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.