Myelin Membrane Research Articles

Conformational choreography of a molecular switch region in myelin basic protein—Molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

The 18.5kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.

Response of Human Oligodendrocyte Progenitors to Growth Factors and Axon Signals

We examined the effects of growth factors and axonal signals on the differentiation of human fetal and adult oligodendrocyte progenitor cells (OPCs) and determined whether these effects translated into enhanced axonal ensheathment. Only small numbers of fetal OPCs grown in defined medium expressed the oligodendroglial lineage markers Olig2 and O4. The combination of platelet-derived growth factor-AA and basic fibroblast growth factor enhanced proliferation of Olig2-positive and O4-positive cells; a combination of brain-derived neurotrophic factor and insulin-like growth factor 1 promoted O4-positive cell differentiation, galactocerebroside expression, and morphological complexity. Coculturing with rodent dorsal root ganglion neurons in defined medium alone enhanced OPC differentiation and myelin basic protein expression. The addition of brain-derived neurotrophic factor/insulin-like growth factor 1 further enhanced differentiation, axonal attachment and ensheathment, and clustering of the contactin-associated protein Caspr and Na+ channels. By contrast, most adult OPCs were O4 positive and Olig2 positive in defined medium; both brain-derived neurotrophic factor/insulin-like growth factor 1 and platelet-derived growth factor-AA/basic fibroblast growth factor promoted their myelin basic protein expression and membrane sheet formation; coculture with dorsal root ganglion neurons further increased myelin basic protein expression. Growth factors also enhanced attachment of adult OPCs to axons, but their capacity to ensheath axons was lower than that of fetal OPCs. These results demonstrate that fetal and adult OPCs show measurable responses to selected growth factors and axon signals that correlate with their capacity for axon ensheathment. The distinct properties of fetal and adult OPCs may be related to differences in their chronological age and initial differentiation states.

Sleep Homeostasis: Finding Odysseus among the Mnesteres

Sleep Homeostasis: Finding Odysseus among the Mnesteres

Cholesterol Defect Is Marked across Multiple Rodent Models of Huntington's Disease and Is Manifest in Astrocytes

Brain cholesterol, which is synthesized locally, is a major component of myelin and cell membranes and participates in neuronal functions, such as membrane trafficking, signal transduction, neurotransmitter release, and synaptogenesis. Here we show that brain cholesterol biosynthesis is reduced in multiple transgenic and knock-in Huntington's disease (HD) rodent models, arguably dependent on deficits in mutant astrocytes. Mice carrying a progressively increased number of CAG repeats show a more evident reduction in cholesterol biosynthesis. In postnatal life, the cholesterol-dependent activities of neurons mainly rely on the transport of cholesterol from astrocytes on ApoE-containing particles. Our data show that mRNA levels of cholesterol biosynthesis and efflux genes are severely reduced in primary HD astrocytes, along with impaired cellular production and secretion of ApoE. Consistently, in CSF of HD mice, ApoE is mostly associated with smaller lipoproteins, indicating reduced cholesterol transport on ApoE-containing lipoproteins circulating in the HD brain. These findings indicate that cholesterol defect is robustly marked in HD animals, implying that strategies aimed at selectively modulating brain cholesterol metabolism might be of therapeutic significance.

Sphingolipids in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the CNS. Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), are target cells in MS. Although the etiology of MS is poorly known, new insights suggest oligodendrocyte apoptosis as one of the critical events followed by glial activation and infiltration of lymphocytes and macrophages. A major breakthrough in delineation of the mechanism of cell death, perivascular cuffing, and glial activation came from elucidation of the sphingolipid signal transduction pathway. The sphingolipid signal transduction pathway induces apoptosis, differentiation, proliferation, and growth arrest depending upon cell and receptor types, and downstream targets. Sphingomyelin, a major component of myelin membrane formed by mature oligodendrocytes, is abundant in the CNS and ceramide, its primary catabolic product released by activation of either neutral or acidic sphingomyelinase, serves as a potential lipid second messenger or mediator molecule modulating diverse cellular signaling pathways. Similarly, under certain conditions, sphingosine produced from ceramide by ceramidase is phosphorylated by sphingosine kinases to sphingosine-1 phosphate, another potent second messenger molecule. Both ceramide and sphingosine-1 phosphate regulate life and death of many cell types including brain cells and participate in pathogenic processes of MS. In this review, we have made an honest attempt to compile recent findings made by others and us relating to the role of sphingolipids in the disease process of MS.

Structural and functional characterization of human peripheral nervous system myelin protein P2.

The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 Å. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces.

An Apolipoprotein E-Mimetic Stimulates Axonal Regeneration and Remyelination after Peripheral Nerve Injury

Elevated apolipoprotein E (apoE) synthesis within crushed sciatic nerves advocates that apoE could benefit axonal repair and reconstruction of axonal and myelin membranes. We created an apoE-mimetic peptide, COG112 (acetyl-RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL-amide), and found that postinjury treatment with COG112 significantly improved recovery of motor and sensory function following sciatic nerve crush in C57BL/6 mice. Morphometric analysis of injured sciatic nerves revealed that COG112 promoted axonal regrowth after 2 weeks of treatment. More strikingly, the thickness of myelin sheaths was increased by COG112 treatment. Consistent with these histological findings, COG112 potently elevated growth associated protein 43 (GAP-43) and peripheral myelin protein zero (P0), which are markers of axon regeneration and remyelination, respectively. Electron microscopic examination further suggested that the apoE-mimetic COG112 may increase clearance of myelin debris. Schwann cell uptake of cholesterol-containing low-density lipoprotein particles was selectively enhanced by COG112 treatment in a Schwann cell line S16. Moreover, COG112 significantly promoted axon elongation in primary dorsal root ganglion cultures from rat pups. Considering that cholesterol and lipids are needed for reconstructing myelin sheaths and axon extension, these data support a hypothesis where supplementation with exogenous apoE-mimetics such as COG112 may be a promising strategy for restoring lost functional and structural elements following nerve injury.

Method of Identifying Natural Antibodies for Remyelination

Natural autoantibodies are part of the normal human immunoglobulin repertoire. These antibodies react to self-antigens, are usually polyreactive with relatively low affinity, and typically are of the IgM isotype. Natural IgMs in mice that stimulated remyelination in central nervous system (CNS) demyelinating disease all shared the characteristics of binding to the surface of live oligodendrocytes and myelinated tracts in living slices of CNS tissue. A screen for human IgMs with similar character resulted in two human natural antibodies, which when injected peripherally into animal models of demyelination induced remyelination. A recombinant human IgM (rHIgM22) that also promoted remyelination in vivo was constructed. Very small doses of this IgM are required for the promotion of remyelination (EC50 is 460 ng per 20-g mouse). It is clear that after peripheral delivery, rHIgM22 enters the CNS and accumulates in CNS lesions. rHIgM22 was tracked in living mice using ferritin-labeled antihuman mu chain antibodies visualized by magnetic resonance imaging and traditional immunocytochemistry. Although the exact antigen recognized by rHIgM22 is not known, all mouse IgMs that promote remyelination bind to myelin membrane lipids, suggesting the antigen for rHIgM22 is similar. We propose that the IgMs bind to CNS cells and reorganize the membrane, initiating a signal that results in oligodendrocyte proliferation and/or protection with an end result of increased myelin. Recombinant natural human antibodies are potentially important therapeutic molecules that may modulate a wide spectrum of human disease.

LEUKOENCEPHALOPATHIES AND LEUKODYSTROPHIES

The leukoencephalopathies encompass a heterogeneous group of disorders that involve the brain white matter. The cause may be acquired or inherited; in the latter case, mutations have been found in genes that encode protein components of the myelin membrane or enzymes implicated in the turnover of myelin. In patients with cognitive dysfunction and white matter lesions evident on MRI, analysis of the type, pattern, and distribution of lesions can enable a presumptive diagnosis, which can be confirmed by biochemical and/or molecular testing. The presence or absence of peripheral neuropathy and/or autonomic dysfunction can be a helpful clue in differentiating individual diagnoses. Often, patients may be suspected of having and being initially treated inappropriately for a case of primary or secondary progressive multiple sclerosis. In a significant number of patients, the diagnosis may not be made, even after an extensive search. Establishing the cause enables counseling regarding prognosis, family planning, monitoring for disease-related complications, and introducing therapy, when available.

Dihydroprogesterone Increases the Gene Expression of Myelin Basic Protein in Spinal Cord of Diabetic Rats

Alterations in myelin membranes, as well as in the expression of myelin proteins have been reported in experimental models of diabetes. Data here reported show for the first time that the mRNA levels of two isoforms of myelin basic protein (MBP), 18.5 and 21.5 kDa, are decreased in the spinal cord of streptozotocin-treated rats and that treatment with a neuroactive steroid, such as progesterone (P), may counteract this effect. Interestingly, metabolism of progesterone into dihydroprogesterone (DHP) by the enzyme 5alpha-reductase seems to exert an important role in such an effect. As here demonstrated, 5alpha-reductase mRNA and DHP levels are reduced by diabetes in spinal cord, but treatment with P, is able to counteract these effects. Moreover, treatment with DHP is able to mimic the effect of P on MBP gene expression. Thus, the effects of P here observed are due to its enzymatic conversion into DHP. Because DHP, like P, interacts with P receptor (PR), the present results may suggest the importance to analyze the effects of PR modulators as tools of therapeutic strategies for diabetic complications occurring in nervous system.

Myelination and the trophic support of long axons

In addition to their role in providing myelin for rapid impulse propagation, the glia that ensheath long axons are required for the maintenance of normal axon transport and long-term survival. This presumably ancestral function seems to be independent of myelin membrane wrapping. Here, I propose that ensheathing glia provide trophic support to axons that are metabolically isolated, and that myelin itself might cause such isolation. This glial support of axonal integrity may be relevant for a number of neurological and psychiatric diseases.

Tyrosine phosphatases Shp1 and Shp2 have unique and opposing roles in oligodendrocyte development

Oligodendrocyte progenitor cells first proliferate to generate sufficient cell numbers and then differentiate into myelin-producing oligodendrocytes. The signal transduction mediators that underlie these events, however, remain poorly understood. The tyrosine phosphatase Shp1 has been linked to oligodendrocyte differentiation as Shp1-deficient mice show hypomyelination. The Shp1 homolog, Shp2, has recently been shown to regulate astrogliogenesis, but its role in oligodendrocyte development remains unknown. Here, we report that Shp2 protein levels were developmentally regulated in oligodendrocytes, with Shp2 phosphorylation being promoted by oligodendroglial mitogens but suppressed by laminin, an extracellular matrix protein that promotes oligodendroglial differentiation. In contrast, oligodendrocyte progenitors were found to be unresponsive to mitogens following Shp2, but not Shp1, depletion. In agreement with previous studies, Shp1 depletion led to decreased levels of myelin basic protein in differentiating oligodendrocytes, as well as reduced outgrowth of myelin membrane sheets. Shp2 depletion in contrast did not prevent oligodendrocyte differentiation but promoted expanded myelin membrane outgrowth. Taken together these data suggest that Shp1 and Shp2 have distinct functions in oligodendrocyte development: Shp2 regulates oligodendrocyte progenitor proliferation and Shp1 regulates oligodendrocyte differentiation. Adhesion to laminin may additionally provide extrinsic regulation of Shp2 activity and thus promote the transition from progenitor to differentiating oligodendrocyte.

Oligodendrocytes and the “Micro Brake” of Progenitor Cell Proliferation

A large fraction of mammalian genes is regulated posttranscriptionally by microRNAs. In the developing nervous system, a small subset of these short RNA molecules is important to orchestrate the rapid switch from OPCs to myelin-forming oligodendrocytes. But putting the miRNA brake on oligodendrocyte gene expression is required throughout life.

Molecular regulation of Nogo-A in neural cells: Novel insights into structure and function

Nogo-A is part of the reticulon family of proteins localized to the myelin and oligodendroglial plasma membranes. Nogo-A specifically initiates signal transduction cascades limiting axonal regrowth following injury and disease in the adult mammalian central nervous system (CNS). Recent novel data support the contention that neuronal Nogo-A plays an important role in regulating cytoskeletal re-organization without the requirement of signaling through its cognate receptor (Nogo receptor). These data, along with the recent findings that the N-terminus of Nogo-A can interact with integrins and that NgR1 interacts with the amyloid precursor protein extracellularly, as well as novel findings showing ubiquitin ligases binding with Nogo-A intracellularly add a layer of complexity to its functional role in the CNS.

SCAP is required for timely and proper myelin membrane synthesis

Myelination requires a massive increase in glial cell membrane synthesis. Here, we demonstrate that the acute phase of myelin lipid synthesis is regulated by sterol regulatory element-binding protein (SREBP) cleavage activation protein (SCAP), an activator of SREBPs. Deletion of SCAP in Schwann cells led to a loss of SREBP-mediated gene expression involving cholesterol and fatty acid synthesis. Schwann cell SCAP mutant mice show congenital hypomyelination and abnormal gait. Interestingly, aging SCAP mutant mice showed partial regain of function; they exhibited improved gait and produced small amounts of myelin indicating a slow SCAP-independent uptake of external lipids. Accordingly, extracellular lipoproteins partially rescued myelination by SCAP mutant Schwann cells. However, SCAP mutant myelin never reached normal thickness and had biophysical abnormalities concordant with abnormal lipid composition. These data demonstrate that SCAP-mediated regulation of glial lipogenesis is key to the proper synthesis of myelin membrane, and provide insight into abnormal Schwann cell function under conditions affecting lipid metabolism.

In VivoQuantification of Myelin Changes in the Vertebrate Nervous System

Destruction or changes associated with myelin membranes in the CNS play a key role in the pathogenesis of multiple sclerosis and other related neurodegenerative disorders. A long-standing goal has been to detect and quantify myelin content in vivo. For this reason, we have developed a myelin-imaging technique based on positron emission tomography (PET). PET is a quantitative imaging modality that has been widely used in clinical settings for direct assessment of biological processes at the molecular level. However, lack of myelin-imaging probes has hampered the use of PET for imaging of myelination in the CNS. Here, we report a myelin-imaging agent, termed Case Imaging Compound (CIC) that readily penetrates the blood-brain barrier and preferentially localizes to myelinated regions of the brain. After radiolabeling with positron-emitting carbon-11, [(11)C]CIC-PET was conducted in longitudinal studies using a lysolethicin-induced rat model of focal demyelination and subsequent remyelination. Quantitative analysis showed that the retention of [(11)C]CIC correlates with the level of demyelination/remyelination. These studies indicate that, for the first time, [(11)C]CIC-PET can be used as an imaging marker of myelination, which has the potential to be translated into clinical studies in multiple sclerosis and other myelin-related diseases for early diagnosis, subtyping, and efficacy evaluation of therapeutic treatments aimed at myelin repair.

Characterizing healthy and diseased white matter using quantitative magnetization transfer and multicomponent T2 relaxometry: A unified view via a four‐pool model

Nuclear magnetic resonance relaxation and magnetization transfer in cerebral white matter can be described using a four-pool model: two for water protons (in separate myelin and intra/extracellular compartments) and two for protons associated with the lipids and proteins of biologic membranes (of myelin and nonmyelin semisolids). This model was used to gain insight into the observations from multicomponent quantitative T(2) relaxometry and quantitative magnetization transfer imaging, both based on simplified white matter models and experimentally feasible in vivo. Using a set of coupled Bloch equations describing the behavior of the magnetization in a four-pool model of white matter, simulations of the quantitative T(2) relaxometry and quantitative magnetization transfer imaging techniques were performed. Pathology-inspired modifications were made to the four-pool model to gauge their impact on quantitative T(2) relaxometry and quantitative magnetization transfer imaging observations. Our results show that changes in the rate of water movement between microanatomic compartments may impact otherwise stable quantitative T(2) relaxometry observations; that the measure of the quantitative magnetization transfer imaging-based semisolid pool population is robust, despite the presence of two distinct semisolid components; and that quantitative magnetization transfer imaging compartment size estimates are not influenced by changes in the T(2) of the intra/extracellular water pool. The four-pool model, while impractical for in vivo characterization, yields important insight into the interpretation of changes observed with these quantitative MRI methods based on simplified models of white matter.

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP-CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein-protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP-CaM interaction, including a 3D model of the complex between full-length proteins.

Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams

The use of coherent anti-Stokes Raman scattering microscopy tuned to the lipid vibration for quantitative myelin imaging suffers from the excitation polarization dependence of this third-order nonlinear optical effect. The contrast obtained depends on the orientation of the myelin membrane, which in turn affects the morphometric parameters that can be extracted with image analysis. We show how circularly polarized laser beams can be used to avoid this complication, leading to images free of excitation polarization dependence. The technique promises to be optimal for in vivo imaging and the resulting images can be used for coherent anti-Stokes Raman scattering optical histology on native state tissue.

Tibetan Medical Interpretation of Myelin Lipids and Multiple Sclerosis

Tibetan medicine integrates diet, lifestyle, herbs, and accessory therapies to increase health and longevity. A comparison of the three humor theory of Tibetan medicine and the three thermodynamic phase properties of myelin lipids exemplifies how integrating medical systems can increase understanding of complex chronic disabling conditions. As a correlative study to microscopically better understand multiple sclerosis (MS) from the view of Tibetan medicine, the physical disruption of central nervous system myelin membranes in MS is interpreted from the theory of the three humors (vital energies) of Tibetan medicine: rLung (Wind), MKhris pa (Bile), and Bad gen (Phlegm). The three classes of myelin lipids--phospholipids, sphingolipids, and cholesterol--are interpreted as one of three humors based on Langmuir isotherm thermodynamic measurements. The nature of rLung is movement or change. Myelin sphingolipids have rLung properties based on thermodynamic observations of changes in phase organization. MKhris pa is fire, energetic. Phospholipids have MKhris pa properties based on thermodynamic observations of being energetic membrane lipids with fast molecular motions and fluid-like properties. The nature of Bad gen is substance and form; it dominates body structure. Cholesterol relates to Bad gen because it dominates membrane structure. We propose a theoretical relationship whereby demyelination in MS is viewed as a continuum of imbalance of the three humors as understood in Tibetan medicine. Myelin lipid data is presented to support this theoretical relationship. Clinically, MS is, in general, a rLung-MKhrispa disorder in women and a Bad gen-MKhrispa disorder in men, with rLung-MKhrispa excess in both genders during exacerbation, inflammation, and demyelination. Studying Tibetan medicine in its traditional context will create an integrative model for the treatment of MS and other chronic conditions.