2, A3, B2, B3, C, D). Hence, the loss of PAK3 function affected the density of differentiated OLs in the white matter during development. To determine if this defect persisted at the adult stage, we analyzed the density of oligodendroglial cells in the same regions, in P60 Pak3 KO and WT mice. Interestingly, the density of Olig2+ cells significantly decreased in the CC of Pak3 KO with respect to WT (Fig. 3A1–B3, E). In the AC, Olig2+ cell density decreased but did not reach significance (Fig. 3C1–D3, F). The density of Olig2+CC1+ differentiated OLs also decreased in Pak3 KO but did not reach significance with respect to WT mice in both CC (Fig. 3A3, B3, E) and AC (Fig. 3C3, D3, F). Next, we performed western blot experiments to analyze MBP on total forebrain lysates from WT and Pak3 KO mice at different postnatal ages to determine the effects of the developmental decrease of Olig2/ CC1 in the Pak3 KO on MBP expression, a major myelin protein. We observed as expected the absence of MBP at the postnatal day 7 (the very few differentiated and myelinated axons are not detected) in both WT and Pak3 KO forebrains (Fig. S1A). We observed a decrease of the quantity of MBP at P15 but it did not reach significance (Fig. S1A, B). We did not also observe a difference in the quantity of MBP at P60 (Fig. S1A, C).

3.3. Myelination is affected in the corpus callosum during development To analyze the consequence of the developmental decrease of mature OLs on myelination in the Pak3 KO mice, we performed electron microscopy at P14 and P60. At P14, we found a significant decrease in the density of myelinated axons in the CC of Pak3 KO mouse (Fig. 4A, B, E, F, I). The density of myelinated axons in the AC was also decreased, although not reaching significance (Fig. 4C, D, G, H, J). We next tested whether this developmental defect in Pak3 KO CC is compensated, like the density of differentiated oligodendrocytes (Olig2 +/CC1 +). We thus compared the density of myelinated axons in both P60 CC and AC WT and Pak3 KO mice (Fig. 5A–J). Compared with controls, we did not observe any defects in the density of myelinated axons in CC and AC in the Pak3 KO mice (Fig. 5A–J). We also observed that there is no obvious effects on axons (Fig. 5 A–H ).

3.4. The decrease of Olig2+ cells density in the corpus callosum is due to a developmental defect of their proliferation

Fig. 1. PAK3 is expressed in mouse oligodendroglial cells in culture. A. Western immunoblot analysis of PAK3 expression in OPCs and OLs (3 DIV in a differentiation medium). Note the strong expression in OPCs by comparison to OLs. Extracts from P7 brain are used as positive control. MDA-MB-231, a breast cancer cell line that does not express PAK3, is used as a negative control. GAPDH is used as a loading control. B. Quantitative analysis of western blots of PAK3 proteins in OPCs and OLs shown in A. Values represent the fold change of relative expression of PAK3 protein (normalized against GAPDH protein) in OLs over OPCs (Value for OPC is considered 100%; **p b 0.01 (Mann-Whitney test). N: number of experiments.

As PAK3 is highly expressed in OPCs (Fig. 1), it might affect their proliferation and hence loss of PAK3 function may lead to the observed decrease of Olig2 + cell density in the CC. To test this hypothesis, we analyzed OPC proliferation using immunolabeling for Olig2 and Ki67 in adjacent sections from WT and Pak3 KO animals at P7 (when OPCs are highly proliferative). Surprisingly, we did not find significant differences in the density of Olig2+ cells and Olig2+/Ki67+ cells in the CC and the AC (Fig. S2A). However at P10, the density of Olig2+/Ki67+ cells is significantly decreased in the CC of the Pak3 KO with respect to WT (Fig. 6A, B, E). The Olig2+/Ki67+ cell density in the AC is not significantly affected in the Pak3 KO (Fig. 6C, D, F). This result is consistent with the absence of decrease of the density of Olig2+ cells in the AC. We also analyzed OPC proliferation at P14 and showed that the density of Olig2 + Ki67 + cells is not significantly different between Pak3 KO and WT in both regions (CC: 76 cells/mm2 ± 20 for WT and 81 ± 24; Student's t-test. p N 0.05; AC: 146 cells/mm2 ± 41 for WT versus 182 ± 41; Student's t-test. p N 0.05).

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Fig. 2. The density of differentiated OLs is decreased in the white matter of Pak3 KO mice at P14 compared to WT. (A1–A3) Photomicrographs of WT P14 CC immunostained with Olig2 (A1) and CC1 (A2), (A3) Merge. (B1–B3) Photomicrographs of Pak3 KO P14 CC immunostained with Olig2 (B1) and CC1 (B2); B3 is the merge. Quantitative evaluation of the density of Olig2+ cells and Olig2+CC1+ cells in the CC is illustrated in C and in D for the AC. Arrows point to differentiated OLs (Olig2+CC1+ cells). Student's t-test *p b 0.05. Number of animals: 3 and number of sections per animal: at least 6. Scale bar (A1–B3): 50 μm.

3.5. Loss of Pak3 expression does not affect OPC morphology, migration nor proliferation in vitro To complement our in vivo studies and rule out possible indirect effects of Pak3 loss-of-function on OL development, we next studied the impact of Pak3 ablation in OPC cultures from Pak3 KO and WT mice. Given that PAK3 has been implicated in GABAergic interneuron development (Cobos et al., 2007), we speculated that PAK3 might control the actin cytoskeleton remodeling in OPC cells. We first compared the morphology of Pak3 KO and WT OPCs in vitro. We performed phalloidin staining, a F-actin marker, to visualize the morphology of OPCs, and Olig2 immunostaining to confirm the oligodendroglial identity of the cells (Fig. S3A, B). We quantified the number of OPC processes and their total length. Our results did not reveal any obvious morphological differences between OPCs from WT and Pak3 KO mice (Fig. S3A–D). Alternatively, the high level of PAK3 expression in OPCs compared to OLs may suggest that PAK3 is implicated in OPC migration and/or proliferation as these two events occur exclusively at this stage (Baumann and Pham-Dinh, 2001; Pfeiffer et al., 1993; Small et al., 1987). We thus analyzed the role of PAK3 in the motility of OPCs. To this aim, we performed time-lapse videomicroscopy of OPC cultures during 8 h (images taken each 5 min) and then performed cell tracking. Our results did not reveal any significant difference in the velocity or total migration distance between WT and Pak3 KO OPCs (Fig. 7A, B). WT and Pak3 KO OPCs had many growth cones and were able to develop processes and retract them to explore their environment. In addition, OPCs from both genotypes were able to display self-repulsion behavior as described previously (Hughes et al., 2013; Pfeiffer et al., 1993) (data not shown). Given that our results clearly indicate that Pak3 loss-of-function in OPCs does not alter their migration speed (Fig. 7A, B), we

subsequently analyzed the effect of PAK3 loss-of-function on OPC proliferation. The rate of proliferation of WT and Pak3 KO OPCS in a proliferating medium was compared at 3 DIV (days in vitro). The percentage of Olig2 + Ki67+ cells among the total number of Olig2+ cells was not significantly different in both genotypes (Fig. 7C–E). Therefore, ablation of PAK3 expression in OPCs does not modify their migration nor their proliferation in vitro. 3.6. PAK3 controls OPC differentiation into mature oligodendrocytes PAK3 has been previously implicated in the differentiation of neural progenitors into neurons in Xenopus and in the differentiation of mouse cortical GABAergic interneurons (Cobos et al., 2007; Souopgui et al., 2002). As oligodendrocyte differentiation is affected in the white matter of Pak3 KO mice, the high expression of PAK3 in OPCs might be essential in the switch from a proliferative to a differentiation stage. To determine the consequence of Pak3 loss-of-function on OPC differentiation into OLs, we compared the differentiation properties of purified OPCs from WT and Pak3 KO mice. We performed phalloidin staining to assess cytoskeleton remodeling during differentiation and MBP staining to measure the degree of differentiation. We quantified the number of MBP + cells among the total number of phalloidin + cells at 3, 4, 5 and 6 days of differentiation, as described in material and methods. At 3DIV OPCs started already their process of differentiation (Fig.8J (Bernard et al., 2012; Yang et al., 2016)). The results are illustrated at 4 DIV and 6 DIV (Fig. 8A–D). Our data revealed that OPC differentiation rate from Pak3 KO was significantly lower as compared to WT OPCs at 3, 4, 5 and 6 days of differentiation, even though this difference decreases over time (see Fig. 8I), suggesting a delay in the oligodendroglial differentiation in the Pak3 KO mice. Nevertheless, the morphology of

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Fig. 3. The density of Olig2+ cells is decreased in Pak3 KO mice at P60. A1–A3. Sections of P60 WT CC immunostained with Olig2 (A1) and CC1 (A2); A3 is the merge. B1–B3. Sections of P60 Pak3 KO brain at the level of CC immunostained with Olig2 (B1) and CC1 (B2); B3 is the merge. C1–C3. Sections of P60 WT AC immunostained with Olig2 (C1) and CC1 (C2); C3 is the merge. D1–D3. Sections of P60 Pak3 KO brain at the level of AC immunostained with Olig2 (D1) and CC1 (D2); D3 is the merge. E–F. Quantitative evaluation of the density of Olig2+ cells and Olig2+/CC1+ cells in the CC (E) and the AC (F). Arrows indicate differentiated OLs (Olig2+CC1+ cells). Student's t-test. *p b 0.05. Number of experiments: 3 and number of sections per animal: at least 6. Scale bar A1–D3: 50 μm.

oligodendrocytes from WT and Pak3 KO mice did not show obvious changes at 3, 4, 5 and 6 DIV as illustrated by phalloidin and MBP stainings (Fig. 8A–D). Next, to confirm the role of PAK3 in oligodendroglial differentiation, we used O4 staining, as an additional marker of oligodendroglial lineage progression (Baumann and Pham-Dinh, 2001) and NG2 for OPCs (Bouslama-Oueghlani et al., 2005). Data are

illustrated at 4 DIV and 6 DIV (Fig. 8E–H). Quantitative analysis at 3, 4, 5 and 6 DIV confirmed the decreased rate of OPC differentiation into OLs (see in Fig. 8J). We noticed a significant decrease in the proportion of O4+ cells, thus confirming the differentiation defects of OPCs in Pak3 KO mice with respect to controls. In addition, we measured the surface of O4+ oligodendrocytes at 3 DIV and showed that the absence of PAK3

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Fig. 4. Myelination is affected in P14 Pak3 KO CC. Ultrastructural analysis of the CC (A, B, E, F)) and the AC (C, D, G, H) in WT mice (A–D) and in Pak3 KO mice (E–H) at P14. (A, E) General view of the CC. (B, F) High magnification of A and E, respectively. (C, G) General view of the AC. (D, H) High magnification of C and G respectively. (I, J) quantitative analysis of the density of myelinated axons in the CC and the AC, respectively. The density of myelinated axons (*) decreases in the Pak3 KO CC by comparison to the WT and does not change in the AC. The scale bars are the same in WT and Pak3 KO. Student's t-test. *p b 0.05. Number of animals: 3.

Fig. 5. Myelination is not affected in the adult Pak3 KO white matter. Ultrastructural analysis of the CC (A, B, E, F)) and the AC (C, D, G, H) in WT mice (A–D) and in Pak3 KO mice (E–H) at P60. (A, E) General view of the CC. (B, F) High magnification of A and E, respectively. (C, G) General view of the AC. (D, H) High magnification of C and G respectively. (I, J) quantitative analysis of the density of myelinated axons in the CC and the AC, respectively. The density of myelinated axons (*) is not significantly different in WT and KO PAK3 white matter (CC and AC). As: astrocyte, OL: oligodendrocyte. The scale bars are the same in WT and Pak3 KO.

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Fig. 6. PAK3 depletion affects OPC proliferation in the CC at P10. Photomicrographs of WT (A, C), Pak3 KO P7 P10 (B, D) CC (A, B) and AC (C, D) immunostained with Olig2 and Ki67 (Merges are presented). Quantitative evaluation of the density of Olig2+ cells and Olig2+/Ki67+ cells in the CC (E) and the AC (F). Arrows indicate proliferating OPCs (Olig2+Ki67+ cells). Student's t-test. **p b 0.005; ***p b 0.0001. Number of experiments: 3 and number of sections per animal: 10.

did not affect the surface of OLs (3996 μm2 ± 340 in WT versus 3972 μm2 ± 499, p N 0.05 Student's test). Overall, our data support a function of PAK3 in OPC differentiation. The decreased number of differentiated OLs in the Pak3 KO could be due to an increase of cell death during differentiation. To rule out this possibility, we studied the effect of Pak3 deletion on oligodendroglial cell survival in vitro. We compared the survival rate of oligodendroglial cells from the same starting OPC culture at different days in vitro. To do so, we counted the number of these cells at 2, 3 and 6 DIV of culture in differentiation media. The rate of survival was not significantly different between WT and Pak3 KO at all the different time points studied (between 2 and 3 DIV: 89.39% for WT versus 93% for Pak3 KO, p N 0.05, Pearson's Chi-squared test; and between 3 and 6 DIV: 45.32% for WT versus 41.56% for Pak3 KO; p N 0.05, Pearson's Chi-squared test). Altogether these results showed that loss of Pak3 function in OPCs does not affect their survival rate. The decrease of the number of differentiated oligodendrocytes in Pak3 KO mice could be explained by the involvement of PAK3 in the regulation of cell proliferation arrest as previously shown for pro-neural cells in Xenopus and mouse pancreatic β cells (Piccand et al., 2014; Souopgui et al., 2002). To test this hypothesis, we compared the proliferation rate of WT and Pak3 null OPCs in differentiation medium. If PAK3 is controlling OPC proliferation arrest, we should expect the proportion of proliferating OPCs in the Pak3 KO OPC cultures to be greater than in the WT. We thus performed double immunostaining for

PDGFRα to label OPCs and Ki67 to stain proliferating cells (Gerdes et al., 1984; Scholzen and Gerdes, 2000). We calculated the proportion of PDGFRα+Ki67+ cells among the total population of PDGFRα+ OPCs at 1 and 3 DIV of differentiation in WT and Pak3 KO cultures. Our results showed that the rate of proliferation in both WT and Pak3 KO is not significantly different at 1 and 3 DIV (Fig. 9A–D). These data indicate that Pak3 loss-of-function does not alter OPC proliferation arrest. . 4. Discussion PAK3 is critical for cognitive functions, truncating mutations of this gene cause non-syndromic intellectual disability and missense mutations are associated with psychotic disorders (Gedeon et al., 2003; Morrow et al., 2008; Peippo et al., 2007a; Rejeb et al., 2009; Rejeb et al., 2008). The causes of intellectual disability and psychosis in patients with Pak3 mutations are still not fully identified even though it is now accepted that the alteration of dendritic spine density and morphology are the main cause of the disease (for review see (Verpelli et al., 2014). So far, many studies have suggested that PAK3 regulates spine formation and refinement during development (Dubos et al., 2012; Kreis and Barnier, 2009). Hypoplasia of the corpus callosum has been described in some patients with Pak3 mutation (Magini et al., 2014). This could be attributed to defects in OLs and myelin. Neurons highly express Pak3 (Kreis et al., 2008; Manser et al., 1995), however astrocytes

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Fig. 7. Loss of Pak3 function does not affect OPC proliferation or migration. A. Quantitative analysis of the migration speed of OPCs (images are acquired each 5 min during 8 h). B. Quantitative analysis of the distance of migration of WT and Pak3 KO OPCs. Note that no significant difference has been observed in the speed and distance of migration (p N 0.05 Mann-Whitney test). C, D. OPCs (3 DIV in proliferation media) are double stained with Olig2 (red) to stain OPCs (in proliferation media only OPCs are present) and Ki67 (green) to stain proliferating cells. (E) Quantitative analysis of the rate of OPC proliferation (Ki67+Olig2+ cells among the total Olig2+ cells). Arrows are showing proliferating OPCs. Note that no significant difference has been observed in the rate of proliferation (p N 0.05 Pearson's Chi-squared test). N: number of animals and n: number of cells.

do not as it has been clearly shown by RT-PCR in cultures (Kreis et al., 2008). Herein, we analyzed the expression of PAK3 in oligodendroglial cells by western-blot. We showed clearly that this protein is expressed

both in OPCs and mature oligodendrocytes. Nielsen and colleagues previously observed that Pak3 mRNA increases during oligodendrocyte differentiation (Nielsen et al., 2006), while, Dugas and colleagues showed a

Fig. 8. PAK3 controls the differentiation of OPCs into oligodendrocytes. OPCs maintained in differentiation media during 4 DIV (A, B, E, F), 6 DIV (C, D, G, H) are stained with phalloidin (green, A–D) to visualize F-actin and MBP (red, A–D) to stain differentiated OLs. In another set of experiments OPCs maintained in differentiation media are stained with NG2 (red, E– H) to label OPCs and O4 (green, E–H) to label pre- and mature OLs. Quantitative analysis of the % of MBP+ cells at 3, 4, 5 and 6 DIV (I) and O4+ cells at 3, 4, 5 and 6DIV (J). Note that the rate of differentiation is lower in Pak3 KO cultures at the different DIVs. (***p b 0.001 Pearson's Chi-squared test). Arrows point differentiated OLs. N: number of experiments and n: number of cells.

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Fig. 9. Pak3 depletion does not affect proliferation arrest of OPCs. A. WT OPCs and B. Pak3 KO OPCs maintained in differentiation media during 3 DIV immunostained with PDGFRα antibody to stain specifically OPCs and with Ki67 antibody to stain proliferating OPCs. C. Quantitative analysis of OPC proliferation in differentiation media at 3 DIV. D Quantitative analysis of the proliferating rate of OPCs in differentiation media at 1 DIV. Note the absence of significant difference in the rate of proliferation between WT and Pak3 KO OPCs (p N 0.05 Pearson's Chisquared test). N: number of experiments.

moderate decline in the amount of Pak3 mRNA during the course of differentiation in vitro (Dugas et al., 2006). In the present study, we clearly demonstrate a significant decrease of PAK3 protein expression during OPC differentiation. This discrepancy could be explained by the fact that mRNA expression is not always predictive of protein expression level (Guo et al., 2008). Interestingly, OLs are affected in mental/psychiatric diseases (Edgar and Sibille, 2012; Fields, 2008; Hall et al., 2014; Takahashi et al., 2011). Whether oligodendroglial cells are the primary cause of some mental/ psychiatric diseases is currently under debate (for review see (Nave and Ehrenreich, 2014). A recent Genome Wide Transcriptional profiling study performed in different regions of Down-Syndrome postmortem brain patients provided strong evidence of oligodendrocyte differentiation and myelination impairment. These defects are cell autonomous as it has been shown in the Ts65DN mouse model of Down Syndrome (Olmos-Serrano et al., 2016). Our analysis of the Pak3 KO mouse brain shows a significant decrease of Olig2+ in the CC both during development and at the adult age in Pak3 null mice. This decrease was also observed in the AC, although not reaching statistical difference between the two genotypes. The decrease of Olig2 + cells density in the CC of the Pak3 mutant, could be explained by a lower proliferation rate of Pak3 null OPCs. In line with this hypothesis, we found that the density of Ki67+ OPCs is highly decreased in the Pak3 KO CC, at P10. Our data also revealed that the densities of differentiated oligodendrocytes and of myelinated axons are significantly decreased in the CC of the Pak3 KO at P14. However in the AC, a significant decrease of the density of differentiated oligodendrocytes has been observed but this leads only to a slight (not significant) reduction of myelinated axons. The difference between these two white matter structures could be due to specific regional regulation of myelination as it has been described in WAVE1 KO and Olig1 KO mice (Kim et al., 2006; Xin et al., 2005). It is also worth to

note that myelination of the CC starts earlier than that of AC in rodents (Downes and Mullins, 2014). Moreover, the duration of myelination is more prolonged in the CC than the AC (Sturrock, 1975; Sturrock, 1980). The absence of difference in MBP expression at P15 in the whole forebrain lysates of WT and Pak3 KO supports the idea that myelination is controlled differently according to the brain structure. Altogether, our data suggest that Pak3 loss-of-function affects the differentiation of OPCs into mature OLs during development. However at the adult stage, the density of differentiated OLs (Olig2+CC1+) and myelinated axons did not reveal obvious differences between Pak3 KO and WT strains. This result suggests that even if the differentiation is decreased during development, in vivo Olig2+ OPCs are able to differentiate and to myelinate axons in the absence of Pak3 function. Given that Pak3 invalidation in this mouse strain is not specific to oligodendroglial cells and that the final number of OLs depends on the neuronal population (Barres and Raff, 1999; Simons and Trajkovic, 2006; Stevens et al., 2002), the defect observed in vivo could be an indirect consequence of affected neuronal population. Especially, Pak3 mRNAs have been detected by in situ hybridization in neurons of the layers II/III and V of the cortex (Manser et al., 1995), which axons form the CC (Innocenti and Clarke, 1984). However, it is worth to note that the ultrastructural analysis did not show any obvious axonal defects. In our study, the role of PAK3 might be compensated by the other members of the group I of the PAK family, as they share high sequence identity (Bokoch, 2003; King et al., 2014; Kreis and Barnier, 2009). In line with our data, Pak3 KO neurons did not show any major morphological defects, but the Pak3 KO mice still exhibit memory defects (Meng et al., 2005). A conditional KO of Pak3 is required to unambiguously determine the contribution of this protein in oligodendroglial and neuronal abnormalities observed in this mutant strain. To assess the direct impacts of Pak3 loss-of-function on OLs differentiation, and rule out possible

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indirect effects of Pak3 deletion in neurons, we studied the effects of Pak3 knockout in OPC cultures, derived from Pak3 null and WT mouse brains. In vitro, we clearly showed a cell autonomous effect of Pak3 on OPC differentiation. Our data also indicated that this effect was not due to an increase of cell death, as the rate of survival was not significantly different between WT and Pak3 KO at all the different time points studied. This defect was also not related to the known function of PAK proteins in the regulation of actin cytoskeleton (Bokoch, 2003), as the morphology of Pak3 KO OPCs and oligodendrocytes was not affected. Our results are in accordance with the hypothesis that proliferation and differentiation steps are not necessary linked as previously suggested (Rosenberg et al., 2007). We showed that even if OL differentiation is affected in Pak3 KO cultures the percentage of Ki67+ OPCs (in the differentiation media) is not different in Pak3 KO cultures with respect to WT. The mechanism by which PAK3 controls differentiation remains unknown. Several hypothesis could be proposed: i) as PAK3 has a nuclear localization signal (NLS) (Kreis and Barnier, 2009), it is possible that nuclear PAK3 is controlling oligodendroglial differentiation through gene expression, ii) PAK3 might control the subcellular localization of its downstream effector LIMK-1, as it has been recently shown that oligodendroglial maturation is dependent on intracellular protein shuttling and that nuclear LIMK-1 accumulation inhibits OPC differentiation (Gottle et al., 2015). The comparison of migration parameters between WT and Pak3 KO OPCs in vitro did not reveal any obvious differences. Both the migration speed and distance were similar in WT and Pak3 KO OPC cultures. Furthermore, in vivo analysis of Pak3 KO brains revealed that OPCs are able to migrate into the different brain regions, even though their density was decreased in the white matter. In agreement with these findings, we did not detect any morphological differences between WT and Pak3 KO OPCs in vitro. Thus, the function of PAK3 in oligodendroglial cells is different from that of GABAergic neurons, in which both morphology and migration are affected by shRNA knock-down of Pak3 expression (Cobos et al., 2007). In conclusion, we showed that oligodendroglial cells, but not myelin, are affected in the Pak3 KO model of ID at the adult stage. However, mature oligodendrocytes and myelin are affected in the CC during development. Although the in vivo results only suggest a role of PAK3 in oligodendrocyte differentiation, the in vitro experiments demonstrate it clearly. We thus identified the intellectual disability protein PAK3 as a new regulator of oligodendrocyte differentiation. Future studies are required to decipher the mechanism by which PAK3 controls OPC differentiation and whether this factor is directly involved in the onset of ID and other psychiatric diseases. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.nbd.2016.12.004. Acknowledgments This work was supported by the Université Pierre et Marie Curie (LBO), the Centre National de la Recherche Scientifique (ID), Grants from ANR-07-NEURO-043-01 (ID), Royal Society International Joint Project Grant (LBO and CW; grant JP090037), La fondation Jérôme Lejeune (JVB, BNO; grants 1070-BJ2012B and 1547-NB2016A) and the Institut des Neurosciences translationnelles de Paris (IHU-A-ICM, ANR-10-IAIUH-06). We thank Richard Schwarzman and Susanne Bolte at the imaging plateform of IFR83, for assistance with live Imaging. We are grateful to Dominique Langui at Imaging platform of the ICM for electron microscopy experiments. We thank Sandrine Guyon and Nathalie Samson from Orsay University for their technical support. References Abraham, H., Vincze, A., Veszpremi, B., Kravjak, A., Gomori, E., Kovacs, G.G., Seress, L., 2012. Impaired myelination of the human hippocampal formation in down syndrome. Int. J. Dev. Neurosci. Off. J. Int. Soc. Dev. Neurosci. 30, 147–158.

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