Peripheral nerve glycoproteins and myelin fine structure during development of rat sciatic nerve

Developmental changes in relative amounts of peripheral nerve proteins and glycoproteins have been correlated with the degree of morphological myelination at various ages during the first 25 postnatal days in rat sciatic nerve. At birth there is virtually no major myelin glycoprotein (P0), but there is a protein which migrates to the same point on sodium dodecyl sulphate (SDS) polyacrylamide gels as the small myelin basic protein (P2). During the time myelin is being formed in the nerve, the P0 protein increases and the P2 protein appears to decrease in relative amount in the nerve. The accumulation of P0 protein in the nerve correlates extremely well with the degree of myelination in sciatic nerve. At 4-6 days postnatal, smooth membrane profiles are observed which are located within axons and in the inner Schwann cell cytoplasm. Such profiles are also observed to fuse with the axolemma-Schwann cell interface. The profiles may represent membrane material being added to or deleted from the axolemma or myelin during myelination.     

CNP overexpression induces aberrant oligodendrocyte membranes and inhibits MBP accumulation and myelin compaction

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is highly enriched in myelin-forming cells where it is concentrated at the cytoplasmic side of all surface membranes except those of compact myelin. Previous studies have provided evidence that CNP is functionally involved in migration or expansion of membranes during myelination. This hypothesis is supported, in part, by the production of aberrant myelin membranes in transgenic mice that have a 6-fold increase in CNP expression. In addition, many myelin lamellae in these CNP-overexpressing mice lacked major dense lines (MDLs). The purpose of the present study was to determine if CNP overexpression altered: (1) oligodendrocyte and myelin membrane production during early stages of myelination, and (2) the ultrastructural distribution of CNP and myelin basic protein (MBP) in myelin membranes. We identified aberrant membrane expanses that extended from premyelinating oligodendrocyte processes, the periaxonal membrane, and the contact point between oligodendrocyte processes and myelin internodes. Myelin membranes without MDLs were deficient in MBP and enriched in CNP. These data support a functional role for CNP during oligodendrocyte membrane expansion and indicate, for the first time, that CNP may help target MBP to compact myelin.     

Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr98

Phosphorylation is one of a number of post-translational modifications resulting in charge microheterogeneity of myelin basic protein (MBP). This phosphorylation is claimed to destabilise the compact myelin sheath by decreasing the interaction of membrane bilayers, thereby creating or maintaining pockets of cytoplasm. To further investigate and localise MBP phosphorylation to discrete regions of the myelin sheath we raised a monoclonal antibody with specificity for a known phosphorylation site in MBP. A synthetic peptide was made by Fmoc peptide chemistry and phosphorylation of Thr98 was achieved on the resin by the global phosphorylation methodology, utilising dibenzyl-N,N-diethylphosphoramidite phosphitylation and t-butylhydroperoxide oxidation. The peptide coupled to tuberculin was used to immunise mice for monoclonal antibody production. The selected hybridoma (Clone P12) secreted an IgG2a antibody which reacted strongly with the phosphorylated immunogen and with phosphorylated fractions of bovine MBP obtained by ion exchange chromatography. The antibody had minimal reactivity with the unphosphorylated peptide; the same peptide phosphorylated at another site Ser102; a preparation of unphosphorylated MBP obtained by ion exchange chromatography; and with an irrelevant phosphorylated protein (histone). Similar phosphorylation state-specific monoclonal antibodies could be made to recognise other specific phosphorylation sites in MBP or other proteins. It is planned to use these antibodies to quantify and locate the extent of MBP phosphorylation in normal and multiple sclerosis myelin.     

Lipid-protein interactions with native and modified myelin basic protein

The basic protein of central nervous system myelin has been shown to form complexes with acidic lipids in vitro. We measured the interaction of myelin basic protein with several charged and neutral lipids in a biphasic chloroform/methanol/water system and investigated the effect of decreasing the electrical charge of the basic amino groups of the myelin basic protein by acetylation. The modified myelin basic protein, which has an average of eight acetyl residues incorporated, was characterised by gel electrophoresis and circular dichroism. Complexes formed between the acetylated myelin basic protein and acidic lipids exhibited a reduction in the amount of lipids bound, a value that could be correlated with the number of modified amino groups. The significance of these experiments with reference to protein-lipid interaction in the myelin membrane is discussed.     

Crystal structure of the extracellular domain from P0, the major structural protein of peripheral nerve myelin

P0, the major protein of peripheral nerve myelin, mediates membrane adhesion in the spiral wraps of the myelin sheath. We have determined the crystal structure of the extracellular domain from P0 (P0ex) at 1.9 A resolution. P0ex is folded like a typical immunoglobulin variable-like domain; five residues at the C-terminus are disordered, suggesting a flexible linkage to the membrane. The requirements for crystallization of P0ex are similar to those for maintaining the native extracellular spacing of adjacent myelin lamellae; thus, given the self-adhesive character of P0ex, the crystal itself may reveal some of the natural interactions that occur between P0 molecules in myelin. The structure leads to the suggestion that P0 extracellular domains may emanate from the membrane surface as tetramers that link to tetramers on the opposing membrane surface, to result in the formation of networks of molecules. We report analytical ultracentrifugation data for P0ex that support this idea.     

Mouse models of myelin diseases

Dys- and demyelination are the common endpoints of several inherited diseases of glial cells, which elaborate myelin and which maintain the myelin sheath very much like an "external" cellular organelle. Whereas some of the genes that are affected by mutations appear to be glial-specific, other genes are expressed in many cell types but their defect is restricted to oligodendrocytes or Schwann cells. Many of the disease genes and their encoded proteins have been studied with the help of mouse models, and a number of different molecular pathomechanisms have emerged which have been summarized in Figure 8. Some of the new concepts in the field, which have been addressed in this review, have only emerged because similar pathomechanisms were discovered for different myelin proteins. Mouse models have clearly helped to address both, the molecular pathology of myelin diseases and the normal function of myelin genes, but as discussed in this review, these questions turned out to be very different. Despite the progress in understanding the role of the abundant myelin proteins, there also remain a number of open questions that concern, among other things, the initial axon-glia recognition, the assembly process of the myelin sheath, and the long-term interaction of axons with their myelinating glia. Finally, animal models of human neurological diseases should not be restricted to the study of pathology, but they should also contribute to the development of experimental treatments. It is encouraging that a few attempts have been made.     

X-ray diffraction study of myelin structure in immature and mutant mice

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency (msd), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking greater immature greater trembler approximately normal adult; and in sciatic nerves: trembler greater immature greater quaking greater msd approximately normal adult. Repeat periods about 3 A less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190-200 A period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is approximately equal to 2 A thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is approximately equal to 5 A thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.     

Definition of conditions for the detection of genotoxic chemicals in the adult rat-liver epithelial cell/hypoxanthine-guanine phosphoribosyl transferase (ARL/HGRPT) mutagenesis assay

1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configuration. 2. A bilayer configuration of the (lyso)phospholipids was also observed after treatment of erythrocyte ghosts with various phospholipases even in the case that 98% of the phospholipid was converted into lysophospholipid (72%) and ceramides (26%). 3. A slightly decreased order of the phosphate group of phospholipid molecules, seen as reduced effective chemical shift anisotropy in the 31P-NMR spectra, was found following the formation of diacyglycerols and ceramides in the membrane of intact erythrocytes. Treatment of ghosts always resulted in an extensive decrease in the order of the phosphate groups. 4. The results allow the following conclusions to made: a. Hydrolysis of phospholipids in intact red cells and ghosts does not result in the formation of non-bilayer configuration of residual phospholipids and lysophospholipids. b. Haemolysis, which is obtained by subsequent treatment of intact cells with sphingomyelinase and phospholipase A2, or with phospholipase C, cannot be ascribed to the formation of non-bilayer configuration of phosphate-containing lipids. c. Preservation of bilayer structure, even after hydrolysis of all phospholipid, shows that other membrane constitutents, e.g. cholesterol and/or membrane proteins play an important role in stabilizing the structure of the erythrocyte membrane. d. A major prerequisite for the application of phospholipases in lipid localization studies, the preservation of a bilayer configuration during phospholipid hydrolysis, is met for the erythrocyte membrane.     

Myelin basic protein is a zinc-binding protein in brain: possible role in myelin compaction

The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.     

Overexpression of 2',3'-cyclic nucleotide 3'-phosphodiesterase in transgenic mice alters oligodendrocyte development and produces aberrant myelination

The function of the intracellular protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) of oligodendrocytes (ODC) is unknown. We have now generated several homozygous transgenic mouse lines in which the human CNP gene is overexpressed up to sixfold, revealing new insights into early stages of myelinogenesis. Although no behavioral phenotype is immediately apparent, abnormalities of ODC and their myelin sheaths are striking. These are manifested as redundant myelin membrane and intramyelinic vacuoles, as well as lack of myelin compaction concordant with failure of the cytoplasmic leaflets of compact myelin to fuse. Further, ODC that overexpress CNP appear to mature earlier in development, resulting in earlier maximum gene expression for myelin basic proteins and proteolipid protein. These results indicate that CNP is an early expressed regulator of cellular events that culminate in CNS myelination.     

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.     

Basis for phospholipid incorporation into peripheral nerve myelin

To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23-24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P0 (Golgi) and myelin basic proteins (more direct).     

The atomic force microscope detects ATP-sensitive protein clusters in the plasma membrane of transformed MDCK cells

Plasma membrane proteins are supposed to form clusters that allow 'functional cross-talk' between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K+ channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be 'glued' with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9 +/- 0.02 nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20 nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-gamma-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form 'functional clusters' in their native environment. The 'physiological' arrangement of the protein molecules within a cluster requires ATP.     

Axonal swellings and degeneration in mice lacking the major proteolipid of myelin

Glial cells produce myelin and contribute to axonal morphology in the nervous system. Two myelin membrane proteolipids, PLP and DM20, were shown to be essential for the integrity of myelinated axons. In the absence of PLP-DM20, mice assembled compact myelin sheaths but subsequently developed widespread axonal swellings and degeneration, associated predominantly with small-caliber nerve fibers. Similar swellings were absent in dysmyelinated shiverer mice, which lack myelin basic protein (MBP), but recurred in MBP*PLP double mutants. Thus, fiber degeneration, which was probably secondary to impaired axonal transport, could indicate that myelinated axons require local oligodendroglial support.     

Postnatal morphologic changes and glial fibrillary acidic protein immunoreactivity in the anteroventral cochlear nucleus of the acoustically-deprived gerbil

This study investigated the morphological changes and glial fibrillary acidic protein immunoreactivity (GFAP-IR) in the anteroventral cochlear nucleus (AVCN) of acoustically-deprived gerbils during postnatal development. The mongolian gerbil, Meriones unguiculatus, had been acoustically deprived on the right side or left side by a surgical ligation of the external auditory canal at postnatal day 12-14. No discernible microcysts were located in the ipsilateral AVCN at one, three, six and nine months after monaural ligation. Also, no discernible microcysts were located in the contralateral AVCN at one and three months after monaural ligation. Numerous microcysts were located in the contralateral AVCN at six months after monaural ligation and were slightly reduced in number at nine months after monaural ligation. Some of the microcysts closely apposed to and connected with the blood vessels through a leakage route or channel. A foamy region was found in the superficial granule cell cap of the AVCN. The foamy region became evident in the ipsilateral AVCN at three months after monaural ligation. However, the foamy region became evident in the contralateral AVCN at three and nine months after monaural ligation. Vacuoles were mainly found in the neuronal cells at the junction of the superficial and deep layers in the AVCN. These vacuoles were found in the contralateral AVCN at one, three, six, and nine months after monaural ligation. However, vacuoles were found in the ipsilateral AVCN only at three months after monaural ligation. Morphological changes of the myelin sheath were found to be more severe in the contralateral AVCN than in the ipsilateral. GFAP-IR was located in the superficial layer of the contralateral AVCN at three and nine months after monaural ligation. However, GFAP-IR was found in the superficial and deep layers of the ipsilateral AVCN at three and nine months after monaural ligation. GFAP-IR was also found in the superficial layers of the ipsilateral AVCN at six months after monoaural ligation. Microcysts are presumably derived from the detachment of the myelin sheath from the retracted axons, protrusion of the myelin sheath, and disruption of the myelin sheath. The major conclusions were that (1) microcysts were greatly reduced following acoustical ligation during postnatal development, and (2) blood vessels and GFAP-immunoreactive astrocytes may be involved in the depletion of microcysts for maintaining the homeostasis of the microenvironment in the cochlear nuclei.     

The pathogenesis of multiple sclerosis. Cytotoxic antibodies against myelin sheath tissue in multiple sclerosis

Cytotoxic antibodies to myelin can be demonstrated by the method of 51Cr-release from chick erythrocytes coated with myelin basic protein. The cytotoxic antibody is inactivated by heating to 56 degrees C and needs complement in order to exert its action. The antibody was determined as IgM and IgG. It has relative specifity and shows cross-reaction with other basic proteins. The cytotoxic antibody was found in only 8% of healthy persons. Patients with multiple sclerosis were positive in 87% of the cases and in acute cases in 94%. In other neurological diseases cytotoxic antibody was present in 64%. The occurrence of cytotoxic antibody to myelin protein is not specific for a particular neurological disorder, especially not for multiple sclerosis. Cytotoxic antibodies arise as a secondary phenomenon, they are not the cause of the disease involved. They appear to be suitable, however, to determine, in association with cellular immunological reactions against myelin which may be regarded as the "primary" immunological processes, the demyelination process in the multiple sclerosis focus.