How Histone Deacetylases Control Myelination

Myelinated axons are a beautiful example of symbiotic interactions between two cell types: Myelinating glial cells organize axonal membranes and build their myelin sheaths to allow fast action potential conduction, while axons regulate myelination and enhance the survival of myelinating cells. Axonal demyelination, occurring in neurodegenerative diseases or after a nerve injury, results in severe motor and/or mental disabilities. Thus, understanding how the myelination process is induced, regulated, and maintained is crucial to develop new therapeutic strategies for regeneration in the nervous system. Epigenetic regulation has recently been recognized as a fundamental contributing player. In this review, we focus on the central mechanisms of gene regulation mediated by histone deacetylation and other key functions of histone deacetylases in Schwann cells and oligodendrocytes, the myelinating glia of the peripheral and central nervous systems.     

Molecular structure of the axolemma of developing axons following altered gliogenesis in rat optic nerve

Axonal and axolemmal development of fibers from rat optic nerves in which gliogenesis was severely delayed by systemic injection of 5-azacytidine (5-AZ) was examined by freeze-fracture electron microscopy. In neonatal (0-2 days) rat optic nerves, all fibers lack myelin, whereas in the adult, virtually all axons are myelinated. The axolemma of neonatal premyelinated fibers is relatively undifferentiated. The P-fracture face (P-face) displays a moderate (approximately 550/micron 2) density of intramembranous particles (IMPs), whereas the E-fracture face (E-face) has few IMPs (approximately 125/micron 2) present. By 14 days of age, approximately 25% of the axons within control optic nerves are ensheathed or myelinated, with the remaining axons premyelinated. The ensheathed and myelinated fibers display increased axonal diameter compared to premyelinated axons, and these larger caliber fibers exhibit marked axonal membrane differentiation. Notably, the P-face IMP density of ensheathed and myelinated fibers is substantially increased compared to premyelinated axolemma, and, at nodes of Ranvier, the density of E-face particles is moderately high (approximately 1300/micron 2), in comparison to internodal or premyelinated E-face axolemma. In optic nerves from 14-day-old 5-AZ-treated rats, few oligodendrocytes are present, and the percentage of myelinated fibers is markedly reduced. Despite delayed gliogenesis, some unensheathed axons within 5-AZ-treated optic nerves display an increased axonal diameter compared to premyelinated fibers. Most of these large caliber fibers also exhibit a substantial increase in P-face IMP density. Small (less than 0.4 micron) diameter unensheathed axons within treated optic nerves maintain a P-face IMP density similar to that of control premyelinated fibers. Regions of increased E-face particle density were not observed. The results demonstrate that some aspects of axolemma differentiation continue despite delayed gliogenesis and the absence of glial ensheathment, and suggest that axolemmal ultrastructure is, at least in part, independent of glial cell association.     

Cellular junctions of myelinated nerves (Review)

Myelinated nerves are specifically designed to allow the efficient and rapid propagation of action potentials. Myelinating glial cells contain several types of cellular junctions that are found between the myelin lamellas themselves in specialized regions of non-compact myelin and between the myelin membrane and the underlying axon. These include most of the junctional specializations found in epithelial cells, including tight, gap and adherens junctions. However, whereas in epithelial cells these junctions are formed between different cells, in myelinating glia these so called autotypic junctions are found between membrane lamellae of the same cell. In addition, myelinating glial cells form a heterotypic septate-like junction with the axon around the nodes of Ranvier and, in the peripheral nerve system, contact the basal lamina, which surrounds myelinating Schwann cells. This short review discusses the structure, molecular composition and function of the junctions present in myelinating cells, concentrating on the axo-glial junction.     

The Nodes of Ranvier: Molecular Assembly and Maintenance

Action potential (AP) propagation in myelinated nerves requires clustered voltage gated sodium and potassium channels. These channels must be specifically localized to nodes of Ranvier where the AP is regenerated. Several mechanisms have evolved to facilitate and ensure the correct assembly and stabilization of these essential axonal domains. This review highlights the current understanding of the axon intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the peripheral nervous system (PNS) and central nervous system (CNS).Axons conduct electrical signals, called action potentials (APs), among neurons in a circuit in response to sensory input, and between motor neurons and muscles. In mammals and other vertebrates, many axons are myelinated. Myelin, made by Schwann cells and oligodendrocytes in the peripheral nervous system (PNS) and central nervous system (CNS), respectively, is a multilamellar sheet of glial membrane that wraps around axons to increase transmembrane resistance and decrease membrane capacitance. Although myelin is traditionally viewed as a passive contributor to nervous system function, it is now recognized that myelinating glia also play many active roles including regulation of axon diameter, axonal energy metabolism, and the clustering of ion channels at gaps in the myelin sheath called nodes of Ranvier. Together, the active and passive properties conferred on axons by myelin, result in axons with high AP conduction velocities, low metabolic demands, and reduced space requirements as compared with unmyelinated axons. Thus, myelin and the clustering of ion channels in axons permitted the evolution of the complex nervous systems found in vertebrates. This review highlights the current understanding of the axonal intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the PNS and CNS.     

Freeze-etching study on membrane ultrastructural changes caused by freezing: cooling rate-dependent intramembrane particle aggregation in erythrocyte stripped ghosts

The changes of membrane ultrastructures by freezing stresses were examined on stripped ghosts which were made by removing almost all peripheral membrane proteins from human erythrocyte membranes. By freezing these stripped ghost membranes showed cooling rate-dependent intramembrane particle (IMP) aggregation. With the cooling rates at and faster than 30,000 degrees C/min, their IMPs were evenly distributed on the fracture faces. However, cooling rates at and slower than 8000 degrees C/min resulted in IMP aggregation. The degree of IMP aggregation increased in parallel with decreasing cooling rates. Without freezing, the IMP aggregation in stripped ghosts could be induced by exposing these ghosts to hypertonic salt solutions, but lowering the temperature did not affect IMP aggregation. The cooling rate-dependent IMP aggregation during freezing was suppressed by adding cryoprotective agents which were known to reduce the salt concentration of the medium during freezing. It is suggested that the IMP aggregation in stripped ghosts by freezing occurs by exposure to concentrated salt solutions during freezing. This result indicates the possibility that IMP aggregation may arise during slow freezing of some biomembranes as a result of an increase in salt concentration rather than as a result of reduction in temperature.     

Glia unglued: how signals from the extracellular matrix regulate the development of myelinating glia

The health and function of the nervous system relies on glial cells that ensheath neuronal axons with a specialized plasma membrane termed myelin. The molecular mechanisms by which glial cells target and enwrap axons with myelin are only beginning to be elucidated, yet several studies have implicated extracellular matrix proteins and their receptors as being important extrinsic regulators. This review provides an overview of the extracellular matrix proteins and their receptors that regulate multiple steps in the cellular development of Schwann cells and oligodendrocytes, the myelinating glia of the PNS and CNS, respectively, as well as in the construction and maintenance of the myelin sheath itself. The first part describes the relevant cellular events that are influenced by particular extracellular matrix proteins and receptors, including laminins, collagens, integrins, and dystroglycan. The second part describes the signaling pathways and effector molecules that have been demonstrated to be downstream of Schwann cell and oligodendroglial extracellular matrix receptors, including FAK, small Rho GTPases, ILK, and the PI3K/Akt pathway, and the roles that have been ascribed to these signaling mediators. Throughout, we emphasize the concept of extracellular matrix proteins as environmental sensors that act to integrate, or match, cellular responses, in particular to those downstream of growth factors, to appropriate matrix attachment.     

Freeze-Fracture Analysis of Plasma Membranes of CHO Cells Stably Expressing Aquaporins 1-5

Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1–5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10–20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in ∼5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13.5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays. Received: 17 March 1998/Revised: 19 June 1998     

N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.     

Effect of photoperiod and cold acclimation upon plasma free fatty acid levels in the white rat.

1. Animals were acclimated at 3 +/- 1 degrees C and at room temperature (22 degrees C) for 3 weeks. 2. At each acclimation temperature animals were maintained under either an 8:16 L:D cycle or a 16:8 L:D cycle. 3. Blood samples were taken before and after exposure to -38 degrees C for 30 min. 4. Free fatty acid levels were greatest in cold acclimated animals which were maintained on a short light cycle. 5. Interaction between acclimation and photoperiod was apparent.     

A screen for mutations in zebrafish that affect myelin gene expression in Schwann cells and oligodendrocytes

Myelin is the multi-layered glial sheath around axons in the vertebrate nervous system. Myelinating glia develop and function in intimate association with neurons and neuron-glial interactions control much of the life history of these cells. However, many of the factors that regulate key aspects of myelin development and maintenance remain unknown. To discover new molecules that are important for glial development and myelination, we undertook a screen of zebrafish mutants with previously characterized neural defects. We screened for myelin basic protein (mbp) mRNA by in situ hybridization and identified four mutants (neckless, motionless, iguana and doc) that lacked mbp expression in parts of the peripheral and central nervous systems (PNS or CNS), despite the presence of axons. In all four mutants electron microscopy revealed that myelin-forming glia were present and had formed loose wraps around axons but did not form compact myelin. We found that addition of exogenous retinoic acid (RA) rescued mbp expression in neckless mutant embryos, which lack endogenous RA synthesis. Timed application of the RA synthesis inhibitor DEAB to wild type embryos showed that RA signalling is required at least 48 h before the onset of myelin protein synthesis in both CNS and PNS.     

Microtubule-associated protein 1B: a neuronal binding partner for myelin-associated glycoprotein.

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia-axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553-562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.     

Components of the plasma membrane of growing axons. II. Diffusion of membrane protein complexes

Intramembrane particles (IMPs) of the plasmalemma of mature, synapsing neurons are evenly distributed along the axon shaft. In contrast, IMPs of growing olfactory axons form density gradients: IMP density decreases with increasing distance from the perikarya, with a slope that depends upon IMP size (Small, R., and K. H. Pfenninger, 1984, J. Cell Biol., 98: 1422-1433). These IMP density gradients resemble Gaussian tails, but they are much more accurately described by the equations formulated for diffusion in a system with a moving boundary (a Stefan Problem), using constants that are dependent upon IMP size. The resulting model predicts a shallow, nearly linear IMP density profile at early stages of growth. Later, this profile becomes gradually transformed into a steep nonlinear gradient as axon elongation proceeds. This prediction is borne out by the experimental evidence. The diffusion coefficients calculated from this model range from 0.5 to 1.8 X 10(-7) cm2/s for IMPs between 14.8 and 3.6 nm, respectively. These diffusion coefficients are linearly dependent upon the inverse IMP diameter in accordance with the Stokes-Einstein relationship. The measured viscosity is approximately 7 centipoise. Our findings indicate (a) that most IMPs in growing axons reach distal locations by lateral diffusion in the plasma membrane, (b) that IMPs-- or complexes of integral membrane proteins--can diffuse at considerably higher rates than previously reported for iso-concentration systems, and (c) that the laws of diffusion determined for macroscopic systems are applicable to the submicroscopic membrane system.     

Freeze-fracture features of epithelioid cells,multinucleated giant cells,and phagocytic macrophages

The freeze-fracture morphology of epithelioid cells, multinucleated giant cells (Langhans' type), and phagocytic macrophages was investigated. The intensely folded and interdigitating surface membranes of epithelioid cells and multinucleated giant cells displayed no specialized areas of cell contact. The size of the intramembranous particles (IMP) and the fact that the area density of IMPs was higher in the cytoplasmic (P) faces than in the external (E) faces of the cell membranes agreed with observations in other eukaryotic cells. The area densities of the IMPs suggest lower transport rates of molecules across the cell membranes of granuloma cells than of certain epithelial cells. Small pits were detected in the surface membranes of the granuloma cells but an extrusion of granules was not observed. The cytoplasmic granules displayed very different sizes and shapes ranging from spherical to rod-shaped. The latter type of granules (probably primary lysosomes) dominated in multinucleated giant cells. The granule membranes were studded with IMPs whose area densities increased with the granule size. Multilamellar bodies with smooth (lipid) fracture faces were found only in phagocytic macrophages. The nuclear pores of the granuloma cells were distributed over the entire surfaces of the nuclei and displayed moderate clustering. The values of the area densities of the nuclear pores were in keeping with the values observed in mammalian and human epithelial or mesenchymal cells, indicating similar exchange rates of molecules between the nucleoplasm and the cytoplasm in these different cell types. In a single phagocytic macrophage the E-face of the inner membrane of the nuclear envelope displayed a network of fine filaments whose nature is at present unknown.     

Oligodendrocytes support axonal transport and maintenance via exosome secretion

Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity.

The long-term integrity of neuronal axons depends on intrinsic mechanisms such as axonal transport and on extrinsic support from adjacent glial cells. This study shows that genetic defects in glia that affect axonal integrity impair the secretion of oligodendrocyte exosomes and their ability to support nutrient-deprived neurons and promote axonal transport.     

The Axonal Membrane Protein Caspr, a Homologue of Neurexin IV, Is a Component of the Septate-like Paranodal Junctions That Assemble during Myelination

We have investigated the potential role of contactin and contactin-associated protein (Caspr) in the axonal–glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homologue of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia.     

Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone

Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition.     

Integrins are necessary for the development and maintenance of the glial layers in the Drosophila peripheral nerve

Peripheral nerve development involves multiple classes of glia that cooperate to form overlapping glial layers paired with the deposition of a surrounding extracellular matrix (ECM). The formation of this tubular structure protects the ensheathed axons from physical and pathogenic damage and from changes in the ionic environment. Integrins, a major family of ECM receptors, play a number of roles in the development of myelinating Schwann cells, one class of glia ensheathing the peripheral nerves of vertebrates. However, the identity and the role of the integrin complexes utilized by the other classes of peripheral nerve glia have not been determined in any animal. Here, we show that, in the peripheral nerves of Drosophila melanogaster, two integrin complexes (αPS2βPS and αPS3βPS) are expressed in the different glial layers and form adhesion complexes with integrin-linked kinase and Talin. Knockdown of the common beta subunit (βPS) using inducible RNAi in all glial cells results in lethality and glial defects. Analysis of integrin complex function in specific glial layers showed that loss of βPS in the outermost layer (the perineurial glia) results in a failure to wrap the nerve, a phenotype similar to that of Matrix metalloproteinase 2-mediated degradation of the ECM. Knockdown of βPS integrin in the innermost wrapping glia causes a loss of glial processes around axons. Together, our data suggest that integrins are employed in different glial layers to mediate the development and maintenance of the protective glial sheath in Drosophila peripheral nerves.     

Behavioral thermoregulation in Hemigrapsus nudus,the amphibious purple shore crab

The thermoregulatory behavior of Hemigrapsus nudus, the amphibious purple shore crab, was examined in both aquatic and aerial environments. Crabs warmed and cooled more rapidly in water than in air. Acclimation in water of 16 degrees C (summer temperatures) raised the critical thermal maximum temperature (CTMax); acclimation in water of 10 degrees C (winter temperatures) lowered the critical thermal minimum temperature (CTMin). The changes occurred in both water and air. However, these survival regimes did not reflect the thermal preferences of the animals. In water, the thermal preference of crabs acclimated to 16 degrees C was 14.6 degrees C, and they avoided water warmer than 25.5 degrees C. These values were significantly lower than those of the crabs acclimated to 10 degrees C; these animals demonstrated temperature preferences for water that was 17 degrees C, and they avoided water that was warmer than 26.9 degrees C. This temperature preference was also exhibited in air, where 10 degrees C acclimated crabs exited from under rocks at a temperature that was 3.2 degrees C higher than that at which the 16 degrees C acclimated animals responded. This behavioral pattern was possibly due to a decreased thermal tolerance of 16 degrees C acclimated crabs, related with the molting process. H. nudus was better able to survive prolonged exposure to cold temperatures than to warm temperatures, and there was a trend towards lower exit temperatures with the lower acclimation (10 degrees C) temperature. Using a complex series of behaviors, the crabs were able to precisely control body temperature independent of the medium, by shuttling between air and water. The time spent in either air or water was influenced more strongly by the temperature than by the medium. In the field, this species may experience ranges in temperatures of up to 20 degrees C; however, it is able to utilize thermal microhabitats underneath rocks to maintain its body temperature within fairly narrow limits.     

The Fine Anatomy of the Optic Nerve of Anurans—An Electron Microscope Study

In the optic nerve of Anurans numerous myelinated and unmyelinated axons appear under the electron microscope as compact bundles that are closely bounded by one or several glial cells. In these bundles the unmyelinated fibers (0.15 to 0.6 µ in diameter) are many times more numerous than the myelinated fibers, and are separated from each other, from the bounding glial cells, or from adjacent myelin sheaths, by an extracellular gap that is 90 to 250 A wide. This intercellular space is continuous with the extracellular space in the periphery of the nerve through the numerous mesaxons and cell boundaries which reach the surface. Numerous desmosomes reinforce the attachments of adjacent glial membranes. The myelinated axons do not follow any preferential course and, like the unmyelinated ones, have a sinuous path, continuously shifting their relative position and passing from one bundle to another. At the nodes of Ranvier they behave entirely like unmyelinated axons in their relations to the surrounding cells. At the internodes they lie between the unmyelinated axons without showing an obvious myelogenic connection with the surrounding glial cells. In the absence of connective tissue separating individual myelinated fibers and with each glial cell simultaneously related to many axons, this myelogenic connection is highly distorted by other passing fibers and is very difficult to demonstrate. However, the mode of ending of the myelin layers at the nodes of Ranvier and the spiral disposition of the myelin layers indicate that myelination of these fibers occurs by a process similar to that of peripheral nerves. There are no incisures of Schmidt-Lantermann in the optic myelinated fibers.