c-Jun N-terminal kinase 1 (JNK1) modulates oligodendrocyte progenitor cell architecture, proliferation and myelination

During Central Nervous System ontogenesis, myelinating oligodendrocytes (OLs) arise from highly ramified and proliferative precursors called oligodendrocyte progenitor cells (OPCs). OPC architecture, proliferation and oligodendro-/myelino-genesis are finely regulated by the interplay of cell-intrinsic and extrinsic factors. A variety of extrinsic cues converge on the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway. Here we found that the germinal ablation of the MAPK c-Jun N-Terminal Kinase isoform 1 (JNK1) results in a significant reduction of myelin in the cerebral cortex and corpus callosum at both postnatal and adult stages. Myelin alterations are accompanied by higher OPC density and proliferation during the first weeks of life, consistent with a transient alteration of mechanisms regulating OPC self-renewal and differentiation. JNK1 KO OPCs also show smaller occupancy territories and a less complex branching architecture in vivo. Notably, these latter phenotypes are recapitulated in pure cultures of JNK1 KO OPCs and of WT OPCs treated with the JNK inhibitor D-JNKI-1. Moreover, JNK1 KO and WT D-JNKI-1 treated OLs, while not showing overt alterations of differentiation in vitro, display a reduced surface compared to controls. Our results unveil a novel player in the complex regulation of OPC biology, on the one hand showing that JNK1 ablation cell-autonomously determines alterations of OPC proliferation and branching architecture and, on the other hand, suggesting that JNK1 signaling in OLs participates in myelination in vivo.


JNK1 KO mice display myelin abnormalities.
In order to address the impact of JNK1 ablation on oligodendroglia, we firstly examined the expression of the myelin marker MBP in the cerebral cortex. We found that JNK1 KO mice display a lower expression of MBP, both in infragranular and supragranular layers of the somatosensory cortex (Fig. 1A,C and Suppl. Fig. 1A,B) and in the CC (Fig. 1B,D). This defect was found at postnatal ages (P7 and P15) and persisted at adult stages (P90). Myelin abnormalities, not only restricted to MBP expression, were also confirmed by observation of WT and JNK1 KO Gallyas-stained sagittal sections (Suppl. Fig. 1C). Former studies on JNK1 KO revealed some extent of axonal degeneration 17 . Thus, we asked whether the observed reduction of MBP and myelin reflected the axonal regressive events. Indeed, the ratio of MBP/healthy axons (as detected by labelling of SMI31, a phosphorylated epitope of neurofilament H, a major component of the axonal cytoskeleton ( Fig. 2A; Yandamuri et al. 27 )) appeared to be reduced in JNK1 KO cortices and CC compared to WT, and the axon densities did not display a major decrease in mutant mice within the analyzed time window ( Fig. 2A-D). These histological results were in line with western blotting (WB) analyses, which confirmed a reduction in the amount of other myelin-associated proteins, such as 2′,3′-Cyclic-Nucleotide 3′-phosphodiesterase (CNPase) and Myelin Oligodendrocyte Glycoprotein (MOG) (Fig. 2E,F and Suppl. Fig. 2A,B). On the whole, these results suggest hypomyelination in JNK1 KO mice. Also, myelin alterations were not merely attributable to a decrease of mature OLs in JNK1 KO mice as the densities of CC1 + OLs were overall comparable to those of WTs in both cerebral cortex and CC (Suppl. Fig. 3A,B).
To assess whether changes in myelin levels in mutants were accompanied by alterations of the axo-myelinic arrangement, we examined the nodal/paranodal region by immunostaining against the paranodal protein CASPR 28,29 . Quantification of CASPR + segments in the CC of adult brains revealed a significant staining increase in JNK1 KO samples (Fig. 1E,F). Moreover, analyses of CASPR + node/paranode length showed a 17% increase in CASPR + segment length in mutants as compared to control mice (Fig. 1G). Of note, this latter feature is frequently found in hypo-/dys-myelinating conditions 30,31 , corroborating the idea of myelin alterations in JNK1 KO mice cortex and CC. JNK1 KO cortical OPCs display enhanced proliferation early after birth and morphological alterations. As a second step, we expanded the investigation on OPCs and assessed their density, proliferation rate and apoptosis at different survival times (P7, P15 and P90). We found a significant increase (about 34%) in the density of PDGFRα + OPCs at P7 and P15 in KO mice compared to WT, both in cerebral cortex and CC ( Fig. 3A-C, representative images at P7) with no changes in cell distribution throughout the cortical layers (Suppl. Fig. 3C). Since the presence of a higher number of OPCs in the JNK1 KO cortex could result from either higher cell proliferation or decreased apoptosis (or a combination of the two), we counted PDGFRα + duplets as a measure of proliferative OPCs 5,32 . At P7, the fraction of OPCs in duplets in JNK1 KO cortices was almost twofold higher than in WT, revealing that mutant OPCs have a higher proliferation rate than WT cells (Fig. 3D,E). Yet, the normal density (Fig. 3B,C) as well as the OPC proliferative fraction (Fig. 3E) of JNK1 KO OPCs appeared restored at adult stages (P90), suggesting a higher susceptibility of young OPCs to JNK1-dependent regulatory mechanisms. These results were also confirmed by analyses of NG2 + /Ki67 + OPCs (Suppl. Fig. 3D).
Conversely, when we examined NG2 + OPCs expressing activated caspase 3 (cCASP3) to detect ongoing apoptosis, co-expressing OPCs were barely detected in both WT and JNK1-KO mice (not shown). Similar results were also obtained by TUNEL staining (Suppl. Fig. 3E). These data point to JNK1 participation in the regulation of OPC proliferation, at least in a developmental time window.
Based on increased OPC density, we hypothesized that the territory occupied by each cell could also be altered in JNK1 KO cortices. This hypothesis was initially tested by the analysis of the Voronoi polygons, a tool  www.nature.com/scientificreports/ to analyze the spatial distribution of cells [33][34][35] . Voronoi analysis suggested that, during early developmental stages (P7-P15), JNK1 KO OPCs occupied a less extended area than WT cells (Fig. 3F-I). To further corroborate these data and better understand the underlying cellular features, we performed morphometric analyses of both OPC somata and branches ( Fig. 3J-L). Analyses at early and adult stages showed that OPC soma areas did not differ between WT and KO cells (Fig. 3K). However, in agreement with the Voronoi results, OPC territory (i.e. the area occupied by the entire OPC extension, including cell ramification) was significantly smaller in JNK1 KO than in WT (Fig. 3L). Yet, this decrease was no longer appreciated at adult stages (Fig. 3L). Nevertheless, at P90, JNK1 KO OPCs displayed a shorter total length of ramifications with no changes in the number of primary ramifications (Fig. 3M,N) and a lower ratio of the number of branches over branch order (Fig. 3O). Thus, mutant OPC processes appeared less complex and overall less extended compared to the WT ones. Taken together, these data indicate that JNK1 may play a role in the OPC proliferation and in the regulation of OPC branching architecture.
Cultured JNK1 KO OPCs reproduce proliferative and morphological alterations found in vivo. In order to disentangle whether JNK1 KO OPC alterations in vivo depended on other cell types or could be explained cell autonomously, we performed cultures of MACS-isolated OPCs derived from P0 WT or JNK1 KO mice and examined cell proliferation, apoptosis and morphology.
At first, we tested the occurrence of possible dysregulated expression of the other JNK isoforms, potentially accounting for compensatory mechanisms or functional alterations. However, levels of JNK2 and JNK3 expression in acutely isolated JNK1 KO cells, as tested by qRT-PCR, were comparable to those of WT cells (Suppl. Fig. 4C-E), thus confirming that we were assessing the consequence of JNK1 abrogation.
In culture, MACS-sorted JNK1 KO OPCs showed higher cell densities per field ( Fig. 4A-D) and a twofold higher proliferation rate compared to WT cells, as revealed by colocalization with the proliferative marker Ki67 (Fig. 4A,B). Of note, while the proliferative fraction of WT cells decreased with increasing cell densities, the proliferative fraction of JNK1 KO OPCs remained constant, irrespective of the number of OPCs (Fig. 4C,D). As regards apoptosis, we found a threefold higher fraction of cCASP3 + JNK1 KO OPCs compared to WT cells and an apoptotic rate decreasing with increasing densities in both KO and WT cells (Suppl. Fig. 4A,B). These data suggest that, although increased in KO cells, apoptosis is similarly regulated in both mutant and WT cells, whereas in mutant OPCs proliferative regulatory mechanisms may be altered as a consequence of JNK1 loss.
Moreover, morphometric analyses on non-proliferative isolated OPCs showed that JNK1 KO OPCs display a reduced ramification complexity compared to WT cells (Fig. 4E,F-J) in the presence of similar soma area (Fig. 4F) and of a slightly higher number (about 12%) of primary ramifications (Fig. 4G). These results reveal that mutant OPCs show alterations independently of the presence of other cell types.
To confirm these results in a distinct experimental model, we investigated the effects of JNK inhibition obtained with the D-JNKI-1 inhibitor 36 on rat OPC cultures. D-JNKI-1 is a cell penetrating peptide that prevents, through a competitive mechanism, the binding of JNK to both the scaffold protein JNK-interacting protein-1 (JIP1) and its substrates [36][37][38] . Of note, D-JNKI-1 does not act exclusively on the binding of JNK1, but also on that of JNK2 and JNK3. Analysis of Ki67 expression revealed a higher proliferative rate in OPCs treated with D-JNKI-1 compared to controls (Fig. 5A,B). Moreover, morphometric analyses highlighted branching alterations resulting in a reduced ramification complexity (Fig. 5C), thus resembling those of MACS-sorted JNK1 KO OPCs, as indicated by Sholl analysis (Fig. 5D).
Altogether these data show that JNK1 KO-related OPC functional and morphological abnormalities occur also independently of other cell types affected by the mutations and suggest that JNK1 is implicated in the regulation of OPC proliferation and process architecture through a cell autonomous mechanism. JNK1 KO OLs do not show overt differentiation defects in vitro but display reduced territory occupancy. In order to study whether JNK1 KO myelin alterations in vivo could be explained by an altered ability of JNK1 KO OLs to differentiate, we cultured MACS-isolated OPCs derived from P0 WT or JNK1 KO in non-proliferative conditions and examined MBP expression as well as cell morphology.
JNK1 KO and WT OLs in culture displayed equivalent capability to express MBP (Fig. 6A,B). Moreover, when we analyzed the frequency of immature vs mature MBP + cells, as distinguished by process complexity and by MBP localization (see "Methods") we found no differences in JNK1 KO vs WT cells (Fig. 6C).
We also investigated the effects of JNK inhibition obtained with D-JNKI-1 36 on cultured rat OLs. Analysis of MBP + OLs confirmed the results obtained for mutant OLs showing that D-JNKI-1 treated cells were able to differentiate, branch and form MBP + lamellae to the same extent of control cells (Fig. 6E-G). However, in both experimental conditions, the cell territory occupied by MBP + lamellae appeared reduced in JNK1 KO and D-JNKI-1 treated OLs (Fig. 6D,H).
Overall, these data show that the germinal ablation/inhibition of JNK1 does not affect the ability of OPCs to differentiate in MBP + OLs.

Discussion
The ERK/MAPK pathway is known to take part in the regulation of OPC architecture, proliferation and oligodendro-/myelino-genesis 11,12 . Among MAPKs, JNK1 contribution to oligodendroglial biology has been only marginally investigated so far. In this study, we found that constitutive JNK1 ablation in KO mice is associated with decreased expression of myelin proteins and myelin/paranodal abnormalities in the cerebral cortex and CC of postnatal and adult mice. Such alterations are accompanied by a transient increase in OPC density and proliferative ability and by a persistent reduction in OPC ramifications complexity. These abnormal features are also present in JNK1 KO OPCs cultures and in WT OPCs cultures treated with D-JNKI-1, indicating that cell

JNK1 and myelination.
In the cerebral cortex of the mutant mice, we observed a lower expression of myelin proteins and longer CASPR + paranodes, suggesting deficits in myelin structure and alterations in myelinating OLs/axon crosstalk. Defective myelin deposition and alterations in the paranode length are two recurrent features of hypo/dysmyelinating conditions linked to primary oligodendroglia pathology 30,31 . In vitro experiments indicate that JNK1 KO does not impair MBP expression or affect major steps of OL differentiation. However, morphological maturation in differentiating OLs appeared affected in mutant and treated cells, as lamellaeoccupied territories were reduced. Based on evidence that OL morphological or cytoskeletal alterations are often associated with reduced myelination 9, 39 , it is conceivable that reduced membrane extension in mutant OLs may contribute to the hypomyelinated phenotype found in vivo. Although we did not observe overt degeneration in JNK1 KO axons at the examined ages and territories, former electron microscopy investigations revealed some extent of axonal degeneration in JNK1 KO mice 17 , and showed that JNK1 takes part in microtubule maintenance and integrity, since earlier ages 16,17 . Microtubule dynamics both in neurons and oligodendrocytes play a fundamental role in OLs/neuron crosstalk, whose integrity is crucial for a correct myelination [40][41][42][43] . On these bases, we cannot exclude subtle microtubule-related alterations in axons, myelin sheath formation and/or OLs/axon crosstalk could all take part in the hypomyelination phenotype observed in vivo in JNK1 KO. Further investigations are needed to clarify this issue.

JNK1 role in OPC proliferation and apoptosis.
We also found that JNK1 KO OPCs display a higher proliferative rate associated with increased density at postnatal developmental stages, with no changes in their distribution through cortical layers. This feature suggests that JNK1 operates as a negative regulator of cell proliferation in OPCs. According to in vitro experiments JNK1 appears to act in a cell autonomous fashion in cell division regulation in OPCs. However, it cannot be excluded that JNK1 KO OPCs could have been primed to an altered regulation of proliferation by environmental signals received at embryonic ages in vivo, so to determine their increased division rate also in purified culture conditions. Notably, our observations apparently clash with the results of former studies showing JNK pathway (although without isoform specifications) as necessary for OPC proliferation upon incubation with the conditioned medium of neuroblastoma cells 44 . However, on the other hand, JNK1 specific inhibition was shown to increase endothelial cell division in controlled conditions 45,46 or to have no effect in a carcinoma cell line 47 . Moreover, in cancer development, JNK1 seems to play a dual role in promoting/inhibiting cell proliferation 48 . Thus, literature data indicate a cell/context dependent role for JNK1 in the modulation of proliferative events.
OPC proliferation is finely tuned by two main mechanisms. One first mechanism appears to operate through an intracellular timer driven by the mitogen PDGF (Platelet-Derived Growth Factor), that determines when individual OPCs should stop dividing to proceed toward differentiation [49][50][51] . One other mechanism implies OPCto-OPC contact-mediated inhibition of cell proliferation through, for instance, Netrin-1 (NT-1) and its receptor Deleted in Colorectal Cancer (DCC) signaling 6,7 . Of note, other sources of these contact-mediated inhibitors are unclear, although neurons have been shown to produce NT-1 7,52 . Former studies have implicated JNK1 activity as a positive regulator of cell cycle progression and a mediator of PDGF actions in OPCs 53 . On the other hand, JNK1 was also reported to mediate NT-1/DCC signaling in neurons, suggesting that similar mechanisms could act also in oligodendroglia and, therefore, that JNK1 ablation could alter contact-mediated OPC proliferation inhibition 54 . In vitro data appear to support this latter hypothesis, as they show that, at difference with WT cells, JNK1 KO OPC proliferative rate is maintained high also in conditions of elevated cell density.  www.nature.com/scientificreports/ Our data further showed that JNK1 KO OPCs proliferation and density in vivo are increased only during developmental stages. Although OPC amplification, self-maintenance and maturation at adult stage are supposed to recapitulate the corresponding developmental processes 55 , to what extent the very same molecular mechanisms subserve these events in the postnatal vs adult CNS is unclear. Age-dependent differences in gene expression and function occur in OPCs. In particular, early OPCs are more proliferative, characterized by a shorter cell cycle and more susceptible to JNK-dependent death 24, 56-58 . Whether and how JNK1 is involved in postnatal vs adult OPC distinct properties remains elusive. We can also speculate that supernumerary JNK1 KO OPCs may be simply eliminated in parallel with the progression of myelination, thereby adjusting the number of OLs to that of the axons (and to limiting amounts of trophic factors provided by axons) 59 , as normally occurs in WT brains 60,61 . JNK1 signaling has also been reported to participate in cell death which could impact on proliferation rates and cell densities. JNK pathway was shown to promote apoptosis in OPCs/OLs under stress conditions [62][63][64] . However, if JNK1 isoform is implicated in physiological cell death is unknown. In in vivo analyses we did not find evidence of an altered apoptosis rate in JNK1 KO OPCs. Conversely, in MACS-sorted JNK1 KO OPCs cultures, we found an increased fraction of apoptotic cells. Such a fraction, similar to what occurs for WT cells, appeared to decline with increasing cell densities, in agreement with an increased production of survival signals at sites with high cellularity. These data overall suggest that the mechanisms underlying the physiological regulation of apoptosis are maintained in mutant cells, and increased apoptosis may simply reflect the increased number of JNK1 KO OPCs. This may imply that, in OPCs, JNK isoforms other than JNK1 regulate this aspect, or can compensate for JNK1 ablation in the physiological regulation of apoptosis. JNK1 role in OPC architecture. Our analyses also provided evidence of a transient alteration of OPC territory occupancy. Voronoi polygons and cell territory analyses (Fig. 3F-I,K,L) show that, at least during development, OPC territory in JNK1 KO is significantly reduced. Although at adult stages this gross OPC alteration seems to be restored, adult JNK1 KO OPCs displayed a reduction in ramification length and branching complexity (Fig. 3J-O). These defects were also recapitulated in cell culture analyses (Figs. 4E-J, 5C-D), confirming www.nature.com/scientificreports/ the cell autonomous role of JNK1. These findings might also reflect the persistence of less complex immature phenotypes associated with the increased proliferative activity of the mutant cells. However, the maintenance of morphological alterations at adult ages, when mutant cell proliferation has declined back to WT levels, supports an involvement of JNK1 in OPC cytoskeletal dynamics independent of proliferative events, as previously found in neurons 17,65 . In keeping with this possibility, one potential JNK1 effector candidate in the regulation of OPC cytoskeleton is the microtubule-associated protein 1B (MAP1B), expressed both in neurons and oligodendrocytes 66 , that regulates microtubule elongation and dynamics. MAP1B is activated by kinases including JNK through phosphorylation 67 , and in neurons is known to support axon outgrowth. Notably, among the JNK isoforms, JNK1 appears to be particularly involved in the process of axonal elongation 68 . In oligodendroglia, MAP1B is expressed in OPCs progressing toward the preoligodendrocyte stages 66, 69, 70 -a transition that involves profound morphological changes-suggesting that its deregulated activation in the absence of JNK1 could participate in the altered branching of mutant OPCs. Another possible target of JNK1 in the regulation of OPC cytoskeleton is mTOR. Both molecules act in parallel or via cross-regulation in many pathological contexts, where JNK seems to positively regulate mTOR activity 71 .
JNK isoform 1 appears to play a predominant role in oligodendroglia. In further support of JNK1 functions in oligodendrocytes, we found that mutant cell proliferative and morphological alterations were recapitulated in WT OPCs treated with D-JNKI-1 36 . This inhibitor is able to block JIP-JNK interaction, thus preventing the phosphorylation of c-Jun, the main downstream target of all JNK isoforms, and of the other JBD targets 38,72 . Thus, this treatment might have revealed a much broader impact on the cells. However, D-JNKI-1 administration well recapitulated the proliferative and morphological phenotype of JNK1 KO OPCs, suggesting that JNK1, among the three JNK isoforms, has a predominant role in the regulation of OPC proliferation, branching and membrane extension. This hypothesis is also supported by qRT-PCR data of MACS-sorted OPCs (Suppl. Fig. 4C-E), revealing the absence of any compensatory upregulation or dysregulated expression the other two JNK isoforms.
In conclusion, our study shows that JNK1 ablation results in persistent myelin abnormalities in vivo and that JNK1 participates in a cell-autonomous manner in the regulation of OPC proliferation, branching architecture and membrane extension at mature stages, unveiling a novel player in the complex regulatory network of OPC biology. Further investigations are needed to disentangle the potential contribution of axonal vs oligodendroglial alterations in the hypomyelination phenotype in vivo. Finally, it is also interesting to note that most of the alterations that we reported in mutant oligodendroglia (i.e. contact-mediated regulation of OPC proliferation, OPC branching architecture and paranodal organization) are regulated by NT-1 signaling 7, 73-75 . Further studies are needed to clarify whether NT-1 acts via JNK1 in oligodendroglia, as formerly shown in neurons 76,77 .

Methods
Experimental animals. For histological analyses and Magnetic-Activated Cell Sorting (MACS) we employed JNK1 KO 78 and age-matched wild-type (WT) mice as controls. Perfusions of juvenile and adult mice were carried out under deep general anaesthesia obtained by intraperitoneal administration of ketamine (100 mg/kg; Ketavet; Bayern; Leverkusen, Germany) supplemented by xylazine (5 mg/kg; Rompun; Bayer). For OPCs cultures, postnatal (P0-P1) mice and rats were anesthetized on melting ice. Groups of 4-5 mice were housed in transparent polycarbonate cages (Tecnoplast, Buggirate, Italy) provided with sawdust bedding, boxes/ tunnels hideout as environmental enrichment and striped paper as nesting material. Food and water were provided ad libitum; environmental conditions were 12 h/12 h light/dark cycle, room temperature 21 °C ± 1 °C and room humidity 55% ± 5%.
The experimental plan was designed according to the guidelines of the NIH, the European Communities Council (2010/63/EU) and the Italian Law for Care and Use of Experimental Animals (DL26/2014). It was also approved by the Italian Ministry of Health (Authorization 1112/2016 prot E669C.20) and the Bioethical Committee of the University of Turin. The study was conducted according to the ARRIVE guidelines.
To test the effect of D-JNKI-1 treatment, primary rat OPCs were isolated by the shaking method from mixed glial cultures obtained from P0-1 Sprague-Dawley rat cortex, as described in 5 . OPCs were plated onto poly-D-lysine (1 µg/ml, Sigma-Aldrich) -coated 12-mm glass coverslips for immunocytochemistry (5 × 10 4 cells/ coverslip) in the proliferative medium (see above). After 1 day in vitro (DIV), the cell permeable JNK-inhibitor D-JNKI-1 (2 µM) 36 was added to the medium until fixation (after 3DIV or 7DIV in proliferative or non-proliferative conditions, respectively). Voronoi analysis of the cell distribution was performed with ImageJ while cell territory and soma area were analyzed with Imaris (Bitplane) software (only cells whose entire extension was completely included in the confocal stack were considered). The number of inspected cells ranged from 46 to 70 cells per individual, with a total of ~ 300/350 cells per genotype. Primary ramifications, ramification length and complexity of branching analyses were performed with the Neurolucida system (MicroBrightfield, Colchester, VT). The analysis of the complexity of branching was performed assigning progressive numbers (i.e. orders) to branches extending directly from the cell soma (order 1) and then to all processes centrifugally emerging from subsequent branches (order > 1), to describe the hierarchy of the branching scheme. Each tree (i.e. each primary ramification (order 1) associated with its branching scheme) was analyzed individually. Plotted values (Figs. 3O,4I) represent the mean of all analyzed trees. OPCs juxtaposed with symmetrical cell somata and decondensed grainy DNA were recognized as duplets of cells that exited cytokinesis after cell division 5,81,82 . As such, OPC duplets were counted in tissue slices to measure OPC proliferative activity 32 . OPC proliferation was also evaluated by counting NG2 + /Ki67 + double positive cells. Adobe Illustrator 6.0 (Adobe Systems, San Jose, CA) was used to assemble the final plates. In all the analyses the experimenter was blind to the genotype of the samples.

In vitro cell counting and morphological analyses.
Expression of Ki67/cCASP3 in cultured OPCs and of MBP in cultured OLs was investigated live in five to eight quadrants localized in central and peripheral areas of each coverslip-as described in 83 -with the Neurolucida software. Results for each quadrant were expressed as a percentage of marker-positive cells over the number of OPCs and averaged across different coverslips. For reconstructing OPC arborizations, 20-30 non-proliferative (Ki67-negative) OPCs/coverslip isolated from other cells were randomly selected and traced live with the Neurolucida software, with a total of ~ 60-70 inspected cells per condition. Cultured MBP + OLs were categorized in immature/mature OLs depending on the localization of MBP + staining (restricted to ramifications for immature cells or further expanded to lamellae-like membranes for mature cells) and on the complexity of their processes (poorly branched for immature cells and complexly branched for mature cells, as described in 84 ). The surface occupancy of MBP + lamelliform OLs was analyzed with ImageJ. The number of inspected cells ranged from 15 to 25 cells per coverslip, with a total of ~ 80/100 cells per condition. In all cell counting and morphological analyses the experimenter was blind to the genotype or treatment of the cells.
Quantitative RT-PCR. Total RNA from MACS-sorted OPCs was extracted with the Direct-zol RNA Miniprep kit (Zymo Research, Irevine, USA), and reverse transcribed to cDNA with the High-Capacity cDNA Archive kit (Applied Biosystems, Thermofisher, Waltham, USA). Quantitative Real Time RT-PCR was performed as described in 85  www.nature.com/scientificreports/ or by combining the RealTime Ready Universal Probe Library (UPL, Roche Diagnostics, Monza, Italy) with the primers indicated in Suppl. Table 1. A relative quantification approach was used, according to the 2 -ddCT method 86 . β-actin was used to normalize expression levels.
Tissue dissection, lysates and western blotting. CC and cortices from P7, P15 and P30 WT and JNK1KO mice, after brain sectioning with Leica vibratome, were obtained by dissection. Tissue lysates were obtained adding RIPA buffer (1% NP40, 150 mM NaCl, 50 mM TRIS HCl pH 8, 5 mM EDTA, 0.01% SDS, 0.005% Sodium deoxycholate, Roche protease inhibitors, PMSF) for 10 min at 4 °C. Samples were homogenized on ice with a pellet pestle (Sigma-Aldrich, Saint Louis, MS, USA) and centrifuged at 1300 rpm at 4 °C. For immunoblots, equal amounts of proteins were resolved by SDS-PAGE and blotted to nitrocellulose membranes, which were then probed with anti-MBP (1:1000 Statistical analyses were carried out with GraphPad Prism 7 (GraphPad software, Inc). The Shapiro-Wilk test was first applied to test for a normal distribution of the data. When normally distributed, unpaired Student's t test (to compare two groups) and Two-way ANOVA test (for multiple group comparisons) followed by Sidak's post hoc analysis were used. Statistics also included Chi-square test (to compare frequencies) and linear regression analysis (to analyze in vitro OPC proliferation and apoptosis in relation to cell density). In all instances, P < 0.05 was considered as statistically significant. Histograms represent mean ± standard error (SE). Statistical differences were indicated with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The list of the applied tests, F values and values for n (animals for in vivo analyses, experiments for in vitro analyses), results of post hoc analyses are included in Supplementary Table 2. Supporting data are available within the article or can be provided upon request.