Peripheral Nervous System Myelin Proteins Research Articles

Myelin protein zero - the structural foundation behind peripheral compact myelin.

Schwann cells are the myelinating glia of the peripheral nervous system (PNS). During PNS development, Schwann cell precursors differentiate into mature Schwann cells after the radial sorting of axons and form myelin around selected axonal segments. This multilayered, highly lipid-rich and insulative membrane structure significantly accelerates nerve impulse propagation, which is a pre-requisite for the efficient function of the PNS. During this differentiation process, protein zero (P0), a single-pass transmembrane protein, is expressed in high amounts. P0 is the most abundant PNS myelin protein, at up to 50% of total protein, and its accumulation in membranes eventually forms stable compact myelin. Failure to do so, like in the case of mutated or truncated protein, results in non-compacted myelin and demyelination. This subsequently causes incurable peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), where CMT alone afflicts nearly 3 million people globally. To understand the compaction and maturation of PNS myelin, and the molecular etiology of disease, uncovering the structure-function relationships of P0 are of great importance. We demonstrate the molecular foundation of P0-mediated membrane stacking using a hybrid structural biology approach: on the extracellular side, immunoglobulin (Ig)-like domains form a zipper-like assembly of apposing protein monomers, whereas on the cytoplasmic side, the short, positively charged P0 tail (P0ct) glues membranes together. P0ct undergoes a substantial folding event during membrane binding, and we provide rationale to why membrane stacking is affected in diseases through functional in vitro mutagenesis studies. Additionally, we speculate on how post-translational modifications in P0ct help P0 locate to its target compartments, without inducing premature membrane stacking. Our results shed light on the essential role of P0 in during myelin maturation, and why other proteins fail to replace P0 when its function is compromised.

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail.

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane.

Friedreich Ataxia: Developmental Failure of the Dorsal Root Entry Zone.

Dorsal root ganglia, dorsal roots (DR), and dorsal root entry zones (DREZ) are vulnerable to frataxin deficiency in Friedreich ataxia (FA). A previously unrecognized abnormality is the intrusion of astroglial tissue into DR. Segments of formalin-fixed upper lumbar spinal cord of 13 homozygous and 2 compound heterozygous FA patients were sectioned longitudinally to represent DREZ and stained for glial fibrillary acidic protein (GFAP), S100, vimentin, the central nervous system (CNS)-specific myelin protein proteolipid protein, the peripheral nervous system (PNS) myelin proteins PMP-22 and P0, and the Schwann cell proteins laminin, alpha-dystroglycan, and periaxin. Normal DREZ showed short, sharply demarcated, dome-like extensions of CNS tissue into DR. The Schwann cell-related proteins formed tight caps around these domes. In FA, GFAP-, S100-, and vimentin-reactive CNS tissue extended across DREZ and into DR over much longer distances by breaching the CNS-PNS barrier. The transition between PNS and CNS myelin proteins was disorganized. During development, neural-crest derived boundary cap cells provide guidance to dorsal root ganglia axons growing into the dorsal spinal cord and at the same time block the inappropriate intrusion of CNS glia into DR. It is likely that frataxin is required during a critical period of permissive (axons) and nonpermissive (astroglia) border-control.

Influence of myelin proteins on the structure and dynamics of a model membrane with emphasis on the low temperature regime.

Myelin is an insulating, multi-lamellar membrane structure wrapped around selected nerve axons. Increasing the speed of nerve impulses, it is crucial for the proper functioning of the vertebrate nervous system. Human neurodegenerative diseases, such as multiple sclerosis, are linked to damage to the myelin sheath through demyelination. Myelin exhibits a well defined subset of myelin-specific proteins, whose influence on membrane dynamics, i.e., myelin flexibility and stability, has not yet been explored in detail. In a first paper [W. Knoll, J. Peters, P. Kursula, Y. Gerelli, J. Ollivier, B. Demé, M. Telling, E. Kemner, and F. Natali, Soft Matter 10, 519 (2014)] we were able to spotlight, through neutron scattering experiments, the role of peripheral nervous system myelin proteins on membrane stability at room temperature. In particular, the myelin basic protein and peripheral myelin protein 2 were found to synergistically influence the membrane structure while keeping almost unchanged the membrane mobility. Further insight is provided by this work, in which we particularly address the investigation of the membrane flexibility in the low temperature regime. We evidence a different behavior suggesting that the proton dynamics is reduced by the addition of the myelin basic protein accompanied by negligible membrane structural changes. Moreover, we address the importance of correct sample preparation and characterization for the success of the experiment and for the reliability of the obtained results.

Proteolipid protein cannot replace P0 protein as the major structural protein of peripheral nervous system myelin.

The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when proteolipid protein (PLP) replaced P0 protein as the most abundant protein of CNS myelin. However, PLP did not replace P0 in peripheral nervous system (PNS) myelin. To investigate the possible consequences of a PLP to P0 shift in PNS myelin, we engineered mice to express PLP instead of P0 in PNS myelin (PLP-PNS mice). PLP-PNS mice had severe neurological disabilities and died between 3 and 6 months of age. Schwann cells in sciatic nerves from PLP-PNS mice sorted axons into one-to-one relationships but failed to form myelin internodes. Mice with equal amounts of P0 and PLP had normal PNS myelination and lifespans similar to wild-type (WT) mice. When PLP was overexpressed with one copy of the P0 gene, sciatic nerves were hypomyelinated; mice displayed motor deficits, but had normal lifespans. These data support the hypothesis that while PLP can co-exist with P0 in PNS myelin, PLP cannot replace P0 as the major structural protein of PNS myelin.

Immune mechanisms in spontaneously occurring CIDP in NOD mice

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune disease affecting the peripheral nervous system (PNS) and is thought to involve both cellular and humoral immunity. Although its etiology remains to be fully elucidated, the use of animal models has provided some important information regarding its pathogenetic mechanisms. The development of a spontaneous autoimmune polyneuropathy (SAP) in B7-2 knockout non-obese diabetic (NOD) mice underscores the importance of co-stimulatory pathways such as B7-1/B7-2:CD28/CTLA-4 molecules in inflammatory neuropathies. These co-stimulatory molecules regulate the balance between pathogenic and regulatory T cells (Tregs). In SAP, pathogenic T cells are directed against myelin protein zero (P0), the most prominent PNS myelin protein that is a member of immunoglobulin gene superfamily.

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.

Comparison of CNS and PNS myelin proteins in the pathology of myelin disorders

This brief overview summarizes some of the ways that the structure, location and function of proteins in myelin sheaths affect the pathological abnormalities brought on by immunological, viral, genetic or other insults occurring in myelin disorders. The composition of compact myelin membranes is novel in comparison to other biological membranes, because of its high lipid content and the presence of several major proteins that are relatively specific for myelin [1,2]. The overall morphological structures of compact central nervous system (CNS) and peripheral nervous system (PNS) myelin are similar, even though they are formed by different cell types, that is, oligodendrocytes and Schwann cells, respectively.

The current status of structural studies on proteins of the myelin sheath (Review)

Myelin, the multilayered membrane structure surrounding axons, provides a unique environment to its proteins, which are either transmembrane proteins or interacting intimately with the membrane surface. Although myelin-specific proteins have been studied for decades, remarkably little is known of their three-dimensional structures. In addition, the exact functions of myelin proteins are to a large extent unknown. In this report, our current knowledge of peripheral nervous system myelin protein structures is reviewed, and the current status of attempts to solve the structures of full-length myelin proteins is evaluated. Furthermore, molecular models for the extracellular domain of the myelin-associated glycoprotein and the putative kinase-like domain of 2',3'-cyclic nucleotide 3'-phosphodiesterase are presented and discussed.

Myelin Po protein mutated at Cys21 has a dominant-negative effect on adhesion of wild type Po.

The homophilic adhesion of the peripheral nervous system myelin protein, Po, holds myelin compact at the extracellular leaflets. Po carries a single immunoglobulin (Ig)-like domain that is stabilized by a disulfide bond between Cys21 and Cys98. We showed previously that Po mutated at Cys21 to Ala (C21A Po), unlike wild type Po, when it was expressed in Chinese hamster ovary (CHO) cells after transfection, was not adhesive. To determine whether C21A Po could influence the adhesion of wild type Po, cells that already expressed wild type Po and that were shown to be adhesive were retransfected with plasmids containing C21A Po. After selection in hygromycin, clones that coexpressed wild type Po and Cys21-mutated Po were identified by polyacrylamide gel electrophoresis in the presence and absence of beta-mercaptoethanol, because only in coexpressors will there be two forms of Po of different apparent molecular weights under nonreducing conditions. Two clones that coexpressed wild type Po and C21A Po and a third clone, which, although it was resistant to hygromycin, expressed only wild type Po, were chosen for further study. An enzyme-linked immunosorbent assay of fixed, unpermeabilized cells showed that all of these clones expressed approximately equivalent amounts of Po at the cell surface. However, in an aggregation adhesion assay, only the cells that expressed wild type Po alone formed large aggregates and dropped in total particle number with time. The cells that coexpressed wild type and C21A Po did not form aggregates, and their adhesive properties were indistinguishable from the control transfected cells, which did not express Po. These results suggest that C21A Po has a dominant-negative effect on adhesion of wild type Po.

Prevention of experimental autoimmune neuritis by nasal administration of P2 protein peptide 57-81.

Experimental autoimmune neuritis (EAN) is a CD4+ T cell-mediated inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-Barre syndrome (GBS) in humans. Both EAN and GBS are associated with upregulated T and B cells responses to PNS myelin proteins including P2 protein, and by changes of the Th1/Th2 cell balance in favor of Th1. Here we report that EAN can be prevented by the dominant neuritogenic peptide 57-81 of the PNS P2 protein when given nasally before immunization of Lewis rats with bovine PNS myelin (BPM) + Freund's complete adjuvant (FCA). P2 peptide-tolerized rats were also resistant to EAN relapse after challenge with BPM. Tolerance to EAN in rats receiving high dose (60 microg/day/rat) P2 peptide nasally was associated with specific T and B cell anergy. This was characterized by the failure of T cells to proliferate in response to PNS myelin antigens, while responsiveness to phytohemagglutinin was retained. Numbers of BPM- and P2 peptide-reactive interferon-gamma mRNA expressing lymph node cells were reduced, while levels of P2 peptide-reactive interleukin 4 and transforming growth factor-beta mRNA-expressing cells were markedly upregulated on day 18 post immunization in the rats receiving high dose P2 peptide nasally. Tolerance to EAN was also associated with lower CD4+ cell infiltration, low-grade inflammation, or the absence of histological evidence of EAN, as well as with low IL-2 receptor and MHC class II molecule expression within the PNS. This is the first study showing that mucosal tolerance is applicable to EAN and, as an extension, could be considered in GBS.

Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13).

We have recently described a novel cDNA, SR13 (Welcher, A. A., U. Suter, M. De Leon, G. J. Snipes, and E. M. Shooter. 1991. Proc. Natl. Acad. Sci. USA. 88:7195-7199), that is repressed after sciatic nerve crush injury and shows homology to both the growth arrest-specific mRNA, gas3 (Manfioletti, G., M. E. Ruaro, G. Del Sal, L. Philipson, and C. Schneider, 1990. Mol. Cell Biol. 10:2924-2930), and to the myelin protein, PASII (Kitamura, K., M. Suzuki, and K. Uyemura. 1976. Biochim. Biophys. Acta. 455:806-816). In this report, we show that the 22-kD SR13 protein is expressed in the compact portion of essentially all myelinated fibers in the peripheral nervous system. Although SR13 mRNA was found in the central nervous system, no corresponding SR13 protein could be detected by either immunoblot analysis or by immunohistochemistry. Northern and immunoblot analysis of SR13 mRNA and protein expression during development of the peripheral nervous system reveal a pattern similar to other myelin proteins. Furthermore, we demonstrate by in situ mRNA hybridization on tissue sections and on individual nerve fibers that SR13 mRNA is produced predominantly by Schwann cells. We conclude that the SR13 protein is apparently exclusively expressed in the peripheral nervous system where it is a major component of myelin. Thus, we propose the name Peripheral Myelin Protein-22 (PMP-22) for the proteins and cDNA previously designated PASII, SR13, and gas3.

Expression of the PMP-22 Gene in Trembler Mutant Mice: Comparison with the Other Myelin Protein Genes

The Trembler mouse suffers from a dominantly inherited autosomal mutation affecting the Schwann cell activities, which results in an abnormal myelination of the peripheral nervous system. Very recently, it has been shown that the mutation is in the PMP-22 gene. However, the level of expression of the mutated gene in trembler mice has not been studied. Therefore, we measured the steady-state levels of mRNA encoding PMP-22 in the sciatic nerve of normal and trembler mice, and we compared these results with the steady-state levels of mRNAs encoding the major peripheral nervous system myelin proteins. Our results show that all the myelin protein genes studied are affected by the trembler mutation but to a different extent, and that the PMP-22 gene is expressed at very low levels in the trembler nerve. This suggests that regulation of the expression of the PMP-22 gene is altered in the Trembler mutant.

Separation of the major proteins of central and peripheral nervous system myelin using reversed-phase high-performance liquid chromatography

A general method to separate the major proteins of rat central and peripheral nervous system myelin has been developed. The key step is the initial quantitative removal of the lipids under conditions where the proteins retain their solubility in HPLC solvents. Lipids are removed by a combination of solvent extraction and column chromatography on Sephadex LH-60 in 2-chloroethanol:10 m m HCl (9:1). Proteins are then separated by reversed-phase (RP) HPLC. Samples are applied to a wide pore reversed-phase C-3 column and eluted with a linear gradient of 10–70% 1-propanol in 0.1% trifluoroacetic acid (0–100% B) over a 60-min period. Myelin basic proteins elute between 25 and 30% B, Wolfgram and other high molecular weight proteins at 35–50% B, proteolipid protein at 65–80% B, and P0 glycoprotein at 55–65% B. This elution pattern is consistent with the known relative hydrophobicity of these proteins. Protein recovery for the entire procedure is greater than 74%. Proteolipid and P0 proteins isolated by HPLC contain 2.3 and 1.1 mol of covalently bound fatty acids, respectively. This fatty acid composition is similar to that previously reported using different isolation procedures. The analysis of central and peripheral nervous system myelin proteins by RP-HPLC permits the isolation of purified proteins for structural and metabolic experiments.

Biosynthesis of the major integral protein of rat peripheral nerve myelin

Schwann cells produce myelin as an extension of their plasma membrane and ensheath nerve fibres in the peripheral nervous system (PNS). Several myelin proteins are common to both central nervous system (CNS) and PNS myelin but the major integral protein (Po) of PNS myelin is peculiar to this membrane (Greenfield et al . , 1973). Po comprises around 60% of the total protein of PNS myelin. SDS/polyacrylamide-gel analysis indicates an apparent M , of 28 00@30 000 and the protein is known to have a single N-linked oligosaccharide chain (Ishaque et al., 1980). Other post-translational modifications include acylation (Agrawal et al . , 1982). phosphorylation (Wiggins & Morell, 1980) and sulphation (Matthieu et al., 1975). The purpose of this work was to identify the subcellular site of synthesis of Po in Schwann cells and determine the mechanisms by which this protein is inserted into membranes before transport to the Schwann cell plasma membrane. mRNA isolated from sciatic nerves of 15-day-old rats was translated in a wheatgerm system and Po was immune precipitated and analysed on SDS-gels (Colman et ul., 1982). The primary translation product had a M , of 30700 compared with an M , of 28500 for the mature protein. This result indicated a cleavable signal sequence in the primary translation product of P,, mRNA. Removal of the single oligosaccharide from mature P,, with endoglycosidase F

Myelin-associated glycoprotein and myelin basic protein are present in central and peripheral nerve myelin throughout phylogeny

This phylogenetic study of central and peripheral nervous system myelin proteins demonstrates that important changes occur in the composition of certain myelin proteins during evolution. Only two components, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) are present in all Gnathostomata representatives investigated. While MBP components varied considerably even among the representatives of a given order, the apparent molecular weight of MAG showed little variation indicating that the conservation of the molecular structure could be important for the function of MAG in glia axon interactions.

Production and characterization of monoclonal antibodies to peripheral and central nervous system myelin.

Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low.

Myelin-associated glycoprotein and myelinating Schwann cell-axon interaction in chronic B,B'-iminodipropionitrile neuropathy.

The myelin-associated glycoprotein (MAG) is a heavily glycosylated integral membrane glycoprotein which is a minor component of isolated rat peripheral nervous system (PNS) myelin. Immunocytochemically MAG has been localized in the periaxonal region of PNS myelin sheaths. The periaxonal localization and biochemical features of MAG are consistent with the hypothesis that MAG plays a role in maintaining the periaxonal space of myelinated fibers. To test this hypothesis, MAG was localized immunocytochemically in 1-micron sections of the L5 ventral root from rats exposed to B,B'-iminodipropionitrile. In chronic states of B,B'-iminodipropionitrile intoxication, Schwann cell periaxonal membranes and the axolemma invaginate into giant axonal swellings and separate a central zone of normally oriented axoplasm from an outer zone of maloriented neurofilaments. Ultrastructurally, the width of the periaxonal space (12-14 nm) in the ingrowths is identical to that found in normally myelinated fibers. These Schwann cell ingrowths which are separated from compact myelin by several micra are stained intensely by MAG antiserum. Antiserum directed against Po protein, the major structural protein of compact PNS myelin, does not stain the ingrowths unless compact myelin is present. These results demonstrate the periaxonal localization of MAG and support a functional role for MAG in maintaining the periaxonal space of PNS myelinated fibers.

Peripheral nervous system myelin and Schwann cell glycoproteins: identification by lectin binding and partial purification of a peripheral nervous system myelin-specific 170,000 molecular weight glycoprotein.

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. II. Immunohistochemical studies in cultures of rat dorsal root ganglion neurons and Schwann cells

Antiserum against rat peripheral nervous system (PNS) myelin contained immunoglobulins which bound preferentially to the extracellular surfaces of myelin-related Schwann cells in intact cultures of dorsal root ganglion (DRG) neurons and Schwann cells, while antiserum against basic protein (BP) from central nervous system myelin or the PNS basic protein P 2 did not. We demonstrate the presence of PNS myelin proteins P 1 (identical to BP) and P 2 by immunoperoxidase techniques in DRG cultures that had been treated to disrupt cellular membranes. These observations suggest that P 1 and P 2 are not exposed on the extracellular surfaces of myelin-related Schwann cells in culture. The results also supported the hypothesis concerning the possible mechanisms by which anti-PNS myelin serum demyelinates DRG cultures, while anti-BP serum and anti-P 2 serum do not.