Antagonistic Functions of MBP and CNP Establish Cytosolic Channels in CNS Myelin.

The myelin sheath is a multilamellar plasma membrane extension of highly specialized glial cells laid down in regularly spaced segments along axons. Recent studies indicate that myelin is metabolically active and capable of communicating with the underlying axon. To be functionally connected to the neuron, oligodendrocytes maintain non-compacted myelin as cytoplasmic nanochannels. Here, we used high-pressure freezing for electron microscopy to study these cytoplasmic regions within myelin close to their native state. We identified 2,'3'-cyclic nucleotide 3'-phosphodiesterase (CNP), an oligodendrocyte-specific protein previously implicated in the maintenance of axonal integrity, as an essential factor in generating and maintaining cytoplasm within the myelin compartment. We provide evidence that CNP directly associates with and organizes the actin cytoskeleton, thereby providing an intracellular strut that counteracts membrane compaction by myelin basic protein (MBP). Our study provides a molecular and structural framework for understanding how myelin maintains its cytoplasm to function as an active axon-glial unit.

Subcortical cytoskeleton periodicity throughout the nervous system.

Superresolution fluorescence microscopy recently revealed a ~190 nm periodic cytoskeleton lattice consisting of actin, spectrin, and other proteins underneath the membrane of cultured hippocampal neurons. Whether the periodic cytoskeleton lattice is a structural feature of all neurons and how it is modified when axons are ensheathed by myelin forming glial cells is not known. Here, STED nanoscopy is used to demonstrate that this structure is a commonplace of virtually all neuron types in vitro. To check how the subcortical meshwork is modified during myelination, we studied sciatic nerve fibers from adult mice. Periodicity of both actin and spectrin was uncovered at the internodes, indicating no substantial differences between unmyelinated and myelinated axons. Remarkably, the actin/spectrin pattern was also detected in glial cells such as cultured oligodendrocyte precursor cells. Altogether our work shows that the periodic subcortical cytoskeletal meshwork is a fundamental characteristic of cells in the nervous system and is not a distinctive feature of neurons, as previously thought.

Substitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding.

Intrinsically-disordered proteins (IDPs) present a complex interplay of conformational variability and multifunctionality, modulated by environment and post-translational modifications. The 18.5-kDa myelin basic protein (MBP) is essential to the formation of the myelin sheath of the central nervous system and is exemplary in this regard. We have recently demonstrated that the unmodified MBP-C1 component undergoes co-operative global conformational changes in increasing concentrations of trifluoroethanol, emulating the decreasing dielectric environment that the protein encounters upon adsorption to the oligodendrocyte membrane [K.A. Vassall et al., Journal of Molecular Biology, 427, 1977-1992, 2015]. Here, we extended this study to the pseudo-deiminated MBP-C8 charge component, one found in greater proportion in developing myelin and in multiple sclerosis. A similar tri-conformational distribution as for MBP-C1 was observed with slight differences in Gibbs free energy. A more dramatic difference was observed by cathepsin D digestion of the protein in both aqueous and membrane environments, which showed significantly greater accessibility of the F42-F43 cut site of MBP-C8, indicative of a global conformational change. In contrast, this modification caused little change in the protein's density of packing on myelin-mimetic membranes as ascertained by double electron-electron resonance spectroscopy [D.R. Kattnig et al., Biochimica et Biophysica Acta (Biomembranes), 1818, 2636-2647, 2012], or in its affinity for Ca(2+)-CaM. Site-specific threonyl pseudo-phosphorylation at residues T92 and/or T95 did not appreciably affect any of the thermodynamic mechanisms of conformational transitions, susceptibility to cathepsin D, or affinity for Ca(2+)-CaM, despite previously having been shown to affect local structure and disposition on the membrane surface.

Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system.

During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.