The large isoform of myelin-associated glycoprotein is scarcely expressed in the quaking mouse brain.

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.

Presence of the plasma membrane proteolipid (plasmolipin) in myelin.

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.

An immunohistochemical study of the topography and cellular localization of three neural proteins in the sheep nervous system.

The peroxidase-anti-peroxidase (PAP) method was used to determine the topography and cellular localization of glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and carbonic anhydrase II (CAII) in the central nervous system (CNS), dorsal root ganglia and dorsal and ventral spinal nerve roots of the sheep. Parallel studies of mouse brain provided comparative data. Several fixatives were compared for their relative merits in preserving marker protein expression: GFAP was well preserved irrespective of the fixative employed; MBP was best preserved in formal sublimate and CAII was best preserved in Carnoy's fluid. In sheep, GFAP expression was seen in protoplasmic and fibrous astrocytes, Bergmann glial cells, a proportion of ependymal cells, amphicytes of spinal ganglia and in a proportion of presumed Schwann cells of dorsal and ventral spinal nerve roots. MBP expression was seen in mature and developing myelin sheaths of the central nervous system and in the cytoplasm of sparse myelinating oligodendroglia of the sub-cortical white matter of the cerebrum. CAII expression was seen in choroid plexus epithelium in all ages of sheep studied and, in a young lamb and an adult sheep, in glia and neuropil of ventral horn grey matter of the spinal cord and in the cytoplasm of white matter glia, presumed fibrous astrocytes, throughout the CNS. Compared with sheep brain, mouse brain showed the following differences in marker protein localization. GFAP was weakly expressed by protoplasmic astrocytes and not expressed in ependyma, oligodendroglia expressing intracytoplasmic MBP were frequent and widespread in neonatal mouse brain, CAII was expressed in myelin and oligodendroglia.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid profile of rat myelin subfractions.

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of "light", "heavy" and "membrane fraction" lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the "membrane fraction" resembles in its lipid and fatty acid composition other cell membranes (particularly oligodentrocytes); ii) "light" and "heavy" myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nonhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the "heavy" subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.

The dysmyelinating mouse mutations shiverer (shi) and myelin deficient (shimld).

Shiverer (shi/shi) is an autosomal recessive mouse mutation that produces a shivering phenotype in affected mice. A shivering gait can be seen from a few weeks after birth until their early death, which occurs between 50 and 100 days. The central nervous system of the mutant mouse is hypomyelinated but the peripheral nervous system appears normal. The myelin of the CNS, wherever present, is not well compacted and lacks the major dense line. Myelin basic protein (MBP), which is associated with the major dense line, is absent, and this is due to a deletion of the major part of the gene encoding MBP. Transgenic shiverer mice that have integrated and express the wild-type mouse MBP transgene no longer shiver and have normal life spans. Conversely, normal mice that have integrated an antisense MBP transgene, shiver. Myelin deficient shimld/shimld is allelic to shiverer (shi/shi) but the mutant mouse is less severely affected. Although MBP is present in the CNS, it is low in quantity and is not developmentally regulated. The gene encoding MBP has been both duplicated and inverted. Transgenic shimld/shimld mice with the wild-type MBP transgene have normal phenotypes.

Thin-layer chromatographic analysis of myelin lipids, their differential O-deacylation by primary alkylamines and their selective staining by thionine. A limited phylogenetic study.

A silica gel thin-layer chromatographic procedure is described for the study of the myelin lipid patterns in a small phylogenetic series of nerve tissue specimens. It involves the selective staining by the thiazine dye thionine and the interpretations were facilitated by a preceding primary alkylamine O-deacylation step. Glycolipids, including sulfatides, and ethanolamine plasmalogens were the principal characterizing lipids.

Phospholipid composition of myelin and synaptosomal proteolipid from vertebrate brain.

1. The phospholipid composition of the main proteolipid complexes of the nervous system was studied in myelin and synaptosomal membranes from brains of representatives of various vertebrate classes. 2. The relative content of acid phospholipids was much higher in proteolipid complexes from myelin and synaptosomal membranes of all vertebrates studied as compared to their content in the initial lipid extract (28-80% and 11-20% of total phospholipid content, respectively). 3. The relative content of acid phospholipids in proteolipid complexes of myelin membranes was much lower in brain of fishes and amphibia as compared to higher vertebrates. 4. The main acid phospholipids of proteolipid complexes was phosphatidylserine, phosphatidic acid being characteristic for myelin proteolipids and diphosphatidyl glycerol for synaptosomal proteolipids of all vertebrates studied.

Arachidonic and docosahexanoic acid content of bovine brain myelin: implications for the pathogenesis of multiple sclerosis.

Lipids were extracted from bovine brain myelin using a mixture of hexane and isopropanol (3:2). Myelin lipids were resolved, using Sep Pak chromatography, into four fractions: Fraction 1 contained neutral lipids, fraction 2, free fatty acids, fraction 3, ethanolamine phospholipids and fraction 4, choline phospholipids. Doscosahexanoic (DHA) and arachidonic (AA) acids in these fractions were measured by RPHPLC. Fraction 2 was analyzed directly, the other three fractions were subjected to alkaline hydrolysis before analysis for DHA and AA. DHA and AA were not found in fraction 1. Both DHA and AA were found in fractions 2 and 3. Only AA was consistently found in fraction 4. These results were confirmed by GC.

Desorption of ions from rat membranes: selectivity of different ionization techniques.

Complex lipid biomarkers, including phosphatidylcholines, cerebrosides and sulfatides, are shown to be desorbed intact from rat brain myelin and rat liver microsomes by liquid secondary ion mass spectrometry, by plasma desorption and by laser desorption. Different polar lipids are favored by the different desorption techniques and as negative or positive ions. These selectivities support current theories about ionization for the different techniques.

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat.

Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.

Decreased myelin lipids in Alzheimer's disease and vascular dementia.

The lipid composition of white matter and myelin from the semioval centre was studied in autopsy material from cases with Alzheimer's disease (AD) (n = 11), vascular dementia (VD) (n = 7), and age-matched controls (n = 11). In AD and VD the white matter content of phospholipids and cholesterol was reduced to 72-76% of the control values (P less than 0.01), the diminution of cerebrosides and sulphatides was more pronounced (55-69%) (P less than 0.001) while the concentration of gangliosides did not change significantly (87-90%). The myelin composition was the same in the 3 groups, suggesting that the white matter involvement is not caused by alteration of the myelin structure. The altered lipid composition in white matter in AD and VD suggests that the myelin sheath is the primary lesion site.

Isolation and characterization of complement receptors CR1 from human peripheral nerves.

Extracts from myelinated and unmyelinated nerves, prepared using Nonidet P-40, contained receptors for C3b/C4b (CR1). Extracts from myelinated nerves inhibited EAC3b rosette formation with peripheral blood leucocytes and agglutinated EAC3b, whereas extract from unmyelinated nerves did not. Rosette formation with EAC3bi or EAC3d was not affected. CR1 in extracts from myelinated nerves also expressed decay-accelerating activity of the alternative pathway C3 convertase and cofactor activity in factor I-mediated cleavage of C3b, whereas CR1 in extract from unmyelinated nerves did not. Monoclonal anti-CR1 antibodies, but not monoclonal anti-CR2 (C3d receptors) or anti-CR3 (C3bi receptors) antibodies inhibited the functional activities. Accordingly, CR1 are the only C3 receptor present in the extracts and only CR1 in myelinated nerve extracts are functionally active. CR1 in both myelinated and unmyelinated nerve extracts had a molecular weight of approximately 190 kDa. The electrophoretic mobility did not change after reduction and the 190 kDa band was stained by concanavalin A, indicating that the CR1 are single-chained glycoproteins. Binding to lentil lectin-Sepharose 4B further sustained the glycoprotein nature of the CR1. Periodic acid abolished functional activities of CR1, whereas trypsin and heat did not, indicating the functional significance of the carbohydrate moiety. That CR1 are functionally active in myelinated nerves, but not in unmyelinated nerves, may therefore be due to differences in the carbohydrate moiety. The cofactor and decay-accelerating activities of CR1 may be of significance in the pathogenesis of demyelinating polyneuropathies by limiting complement activation.

Axon-myelin relationships in rat cranial nerves III, IV, and VI: a morphometric study of large- and small-fibre classes.

The primary objectives of this study were to determine (1) if quantitative axon-myelin relationships are similar for large- and for small-fibre classes within individual nerves and (2) if the same axon-myelin relationships hold for equivalent fibre classes in closely similar nerves. The oculomotor, trochlear, and abducent nerves of the rat were examined since they each contain distinct large- and small-fibre classes and are similar in a wide range of anatomical and developmental respects. Accordingly, morphometric analyses of axon-myelin relationships were performed separately on large and small fibres of each of the three nerves. Within each nerve, the setting of the relationship between the two parameters was found to be different for the two fibre classes: Scatterplots relating sheath thickness to axon perimeter for large fibres were shifted upwards relative to those for small fibres. These differences were also reflected in the positions of the regression lines fitted to the plots and in the g-ratios. Significant differences were found between nerves in relation to their large fibres: Those of the abducent nerve had significantly thicker sheaths, those of the oculomotor nerve had significantly smaller axon perimeters, and the myelin sheath-axon perimeter relationship of the abducent nerve differed significantly from that of the other two. This study therefore shows that morphometric axon-myelin relationships may differ significantly between equivalent fibre classes of nerves that are closely similar in respect of morphological class, central origin, peripheral distribution, developmental environment, and function.

Recovery of proteolipid protein in mice heterozygous for the jimpy gene.

We have measured levels and synthesis of proteolipid protein (PLP) and its transport into myelin in female mice heterozygous for the jimpy gene and in their normal female littermates. In both cord and cerebrum, jimpy carriers show deficits in PLP during development followed by compensation in adulthood. Recovery of PLP occurs earlier in cord than in brain. At 13 days levels of PLP in carriers compared to controls are reduced to 0.60 and 0.44, respectively, in cord and cerebrum. By 100 days, normal levels of PLP are attained in cord (1.13) whereas levels of PLP in cerebrum are only 0.78 of control. By 200 days full recovery occurs in cerebrum, with a ratio of 1.21, suggesting a possible over-compensation. The yield of myelin from cerebrum was reduced to 0.78 in carriers compared to controls at 17 days. In brain slices, incorporation of [3H]leucine into homogenate PLP from carriers is the same as in controls, whereas [3H]leucine incorporation into myelin PLP is reduced to 0.68 of control. These results indicate that synthesis of PLP in the carriers is normal at 17 days, but transport of PLP into myelin is reduced. Similarly, acylation of homogenate PLP is normal, whereas acylation of myelin PLP is reduced, as measured by incorporation of [3H]palmitic acid. Transport of PLP into myelin was compared to transport of MBP; transport of both proteins was equally decreased as indicated by the similar ratio of labeled PLP to MBP in myelin from carriers compared to noncarriers.(ABSTRACT TRUNCATED AT 250 WORDS)

Marchi-positive myelinoid bodies at the transition between the central and the peripheral nervous system in some vertebrates.

The CNS-PNS (central nervous system-peripheral nervous system) transitional region of cranial and spinal nerve roots in some vertebrate species was analysed with respect to the occurrence and the distribution of myelinoid Marchi-positive bodies. Both cranial and spinal nerve roots contained more Marchi-positive bodies in their CNS than in their PNS segments. An accumulation of Marchi-positive bodies was usually noted just central to the CNS-PNS borderline. Comparisons between calibre spectra and Marchi index in the cat revealed a particularly high number of Marchi-positive bodies in nerve roots with a high content of myelinated fibres with diameters greater than or equal to 5 microns. Marchi-positive bodies were absent in CNS tissue lacking myelinated nerve fibres. CNS borderline internodes measuring between 200 and 300 microns in length were noted in fibres as thick as 15 microns in feline S1 ventral and dorsal roots. The general picture was similar in all analysed species. Noteworthy however, was the small difference in number of Marchi-positive bodies between CNS and PNS tissue in Xenopus. The chicken contained many myelinoid bodies of similar size and texture as the Marchi-positive bodies but without the Marchi-positive staining properties. The results show that normally occurring Marchi-positive bodies in the CNS are more numerous along paranodal segments than along mid-internodal segments of myelinated nerve fibres and thus support the hypothesis that Marchi-positive bodies are preferentially derived from paranodal myelin.

Buoyant density and lipid composition of purified myelin of aging human brain.

Purified myelin of human brain from 15 young adult (below 50 years of age) and old (above 70 years of age) autopsy cases each was examined by isopycnic centrifugation in continuous sucrose gradients, and for lipid composition. The mean buoyant density of myelin was the same in both groups. Apparent features of old age were a wide range of density values, less compact myelin bands, and the dissociation of myelin into two bands in six of 15 old cases. Lipid analyses of randomly selected myelin samples of each group revealed an inverse relationship between the total lipid to protein ratio and density of myelin. In old age total lipids decreased by an average 10 mol lipid per mol protein. This decrease was accounted for by cholesterol, phosphatidylserine and cerebrosides. Changes in fatty acid moieties mainly affected sphingolipids. C20:0 and C24:0 of sphingomyelin increased, as did even more markedly the more hydrophilic OH-fatty acids of cerebrosides. Correlations with buoyant density existed for the ratios of cholesterol to protein in young adult cases, and those of galactolipids to protein in old cases. The results suggest that old age is associated with impaired stability and altered lipid composition of myelin.

Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry.

We have developed a simple method involving high-performance thin layer chromatographic separation of total brain and myelin lipids. Only two solvent systems consisting of chloroform: methanol:acetic acid and water at different concentrations were needed. The plate was then stained with three sequential procedures to visualize phospholipids, cholesterol and galactolipids. Densitometric procedure at each step of staining was utilized to obtain quantitative analysis of brain and myelin samples.

Topology of proteolipid protein in the myelin membrane of central nervous system. A study using antipeptide antibodies.

Peptides according to amino-acid sequences of the N- and C-terminus of lipophilin (proteolipid protein, PLP) (Gly1-Phe15 = 1; Thr261-Phe276 = 6) and of the other four hydrophilic domains (Glu37-Leu60 = 2; Arg97-Leu112 = 3; Gly119-Gly127 = 3A; Trp144-Tyr156 = 3B; Lys191-Ala203 = 4; Asn222-Phe232 = 5) have been synthesized by the solid-phase Fmoc method, linked covalently to keyhole limpet hemocyanin (KLH) and used as antigens. Monospecific antibodies against these antigens were isolated by affinity chromatography. Each antibody recognized its epitope in isolated partially delipidated PLP with the ELISA technique, western blot, thin sections of paraffin embedded rat brains and in the plasma membrane of appropriately fixed/permeabilized rat oligodendrocytes in culture. After fixation with formaldehyde antipeptide 3A antibody stained intact non-permeabilized cells. Therefore the epitope 3A must be located on the extracellular surface of the membrane. This is in full support of our previous biochemical results on the orientation of lipophilin in the myelin membrane.

Orientation of proteolipid protein in myelin: comparison of models with X-ray diffraction measurements.

Three models have been proposed for the arrangement of proteolipid protein (PLP) in the myelin membrane. We have tested these models by determining to what extent each is consistent with the membrane-membrane interactions and electron density profile of central nervous system myelin obtained from X-ray diffraction. Equilibrium periods and membrane separations were calculated from the proposed organization of lipids and proteins in the membrane, and compared with values obtained experimentally as a function of pH and ionic strength. The orientation of the proteins was also used to calculate electron density levels in the cytoplasmic and extracellular spaces. We found that the Stoffel and Hudson models for PLP were more consistent than the Laursen model with the range of pH over which the intermembrane separation at the extracellular apposition is a minimum. The Hudson model also fits better the swollen periods observed at alkaline pH. The Hudson PLP model has many more residues in the extracellular side of the membrane than does either of the other models, resulting in higher electron density in the extracellular space compared to the cytoplasmic space. Such an asymmetric distribution of electron density is offset by the electron density of myelin basic protein which is localized in the cytoplasmic space. The resulting similar levels of electron density at the two appositions are like those in profiles calculated from the X-ray data.