Myelin: Methods for Purification and Proteome Analysis.

Molecular characterization of myelin is a prerequisite for understanding the normal structure of the axon/myelin-unit in the healthy nervous system and abnormalities in myelin-related disorders. However, reliable molecular profiles necessitate very pure myelin membranes, in particular when considering the power of highly sensitive "omics"-data acquisition methods. Here, we recapitulate the history and recent applications of myelin purification. We then provide our laboratory protocols for the biochemical isolation of a highly pure myelin-enriched fraction from mouse brains and for its proteomic analysis. We also supply methodological modifications when investigating posttranslational modifications, RNA, or myelin from peripheral nerves. Notably, technical advancements in solubilizing myelin are beneficial for gel-based and gel-free myelin proteome analyses. We conclude this article by exemplifying the exceptional power of label-free proteomics in the mass-spectrometric quantification of myelin proteins.

Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin.

Myelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS.

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.

Septin/anillin filaments scaffold central nervous system myelin to accelerate nerve conduction.

Myelination of axons facilitates rapid impulse propagation in the nervous system. The axon/myelin-unit becomes impaired in myelin-related disorders and upon normal aging. However, the molecular cause of many pathological features, including the frequently observed myelin outfoldings, remained unknown. Using label-free quantitative proteomics, we find that the presence of myelin outfoldings correlates with a loss of cytoskeletal septins in myelin. Regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2)-levels, myelin septins (SEPT2/SEPT4/SEPT7/SEPT8) and the PI(4,5)P2-adaptor anillin form previously unrecognized filaments that extend longitudinally along myelinated axons. By confocal microscopy and immunogold-electron microscopy, these filaments are localized to the non-compacted adaxonal myelin compartment. Genetic disruption of these filaments in Sept8-mutant mice causes myelin outfoldings as a very specific neuropathology. Septin filaments thus serve an important function in scaffolding the axon/myelin-unit, evidently a late stage of myelin maturation. We propose that pathological or aging-associated diminishment of the septin/anillin-scaffold causes myelin outfoldings that impair the normal nerve conduction velocity.