Antagonistic actions of PAK1 and NF2/Merlin drive myelin membrane expansion in oligodendrocytes.

In the central nervous system, the formation of myelin by oligodendrocytes (OLs) relies on the switch from the polymerization of the actin cytoskeleton to its depolymerization. The molecular mechanisms that trigger this switch have yet to be elucidated. Here, we identified P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. Our results demonstrate that PAK1 accumulates in OLs in a kinase-inhibited form, triggering actin disassembly and, consequently, myelin membrane expansion. Remarkably, proteomic analysis of PAK1 binding partners enabled the identification of NF2/Merlin as its endogenous inhibitor. Our findings indicate that Nf2 knockdown in OLs results in PAK1 activation, actin polymerization, and a reduction in OL myelin membrane expansion. This effect is rescued by treatment with a PAK1 inhibitor. We also provide evidence that the specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo. Overall, our data indicate that the antagonistic actions of PAK1 and NF2/Merlin on the actin cytoskeleton of the OLs are critical for proper myelin formation. These findings have broad mechanistic and therapeutic implications in demyelinating diseases and neurodevelopmental disorders.

Co-culture of exogenous oligodendrocytes with unmyelinated cerebella: Revisiting ex vivo models and new tools to study myelination.

Common in vitro models used to study the mechanisms regulating myelination rely on co-cultures of oligodendrocyte precursor cells (OPCs) and neurons. In such models, myelination occurs in an environment that does not fully reflect cell-cell interactions and environmental cues present in vivo. To avoid these limitations while specifically manipulating oligodendroglial cells, we developed a reliable ex vivo model of myelination by seeding OPCs on cerebellar slices, deprived of their endogenous oligodendrocytes. We showed that exogenous OPCs seeded on unmyelinated cerebella, efficiently differentiate and form compact myelin. Spectral confocal reflectance microscopy and electron microscopy analysis revealed that the density of compacted myelin sheaths highly increases all along the culture. Importantly, we defined the appropriate culture time frame to study OPC differentiation and myelination, using accurate quantification resources we generated. Thus, this model is a powerful tool to study the cellular and molecular mechanisms of OPC differentiation and myelination. Moreover, it is suitable for the development and validation of new therapies for myelin-related disorders such as multiple sclerosis and psychiatric diseases.

The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation.

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.

Clathrin is required for Scar/Wave-mediated lamellipodium formation.

The Scar/Wave complex (SWC) generates lamellipodia through Arp2/3-dependent polymerisation of branched actin networks. In order to identify new SWC regulators, we conducted a screen in Drosophila cells combining proteomics with functional genomics. This screen identified Clathrin heavy chain (CHC) as a protein that binds to the SWC and whose depletion affects lamellipodium formation. This role of CHC in lamellipodium formation can be uncoupled from its role in membrane trafficking by several experimental approaches. Furthermore, CHC is detected in lamellipodia in the absence of the adaptor and accessory proteins of endocytosis. We found that CHC overexpression decreased membrane recruitment of the SWC, resulting in reduced velocity of protrusions and reduced cell migration. By contrast, when CHC was targeted to the membrane by fusion to a myristoylation sequence, we observed an increase in membrane recruitment of the SWC, protrusion velocity and cell migration. Together these data suggest that, in addition to its classical role in membrane trafficking, CHC brings the SWC to the plasma membrane, thereby controlling lamellipodium formation.

RNAi depleted Drosophila cell extracts to dissect signaling pathways leading to actin polymerization.

Dissection of signal transduction pathways leading to actin polymerization has been performed in cytosolic extracts. In such assays, the implication of an effector molecule is demonstrated by the loss of actin polymerization upon its depletion and the restoration of actin polymerization upon its add-back. Two major limitations in the wide use of this approach have been the availability of immunodepleting antibodies and the functional redundancy for many classes of effector molecules encoded by vertebrate genomes. To circumvent these limitations, we developed extracts derived from S2 Drosophila cells, which are competent for actin polymerization. In this system, depleted extracts are simply obtained from cells cultured with long double stranded RNAs in the medium. We validated the method by showing that beads coated with the C-terminal domain of Wave2 were no longer able to trigger actin polymerization in an extract depleted of the Arp2/3 complex. We also examined the complete set of Drosophila small GTPases of the Rho family for their ability to polymerize actin in such extracts, and found that only dCdc42 was able to induce actin polymerization. Using RNAi depleted extract, we confirmed that dCdc42 triggers actin polymerization in a Wasp dependent manner.

The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination.

Axonal regeneration in the mammalian CNS is a property of immature neurons that is lost during development. Using organotypic culture of cerebellum, we have shown that in vitro Purkinje cells lose their regenerative capacity in parallel with the process of myelination. We have investigated whether myelination is involved in the age-dependent loss of regeneration of these neurons. By applying a high dose of bromodeoxyuridine in the culture medium of newborn cerebellar slices during the first 3 d in vitro, we have succeeded in obtaining cultures with oligodendrocyte depletion, together with a lack of ameboid microglia and enhancement of Purkinje cell survival. These cultures, after 14 d in vitro, are completely devoid of myelin. We have compared the ability of Purkinje cells to regenerate their axons in the presence or absence of myelin. Purkinje cells in cerebellar explants taken at birth, treated with bromodeoxyuridine and axotomized after 7 d in vitro, survive better than similar neurons in untreated cultures. However, despite the lack of myelin and the enhanced survival, Purkinje cells do not regenerate, whereas they do regenerate when the axotomy is done at postnatal day 0. Thus, the Purkinje cell developmental switch from axonal regeneration to lack of regeneration does not appear to be regulated by myelin.

Purkinje cell maturation participates in the control of oligodendrocyte differentiation: role of sonic hedgehog and vitronectin.

Oligodendrocyte differentiation is temporally regulated during development by multiple factors. Here, we investigated whether the timing of oligodendrocyte differentiation might be controlled by neuronal differentiation in cerebellar organotypic cultures. In these cultures, the slices taken from newborn mice show very few oligodendrocytes during the first week of culture (immature slices) whereas their number increases importantly during the second week (mature slices). First, we showed that mature cerebellar slices or their conditioned media stimulated oligodendrocyte differentiation in immature slices thus demonstrating the existence of diffusible factors controlling oligodendrocyte differentiation. Using conditioned media from different models of slice culture in which the number of Purkinje cells varies drastically, we showed that the effects of these differentiating factors were proportional to the number of Purkinje cells. To identify these diffusible factors, we first performed a transcriptome analysis with an Affymetrix array for cerebellar cortex and then real-time quantitative PCR on mRNAs extracted from fluorescent flow cytometry sorted (FACS) Purkinje cells of L7-GFP transgenic mice at different ages. These analyses revealed that during postnatal maturation, Purkinje cells down-regulate Sonic Hedgehog and up-regulate vitronectin. Then, we showed that Sonic Hedgehog stimulates the proliferation of oligodendrocyte precursor cells and inhibits their differentiation. In contrast, vitronectin stimulates oligodendrocyte differentiation, whereas its inhibition with blocking antibodies abolishes the conditioned media effects. Altogether, these results suggest that Purkinje cells participate in controlling the timing of oligodendrocyte differentiation in the cerebellum through the developmentally regulated expression of diffusible molecules such as Sonic Hedgehog and vitronectin.

Heterogeneity of NG2-expressing cells in the newborn mouse cerebellum.

The function and origin of NG2+ cells in the adult brain are still controversial. A large amount of data is available which strongly indicates that adult NG2-expressing cells form a heterogeneous population, constituted by oligodendrocyte precursor cells (OPCs) and a fourth novel type of glial cells named the synantocytes. Whether these two populations derive from the progressive maturation of perinatal NG2+ OPCs or are generated as separate populations is not known. We used organotypic cultures of newborn mouse cerebellum depleted, by anti-mitotic drug treatment, of their NG2+ cells with perinatal features (high proliferating rate and high oligodendrocytic differentiation ability). In these cultures, despite the lack of myelin after 14 days in vitro, numerous NG2+ cells remained. We show that these BrdU-resistant cells were able to slowly divide, as adult NG2+ cells do. Although many of these cells expressed O4, only a very small fraction of them was further engaged in oligodendrocyte lineage, as they had an extremely poor capacity to generate myelin sheaths to the Purkinje cell axons. These results support the view that at least two distinct populations of NG2+ cells coexist in the cerebellum from birth: one with the young OPC characteristics, another with adult NG2+ cell characteristics. Thus, a fraction of adult NG2+ cells do not derive from the maturation of perinatal OPCs.