Serum ganglioside patterns in multiple sclerosis

The relative distribution of gangliosides was determined in the serum of 37 patients with multiple sclerosis (MS) and of 30 healthy subjects. There was a significant increase of GM1 and GD1a, and a decrease of GM3 proportion in the serum of relapsing-remitting MS patients (RRMS) during their first MS attack. The RRMS patients in relapse with a long duration of the disease had a significant decrease of GM1 and an increase of GD1a portion in the serum. An increase of GD1a, one of the major brain neuron ganglioside fraction, suggested the neuron injury in the early and with a long duration RRMS. The finding of an increase of GM1, the main human myelin ganglioside, during the first MS attack in RRMS patients confirms previous evidence for the possible involvement of gangliosides in the early pathological course of demyelination in MS.     

Differences in Susceptibility of MBP Charge Isomers to Digestion by Stromelysin-1 (MMP-3) and Release of an Immunodominant Epitope

Charge microheterogeneity of myelin basic protein is known to affect its conformation and function. Here, the citrullinated myelin basic protein charge isomer, component-8, was shown to be more susceptible to stromelysin-1 cleavage than myelin basic protein component-1. Since levels of component-8 are increased in multiple sclerosis brain, the increased susceptibility of component-8 to proteolytic digestion may play a role in the pathogenesis of multiple sclerosis. Interestingly, component-1 isolated from multiple sclerosis patients was digested at a faster rate by stromelysin-1 than component-1 isolated from normal individuals. The reason for this difference is not clear, but likely reflects conformational differences between the two proteins as a result of post-translational modifications. Stromelysin-1 was able to cleave myelin basic protein in the presence of lipids and within the context of myelin and released several peptides including peptides containing the immunodominant epitope.     

Myelin changes induced by incubation of brain slices with serum

Forebrain and brain stem slices prepared from adult rats were incubated with pooled normal human serum. Following the incubation, the tissue was homogenized and the fraction floating on 0.32 M sucrose as well as two myelin subfractions (light and heavy) were isolated. Addition of serum into the incubation medium increased generation of the floating fraction by the cerebral slices. Changes in the myelin membrane were also observed. Thus, myelin isolated from forebrain slices revealed pronounced increase in the buoyant density of its particles and loss of basic protein. Furthermore, in spite of the intensive washing employed during the isolation procedure, some serum proteins were found firmly attached to the membraneous fractions. The demonstration of the myelin alterations in the living cerebral tissue exposed to serum during incubation may contribute to understanding the pathogenesis of multiple sclerosis.     

Effect of electroconvulsive therapy on serum myelin basic protein immunoreactivity.

A sensitive radioimmunoassay that can detect brain damage in cases of head injury and stroke was applied to blood samples from 13 patients before and after they received multiple treatments with electroconvulsive therapy for psychiatric disorder. None of the patients showed a significant increase in serum myelin basic protein immunoreactivity. As increased serum myelin basic protein immunoreactivity may reflect myelin damage it is apparent that in these patients electroconvulsive therapy did not cause measureable breakdown of myelin.     

Myelin basic protein-stimulated rosette-forming T cells in multiple sclerosis

A subpopulation of T lymphocytes sensitized to human myelin basic protein in peripheral blood of patients with multiple sclerosis, central nervous system (CNS) tumors, and cerebrovascular accidents was demonstrated by the antigen-stimulated, rosette-forming T cell assay. A significant increase in the percent of active rosette-forming T cells was detected after in vitro exposure of peripheral blood lymphocytes to human myelin basic protein but not to histones. In contrast, peripheral blood lymphocytes from healthy controls and from patients with benign and malignant breast diseases were unresponsive to stimulation by either antigen. These results demonstrate a functionally active T-lymphocyte subpopulation sensitized to myelin basic protein in patients with multiple sclerosis and in patients with certain other CNS diseases.     

White Matter Proteins in Multiple Sclerosis

Abstract: The SDS-soluble membrane proteins of plaques and of macroscopically normal white matter from multiple sclerosis brain were investigated by gradient polyacrylamide gel electrophoresis (PAGE). Eleven protein bands were analyzed in detail. The extensive loss of myelin proteins in plaque samples was accompanied by changes in at least three other non-myelin proteins, besides glial fibrillary acidic protein (GFAP), which probably reflect gliosis. Densitometric analysis of the PAGE patterns of membrane fractions from MS and control white matter revealed significant quantitative differences in a number of protein bands. A reduction in myelin basic protein (BP) was associated with an equally significant increase in a high-molecular-weight peptide fragment which may prove to be a breakdown product of BP. Small but highly significant differences in the Wolfgram protein and in one non-myelin protein were also a consistent feature of the normal-appearing white matter samples. The problem of defining normal white matter in multiple sclerosis brain is discussed in relation to the results of the present study, which suggest that one of the early events in the pathogenesis of the disease prior to frank demyelination is an alteration in the protein components of the myelin sheath and possibly of glial cells.     

Regulation of protein kinase C activity by gangliosides

The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.     

Subacute sclerosing panencephalitis and multiple sclerosis: in vitro measles immunity and sensitization to myelin basic protein.

Three children with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS) and 12 healthy persons were studied by the macrophage migration inhibition factor (MIF) assay with measles and rubella antigens and with myelin basic protein. For the SSPE patients the mean migration indexes +/- standard deviation were 44.1 +/- 10.9 for measles antigen, 38.7 +/- 12.3 for rubella antigen and 49.8 +/- 25.7 for myelin basic protein; for the MS patients the indexes were 103.0 +/- 10.6, 93.8 +/- 15.0 and 89.3 +/- 19.9; and for the healthy subjects the indexes were 68.8 +/- 22.6, 77.7 +/- 31.3 and 100.1 +/- 6.5. The results of this study showed increased cellular immunity to measles and rubella in SSPE patients as compared with healthy persons, and absence of immunity to measles in MS patients. Patients with MS showed hypersensitivity to myelin basic protein during clinical exacerbations that was not associated with changes in immunity to measles, whereas all SSPE patients showed a significant response regardless to stage of illness.     

Myelin-associated glycoprotein (Siglec-4) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides

Myelin-associated glycoprotein (MAG, Siglec-4) is a quantitatively minor membrane component expressed preferentially on the innermost myelin wrap, adjacent to the axon. It stabilizes myelin-axon interactions by binding to complementary ligands on the axolemma. MAG, a member of the Siglec family of sialic acid-binding lectins, binds specifically to gangliosides GD1a and GT1b, which are the major sialoglycoconjugates on mammalian axons. Mice with a disrupted Galgt1 gene lack UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and fail to express complex brain gangliosides, including GD1a and GT1b, instead expressing a comparable amount of the simpler gangliosides GM3, GD3, and O-acetyl-GD3. Galgt1-null mice produce similar amounts of total myelin compared to wild-type mice, but as the mice age, they exhibit axon degeneration and dysmyelination with accompanying motor behavioral deficits. Here we report that Galgt1-null mice display progressive and selective loss of MAG from the brain. At 1.5 months of age, MAG expression was similar in Galgt1-null and wild-type mice. However, by 6 months of age MAG was decreased approximately 60% and at 12 months of age approximately 70% in Galgt1-null mice compared to wild-type littermates. Expression of the major myelin proteins (myelin basic protein and proteolipid protein) was not reduced in Galgt1-null mice compared to wild type. MAG mRNA expression was the same in 12-month-old Galgt1-null compared to wild-type mice, an age at which MAG protein expression was markedly reduced. We conclude that the maintenance of MAG protein levels depends on the presence of complex gangliosides, perhaps due to enhanced stability when MAG on myelin binds to its complementary ligands, GD1a and GT1b, on the apposing axon surface.     

A membrane receptor for gangliosides is associated with central nervous system myelin

A binding protein specific for major neuronal gangliosides was detected on rat brain membranes using a synthetic ganglioside-protein conjugate, 125I-(GT1b)4BSA (bovine serum albumin derivatized with 4 mol of ganglioside GT1b/mol of protein), as a radioligand. Specific binding of the ligand displayed marked regional variation within the brain, with white matter-enriched regions demonstrating the highest binding activity. Autoradiographic localization of 125I-(GT1b)4BSA binding to tissue sections revealed selective association with myelinated pathways throughout the brain. The ligand also bound preferentially to brain subcellular fractions enriched in myelin, even after removal of axolemma. In contrast, peripheral nerve myelin had little binding activity. The myelin-associated ganglioside receptor detected by 125I-(GT1b)4BSA binding appears to be a novel oligodendroglial membrane protein which preferentially recognizes neuronal gangliosides.     

Ganglioside-modulated protein phosphorylation in myelin

Gangliosides have profound effects on the phosphorylation of several proteins in myelin. Addition of polysialogangliosides to purified guinea pig brain myelin enhanced the endogenous phosphorylation of a 62-kDa phosphoprotein, but completely inhibited the phosphorylation of myelin basic protein (MBP) (18.5 kDa). The ganglioside-stimulated phosphorylation of the 62-kDa protein was dose-dependent and -specific. Asialo-GM1, ceramide trihexosides, N-acetylneuraminic acid, or colominic acid alone could not mimic this effect, suggesting that the activation process requires both the hydrophobic head group and the anionic character of the gangliosides. Studies on the time course of this reaction revealed that it was a rapid and reversible process and was affected only very slightly by Ca2+. Thus, the stimulatory effect of gangliosides may not involve Ca2+-gangliosides complexes or proteolysis, but may be mediated through an activation of a ganglioside-dependent protein kinase or due to substrate protein-glycolipid interaction. Modulation of the phosphorylation of MBP by gangliosides varies with the states of phosphorylation of this protein. Prior addition of ganglioside to myelin inhibited the phosphorylation of MBP. However, addition of gangliosides to myelin subsequent to maximal phosphorylation of MBP retarded the dephosphorylation of this protein. Phosphorylation of isolated MBP by protein kinase C was stimulated by gangliosides, provided phosphatidylserine was present. In contrast, the glycolipid inhibited the phosphorylation of a unique site catalyzed by cAMP-dependent protein kinase. This site was distinct from those phosphorylated by protein kinase C and was also sensitive to chymotryptic cleavage. Although the exact physiological significance of protein phosphorylation in myelin has yet to be established, gangliosides may play an important role in the modulation of this reversible post-translational modification mechanism.     

Endogenous Interferon-β-Inducible Gene Expression and Interferon-β-Treatment Are Associated with Reduced T Cell Responses to Myelin Basic Protein in Multiple Sclerosis

Autoreactive CD4⁺ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels of interferon-β-inducible genes with an increased expression of interleukin-10 and a milder disease course in untreated multiple sclerosis patients, whereas other studies reported an association with a poor response to treatment with interferon-β. In the present study, we found that untreated multiple sclerosis patients with an increased expression of interferon-β-inducible genes in peripheral blood mononuclear cells and interferon-β-treated multiple sclerosis patients had decreased CD4⁺ T-cell reactivity to the autoantigen myelin basic protein ex vivo. Interferon-β-treated multiple sclerosis patients had increased IL10 and IL27 gene expression levels in monocytes in vivo. In vitro, neutralization of interleukin-10 and monocyte depletion increased CD4⁺ T-cell reactivity to myelin basic protein while interleukin-10, in the presence or absence of monocytes, inhibited CD4⁺ T-cell reactivity to myelin basic protein. Our findings suggest that spontaneous expression of interferon-β-inducible genes in peripheral blood mononuclear cells from untreated multiple sclerosis patients and treatment with interferon-β are associated with reduced myelin basic protein-induced T-cell responses. Reduced myelin basic protein-induced CD4⁺ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.     

Direct leukocyte migration inhibition by myelin basic protein in exacerbations of multiple sclerosis.

Studies in 13 normal subjects, 9 patients with multiple sclerosis (MS) within 3 weeks of exacerbation and 16 others 1 to 6 months after onset were carried out for evidence of cell-mediated hypersensitivity to myelin basic protein. Ten patients with stroke and 10 with Guillain-Barré syndrome were studied as additional controls. Peripheral leukocytes obtained by leukapheresis were packed into capillary tubes and allowed to migrate out onto glass in the presence or absence of myelin basic protein. Cells of patients within 3 weeks of an MS episode gave a mean migration index of 68 +/- 9%, and those 1 to 6 months after onset, 93 +/- 21%. For the entire MS group the mean index was 88 +/- 20%, for those with Guillain-Barré, 103 +/- 7%; and for the stroke patients, 107 +/- 11%. Results for the acutely ill MS patients were significant (P less than 0.005). The data are similar to those obtained using the migration inhibition factor assay but show that sensitized lymphocytes also elaborate a second mediator during acute exacerbations of illness. These observations strengthen evidence that sensitization to this potent encephalitogen occurs simultaneously with exacerbations of clinical illness.     

The Presence of Aldehyde-Reacted Proteins in Normal and Multiple Sclerosis White Matter

The incorporation of tritium from NaB3H4 into the major protein components of myelin and the presence of weak fluorescence emission bands at wavelengths of approximately 440 and 500 nm from sodium dodecyl sulfate-solubilized, delipidated white matter are indicative of the presence of the products of aldehyde reactions with proteins. The incorporation of tritium from NaB3H4 into myelin proteins was confirmed by reaction with purified components of myelin basic protein or with lipophilin, a purified fraction of proteolipid protein. From the extent of tritium incorporation into the purified proteins, it is estimated that approximately 0.2 mol of tritium is incorporated/mol of myelin basic protein and approximately 0.4 mol of tritium/mol of proteolipid protein. There is approximately 50% greater incorporation of tritium into a more degraded, less positively charged form of the basic protein. The incorporation of tritium into normal and multiple sclerosis white matter was compared. There is a small but statistically significant difference in the percentage of the total counts incorporated into the major protein fractions for the two groups, with the multiple sclerosis samples showing a higher percentage of the counts in the Wolfgram protein and a lower percentage in the myelin basic protein compared with the normal samples.     

Antibody to myelin constituents: A possible factor in induction of cell-mediated demyelination

Results from this laboratory have demonstrated that¹⁴C-labeled myelin opsonized with antibodies raised to purified CNS myelin in rabbit is phagocytized by cultured macrophages in larger amounts than untreated myelin or myelin opsonized with preimmune serum. The cultured macrophages produced high amounts of radioactive cholesterol ester and triglyceride from the antibody-treated myelin while much less was formed from preimmune serum-treated or untreated myelin. Antiserum to galactocerebroside also greatly enhanced the formation of radioactive cholesterol ester, while that to myelin basic protein as well as to other myelin constituents had little or no effect. Serum from Lewis rats with acute EAE 13–14 days after immunization with whole CNS myelin also stimulated radioactive cholesterol ester formation compared to serum from Freund's adjuvant-injected controls (FAC). Serum from EAE rats as a result of myelin basic protein injection was as active as that from rats with whole myelin injection. No galactocerebroside antibody could be demonstrated in the EAE sera, although a strong immunostaining to myelin basic protein and proteolipid protein was demonstrated. IgG prepared from EAE serum also showed stimulatory effects compared to IgG from FAC serum, but much of the activity was lost, and the possibility that other factors may be involved is discussed. These experiments provide evidence that myelin phagocytosis and digestion by macrophages is enhanced by the presence of antibody to myelin. In EAE this antibody may leak into CNS with the breakdown of the blood-brain barrier. A humoral involvement in demyelination in EAE is implicated, and these findings may be extended eventually to the demyelinative mechanism in multiple sclerosis where IgG is found in large amounts in the CNS.Special Issue Dedicated to Dr. Abel Lajtha.     

Acrolein induces myelin damage in mammalian spinal cord

Myelin damage can lead to the loss of axonal conduction and paralysis in multiple sclerosis and spinal cord injury. Here, we show that acrolein, a lipid peroxidation product, can cause significant myelin damage in isolated guinea pig spinal cord segments. Acrolein-mediated myelin damage is particularly conspicuous in the paranodal region in both a calcium dependent (nodal lengthening) and a calcium-independent manner (paranodal myelin splitting). In addition, paranodal protein complexes can dissociate with acrolein incubation. Degraded myelin basic protein is also detected at the paranodal region. Acrolein-induced exposure and redistribution of paranodal potassium channels and the resulting axonal conduction failure can be partially reversed by 4-AP, a potassium channel blocker. From this data, it is clear that acrolein is capable of inflicting myelin damage as well as axonal degeneration, and may represent an important factor in the pathogenesis in multiple sclerosis and spinal cord injury.     

Multiple Sclerosis Brain Immunoglobulins Stimulate Myelin Basic Protein Degradation in Human Myelin: A New Cause of Demyelination

Membrane-bound proteolysis may be implicated in the pathogenesis of demyelinating disorders including multiple sclerosis (MS). We previously found that the extent of myelin basic protein (MBP) degradation by the calcium-activated neutral protease did not differ for isolated human control myelin or MS myelin. Hence we suggested that, if involved in demyelination, the myelin neutral protease must be activated in vivo by an increased availability of free calcium. The postulate was therefore tested that immunoglobulin (Ig) binding to myelin results in activation of the myelin neutral protease, possibly through release of free calcium from calcium-binding sites of myelin. Isolated myelin from the brains of controls and patients with MS were incubated with purified Igs eluted from the brains of patients with MS or controls and degradation of MBP was assessed by quantitative electroimmunoblotting. Such degradation was significantly greater in myelin incubated in the presence of MS Igs than in myelin incubated without added Igs or in the presence of control Igs. Furthermore, the degree of MBP degradation in myelin incubated with control Igs was similar to that observed in myelin incubated without added Igs. Accordingly, it is suggested that Ig in MS brain potentiates myelin breakdown. Moreover activation of membrane-bound proteolysis by Ig binding to myelin appears to represent a hitherto undescribed pathway for demyelination in MS.     

The Immunological Identification of Brain Proteins on Cellulose Nitrate in Human Demyelinating Disease

Abstract: An immunological technique has been employed to identify proteins, separated in polyacrylamide gels, which show changes in brain samples from cases of multiple sclerosis and subacute sclerosing panencephalitis. Sodium dodecylsulphate-treated proteins in particulate and soluble fractions were separated in polyacrylamide slab gels, transferred electrophoretically onto cellulose nitrate sheets, incubated with specific antisera and visualized by an immunoperoxidase method. Protein bands showing changes were identified using antisera raised against the myelin basic and Wolfgram proteins, the neurofilament triplet proteins, tubulin and glial fibrillary acidic protein. In addition to the loss of myelin proteins, decreases in the neurofilament proteins and in tubulin were seen in both multiple sclerosis and subacute sclerosing panencephalitis samples. The distribution of glial fibrillary acidic protein polypeptides in the particulate and soluble fractions of plaque samples appeared to vary according to the degree of fibrosis. Changes in the levels of the myelin-associated glycoprotein, the lower molecular weight component of the Wolfgram protein, albumin and immunoglobulin G, none of which were visualized by protein staining, were also seen. This immunological technique has allowed a closer examination of changes occurring in brain protein spectra in multiple sclerosis and subacute sclerosing panencephalitis.     

Polymorphism of length of tetranucleotide repeat from the 5'-side from the myelin basic protein gene in multiple sclerosis in Russians

The myelin basic protein gene (MBP) can confer the susceptibility to multiple sclerosis, because its protein product is the main protein component of myelin of the central nervous system and a potential autoimmune antigen in the disease. A possible association of multiple sclerosis with alleles and genotypes of a microsatellite repeat (TGGA)n, located to the 5' side from the first exon of MBP in ethnic Russians (126 patients with reliable multiple sclerosis and 142 healthy controls from Central Russia) was analyzed using the case-control method. Upon separation of the tetranucleotide repeat site amplification products in 1.5% agarose gel, one can see two distinct bands that can be analyzed as two allele groups (A and B). The distribution of allele A and B group frequencies as well as frequency of allele group B and genotype A/A reliably differs in multiple sclerosis patients and healthy controls. Alleles A and the A/A genotype are associated with the development of multiple sclerosis. We also analyzed the association of multiple sclerosis with combined bearing of alleles and genotypes A and B of MBP and groups of alleles of the DRB1 gene of the major histocompatibility complex that correspond to serospecificities DR1-DR18. The comparison of subgroups of multiple sclerosis patients and healthy individuals, formed on the basis of the DRB1 phenotype, has shown a reliable increase in the frequency of allele B in healthy individuals and the genotype A/A frequency in patients, only among DR4- and DR5-positive individuals. No reliable difference was found in the MBP allele and genotype distribution between multiple sclerosis patients and healthy individuals in combined groups of (DR4,DR5)-negative individuals, i.e., no carriers of any phenotype except DR4 and DR5 were revealed. Thus, MBP or some other nearby gene is involved in the multiple sclerosis development in Russians, predominantly (or exclusively) among DR4 and DR5 carriers. In this case, without stratification of analyzed individuals by the MBP alleles, multiple sclerosis is reliably associated only with DR2(15), but not of DR4 and DR5 alleles of DRB1. The results obtained are in favor of the genetic heterogeneity of multiple sclerosis, and suggest the possibility of epistatic interactions between the MBP and DRB1 genes.     

Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand

Myelin-specific T lymphocytes are considered essential in the pathogenesis of multiple sclerosis. The myelin basic protein peptide (a.a. 83-99) represents one candidate antigen; therefore, it was chosen to design an altered peptide ligand, CGP77116, for specific immunotherapy of multiple sclerosis. A magnetic resonance imaging-controlled phase II clinical trial with this altered peptide ligand documented that it was poorly tolerated at the dose tested, and the trial had therefore to be halted. Improvement or worsening of clinical or magnetic resonance imaging parameters could not be demonstrated in this small group of individuals because of the short treatment duration. Three patients developed exacerbations of multiple sclerosis, and in two this could be linked to altered peptide ligand treatment by immunological studies demonstrating the encephalitogenic potential of the myelin basic protein peptide (a.a. 83-99) in a subgroup of patients. These data raise important considerations for the use of specific immunotherapies in general.