Loss of electrostatic cell-surface repulsion mediates myelin membrane adhesion and compaction in the central nervous system.
ABSTRACT: During the development of the central nervous system (CNS), oligodendrocytes wrap their plasma membrane around axons to form a multilayered stack of tightly attached membranes. Although intracellular myelin compaction and the role of myelin basic protein has been investigated, the forces that mediate the close interaction of myelin membranes at their external surfaces are poorly understood. Such extensive bilayer-bilayer interactions are usually prevented by repulsive forces generated by the glycocalyx, a dense and confluent layer of large and negatively charged oligosaccharides. Here we investigate the molecular mechanisms underlying myelin adhesion and compaction in the CNS. We revisit the role of the proteolipid protein and analyze the contribution of oligosaccharides using cellular assays, biophysical tools, and transgenic mice. We observe that differentiation of oligodendrocytes is accompanied by a striking down-regulation of components of their glycocalyx. Both in vitro and in vivo experiments indicate that the adhesive properties of the proteolipid protein, along with the reduction of sialic acid residues from the cell surface, orchestrate myelin membrane adhesion and compaction in the CNS. We suggest that loss of electrostatic cell-surface repulsion uncovers weak and unspecific attractive forces in the bilayer that bring the extracellular surfaces of a membrane into close contact over long distances.
Project description:Myelination of axons in the CNS by oligodendrocytes is a process critical to rapid and efficient impulse conduction. A new role for the myelin proteolipid protein (PLP), the most abundant protein of CNS myelin, has been identified, in studies showing PLP interaction with signaling proteins in oligodendrocytes. In particular, these studies suggest that the PLP protein may be involved in signaling through integrins in oligodendrocytes. Stimulation of muscarinic acetylcholine receptors on oligodendrocytes induced formation of a tripartite complex containing PLP, calreticulin, and alpha(v)-integrin. PLP interacted directly with the cytoplasmic domain of the alpha(v)-integrin. Complex formation was mediated by phospholipase C and Ca2+ binding to the high affinity binding site on calreticulin. This complex appears important for binding of fibronectin to oligodendrocytes. These data establish a novel function for PLP as a part of the integrin signaling complex in oligodendrocytes and suggest that neurotransmitter-mediated integrin receptor signaling may be involved in myelinogenesis.
Project description:Gap junctions (GJs) are important for maintenance of CNS homeostasis. GJ proteins, connexin 43 (Cx43) and connexin 47 (Cx47), play a crucial role in production and maintenance of CNS myelin. Cx43 is mainly expressed by astrocytes in the CNS and forms gap junction intercellular communications between astrocytes-astrocytes (Cx43-Cx43) and between astrocytes-oligodendrocytes (Cx43-Cx47). Mutations of these connexin (Cx) proteins cause dysmyelinating diseases in humans. Previously, it has been shown that Cx43 localization and expression is altered due to mouse hepatitis virus (MHV)-A59 infection both in vivo and in vitro; however, its mechanism and association with loss of myelin protein was not elaborated. Thus, we explored potential mechanisms by which MHV-A59 infection alters Cx43 localization and examined the effects of viral infection on Cx47 expression and its association with loss of the myelin marker proteolipid protein. Immunofluorescence and total internal reflection fluorescence microscopy confirmed that MHV-A59 used microtubules (MTs) as a conduit to reach the cell surface and restricted MT-mediated Cx43 delivery to the cell membrane. Co-immunoprecipitation experiments demonstrated that Cx43-?-tubulin molecular interaction was depleted due to protein-protein interaction between viral particles and MTs. During acute MHV-A59 infection, oligodendrocytic Cx47, which is mainly stabilized by Cx43 in vivo, was down-regulated, and its characteristic staining remained disrupted even at chronic phase. The loss of Cx47 was associated with loss of proteolipid protein at the chronic stage of MHV-A59 infection.
Project description:Clostridium perfringens epsilon toxin (?-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ?-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ?-toxin causes pathological changes to white matter. In primary CNS cultures, ?-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ?-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ?-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ?-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ?-toxin; oligodendrocyte progenitor cells are not. ?-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ?-toxin. In addition, ?-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ?-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ?-toxin in the CNS. (i) ?-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ?-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ?-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ?-toxin can be abrogated by an ?-toxin neutralizing antibody.Our intestinal tract is host to trillions of microorganisms that play an essential role in health and homeostasis. Disruption of this symbiotic relationship has been implicated in influencing or causing disease in distant organ systems such as the brain. Epsilon toxin (?-toxin)-carrying Clostridium perfringens strains are responsible for a devastating white matter disease in ruminant animals that shares similar features with human multiple sclerosis. In this report, we define the mechanism by which ?-toxin causes white matter disease. We find that ?-toxin specifically targets the myelin-forming cells of the central nervous system (CNS), oligodendrocytes, leading to cell death. The selectivity of ?-toxin for oligodendrocytes is remarkable, as other cells of the CNS are unaffected. Importantly, ?-toxin-induced oligodendrocyte death results in demyelination and is dependent on expression of myelin and lymphocyte protein (MAL). These results help complete the mechanistic pathway from bacteria to brain by explaining the specific cellular target of ?-toxin within the CNS.
Project description:During vertebrate brain development, axons are enwrapped by myelin, an insulating membrane produced by oligodendrocytes. Neuron-derived signaling molecules are temporally and spatially required to coordinate oligodendrocyte differentiation. In this study, we show that neurons regulate myelin membrane trafficking in oligodendrocytes. In the absence of neurons, the major myelin membrane protein, the proteolipid protein (PLP), is internalized and stored in late endosomes/lysosomes (LEs/Ls) by a cholesterol-dependent and clathrin-independent endocytosis pathway that requires actin and the RhoA guanosine triphosphatase. Upon maturation, the rate of endocytosis is reduced, and a cAMP-dependent neuronal signal triggers the transport of PLP from LEs/Ls to the plasma membrane. These findings reveal a fundamental and novel role of LEs/Ls in oligodendrocytes: to store and release PLP in a regulated fashion. The release of myelin membrane from LEs/Ls by neuronal signals may represent a mechanism to control myelin membrane growth.
Project description:Paranodal axoglial junctions are critical for maintaining the segregation of axonal domains along myelinated axons; however, the proteins required to organize and maintain this structure are not fully understood. Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are proteins enriched at paranodes that are expressed by neurons and oligodendrocytes. To identify the specific function of DCC expressed by oligodendrocytes in vivo, we selectively eliminated DCC from mature myelinating oligodendrocytes using an inducible cre regulated by the proteolipid protein promoter. We demonstrate that DCC deletion results in progressive disruption of the organization of axonal domains, myelin ultrastructure, and myelin protein composition. Conditional DCC knock-out mice develop balance and coordination deficits and exhibit decreased conduction velocity. We conclude that DCC expression by oligodendrocytes is required for the maintenance and stability of myelin in vivo, which is essential for proper signal conduction in the CNS.
Project description:Myelination by oligodendrocytes in the central nervous system requires coordinated exocytosis and endocytosis of the major myelin protein, proteolipid protein (PLP). Here, we demonstrated that a small GTPase, Rab27b, is involved in PLP trafficking in oligodendrocytes. We showed that PLP co-localized with Rab27b in late endosomes/lysosomes in oligodendrocytes. Short hairpin-mediated knockdown of Rab27b not only reduced lysosomal exocytosis but also greatly diminished the surface expression of PLP in oligodendrocytes. In addition, knockdown of Rab27b reduced the myelin-like membranes induced by co-culture of oligodendrocytes and neurons. Our data suggest that Rab27b is involved in myelin biogenesis by regulating PLP transport from late endosomes/lysosomes to the cell membrane in oligodendrocytes.
Project description: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.
Project description:Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, thereby allowing rapid propagation of action potentials. Despite the considerable clinical importance of myelination, little is known about the molecular mechanisms that enable oligodendrocytes to generate their specialized membrane wrapping. Here, we used microarray expression profiling of oligodendrocyte-ablated mutant mice to identify new glial molecules that are involved in CNS myelination. This effort resulted in the identification of Ermin, a novel cytoskeletal molecule that is exclusively expressed by oligodendrocytes. Ermin appears at a late stage during myelination, and in the mature nerves, it is localized to the outer cytoplasmic lip of the myelin sheath and the paranodal loops. In cultured oligodendrocytes, Ermin becomes visible in well differentiated MBP-positive cells, where it is concentrated at the tip of F-actin-rich processes (termed "Ermin spikes"). Ectopic expression of Ermin, but not of a mutant protein lacking its actin-binding domain, induced the formation of numerous cell protrusions and a pronounced change in cell morphology. Our results demonstrate that Ermin is a novel marker of myelinating oligodendroglia and suggest that it plays a role in cytoskeletal rearrangements during the late wrapping and/or compaction phases of myelinogenesis.
Project description:Conduction velocity (CV) of myelinated axons has been shown to be regulated by oligodendrocytes even after myelination has been completed. However, how myelinating oligodendrocytes regulate CV, and what the significance of this regulation is for normal brain function remain unknown. To address these questions, we analyzed a transgenic mouse line harboring extra copies of the myelin proteolipid protein 1 (plp1) gene (plp1(tg/-) mice) at 2 months of age. At this stage, the plp1(tg/-) mice have an unaffected myelin structure with a normally appearing ion channel distribution, but the CV in all axonal tracts tested in the CNS is greatly reduced. We also found decreased axonal diameters and slightly abnormal paranodal structures, both of which can be a cause for the reduced CV. Interestingly the plp1(tg/-) mice showed altered anxiety-like behaviors, reduced prepulse inhibitions, spatial learning deficits and working memory deficit, all of which are schizophrenia-related behaviors. Our results implicate that abnormalities in the neuron-glia interactions at the paranodal junctions can result in reduced CV in the CNS, which then induces behavioral abnormalities related to schizophrenia.
Project description:Mice lacking the expression of proteolipid protein (PLP)/DM20 in oligodendrocytes provide a genuine model for spastic paraplegia (SPG-2). Their axons are well myelinated but exhibit impaired axonal transport and progressive degeneration, which is difficult to attribute to the absence of a single myelin protein. We hypothesized that secondary molecular changes in PLP(null) myelin contribute to the loss of PLP/DM20-dependent neuroprotection and provide more insight into glia-axonal interactions in this disease model. By gel-based proteome analysis, we identified >160 proteins in purified myelin membranes, which allowed us to systematically monitor the CNS myelin proteome of adult PLP(null) mice, before the onset of disease. We identified three proteins of the septin family to be reduced in abundance, but the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 2 (SIRT2) was virtually absent. SIRT2 is expressed throughout the oligodendrocyte lineage, and immunoelectron microscopy revealed its association with myelin. Loss of SIRT2 in PLP(null) was posttranscriptional, suggesting that PLP/DM20 is required for its transport into the myelin compartment. Because normal SIRT2 activity is controlled by the NAD+/NADH ratio, its function may be coupled to the axo-glial metabolism and the long-term support of axons by oligodendrocytes.