Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

An Antibody Specific for Component 8 of Myelin Basic Protein from Normal Brain Reacts Strongly with Component 8 from Multiple Sclerosis Brain

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cleavage of Myelin Basic Protein by Neutral Protease Activity of Human White Matter and Myelin

Polypeptides arising from neutral in vitro proteolysis of myelin basic protein (MBP) of human brain were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At pH 7 a marked breakdown of MBP resulted in the formation of 8-12 polypeptides ranging from 6 to 17 kd in molecular weight. As neutral proteolytic activity was not eliminated by either gel filtration or cation-exchange chromatography acid-soluble protease(s) involved probably have a size and electric charge similar to that of MBP. The enzymatic nature of neutral proteolysis was ascertained by heat inactivation and inhibition by alpha 2-macroglobulin. Incomplete inhibition of proteolysis and the failure of small peptides (less than 6 kd) to show up on electrophoresis seem to suggest that MBP was degraded by exopeptic proteases as well. Acid extracts of purified myelin yielded polypeptides similar to those of MBP of delipidated white matter. The results are consistent with a sequential limited proteolysis of MBP by neutral proteases probably associated with myelin and possibly related to the in situ catabolism of MBP in man.     

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.     

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.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Size and Surface Charge Properties of Myelin Vesicles from Normal and Diseased (Multiple Sclerosis) Brain

Differences have been observed between myelin vesicles prepared from normal human central nervous system and from white matter of patients who died with multiple sclerosis (MS). The mean cross-sectional area of the vesicles was 5.69 +/- 0.17 micron 2 from normal myelin and 3.71 +/- 0.28 micron 2 for diseased myelin. Vesicle size was reduced to 4.08 +/- 0.21 micron 2 when normal myelin vesicles were prepared in the presence of 0.1 mM EDTA. The presence of Ca2+ during the preparation of the vesicles had no effect on the mean cross-sectional area. In the case of MS myelin vesicles, 0.1 mM EDTA had no effect on vesicle size, whereas the presence of Ca2+ increased the vesicle size from 3.71 +/- 0.28 to 5.40 +/- 0.31 micron 2. Electrokinetic analysis revealed that the electrophoretic mobility of normal myelin vesicles was -5.169 +/- 0.193 X 10(-8) compared with -6.093 +/- 0.202 X 10(-8) m2 s-1 V-1 for the MS myelin vesicles. The presence of 0.1 mM EDTA increased the electrophoretic mobility of the normal vesicles to -6.483 +/- 0.151 X 10(-8) m2 s-1 V-1 but did not significantly affect that of the MS vesicles. Addition of 0.1 mM Ca2+ decreased the electrophoretic mobility of both normal and MS vesicles to similar mobilities. From these data, the surface charge densities were calculated for both normal and MS myelin vesicles and found to be -2.93 and -5.39 mV m-1, respectively. The phase transition temperature determined by wide-angle x-ray diffraction studies was 63 degrees C for normal myelin vesicles and 43 degrees C for MS myelin vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Role of Citrullinated Proteins Suggests a Novel Mechanism in the Pathogenesis of Multiple Sclerosis

The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer’s, Parkinson’s, and Huntington’s. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS. Special issue dedicated to Anthony Campagnoni.     

2'', 3''-Cyclic Nucleotide 3''-Phosphohydrolase and Lipids of Myelin from Multiple Sclerosis and Normal Brains

Abstract: A study of purified myelin samples from normal-appearing white matter of 10 multiple sclerosis (MS) brains was undertaken and the results were compared with 10 age-matched control brains. Statistical evaluations were carried out with Student's r-test for differences. In pathological samples the yield of myelin came to only two-thirds of the corresponding controls. Enzyme assays of the 2', 3'-cyclic 3'-phosphohydrolase revealed an obviously significant reduction of specific activity to one-half in MS myelins. In myelin the contents of protein, lipid classes as cholesterol, glycolipids and phospholipids did not differ significantly. No cholesterol esters or any lysophospholipid were detectable either in MS or in controls. Within the individual phospholipids the main components were in the same order, while a significant decrease of the acidic representatives and of sphingomyelin occurred. Analysis of the fatty acid pattern of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the aldehydes from the last, revealed quite similar values with no significant differences, except C22: 4 fatty acid in the PE fraction and C20: 1 fatty acid in PS, which were reduced in MS myelin samples.     

Cell-Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice

Total polyribosomes were isolated from the brains of 16-20 day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shil/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 +/- 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and less than 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an "assembly" or "synthesis" defect.     

Evidence of Nitrosative Damage in the Brain White Matter of Patients with Multiple Sclerosis

Nitric oxide (NO) has been implicated in the pathophysiology of both experimental autoimmune encephalomyelitis and multiple sclerosis (MS). NO-mediated protein damage in MS appears to be confined to large plaques where 3-nitrotyrosine has been detected. To determine whether nitrosative damage takes place beyond visible MS plaques, the occurrence of various NO-triggered protein modifications in normal-appearing white matter (NAWM) of eight MS brains was assessed and compared to that in white matter (WM) of four control brains. As determined by amino acid analysis and western blotting, no evidence of tyrosine nitration was found in the MS samples studied, suggesting that they did not contain appreciable amounts of plaque-derived material. The amino acid composition of total myelin proteins and proteolipid protein (PLP) was also unaltered in the diseased tissue, as was the fatty acid composition of PLP. In addition, we detected no changes in the number of protein free thiols suggesting that oxidation do not occur to any appreciable extent. However, the levels of nitrite in MS-NAWM were higher than those in control WM, while in the MS-gray matter (GM) the concentration of this ion was unaltered. Furthermore, five of the MS samples analyzed, and the same as those with high levels of glial fibrilary acidic protein, showed increased amounts of protein nitrosothiols as determined by the biotin switch method. S-nitrosation of GM proteins was again normal. There was no indication of N-nitrosation of tryptophan and N-terminal amino groups in both control and MS tissue. Overall, the data suggests that WM, but not GM, from MS brains is subjected to considerable nitrosative stress. This is the first report to present direct evidence of increased protein S-nitrosation and nitrite content in the brain parenchyma of MS patients.     

Increased Perfusion in Normal Appearing White Matter in High Inflammatory Multiple Sclerosis Patients

PurposeAlthough cerebral perfusion alterations have long been acknowledged in multiple sclerosis (MS), the relationship between measurable perfusion changes and the status of highly active MS has not been examined. We hypothesized that alteration of perfusion can be detected in normal appearing white matter and is increased in high inflammatory patients.ResultsThirteen patients were classified as high-inflammatory. Compared to low-inflammatory patients, the high-inflammatory group demonstrated significantly higher CBV (p = 0.001) and CBF (p = 0.014) values. A mixed model analysis to assess independent variables associated with CBV and CBF revealed that white matter lesion load and atrophy measurements had no significant influence on CBF and CBV.ConclusionThis work provides evidence that high inflammatory lesion load is associated with increased CBV and CBF, underlining the role of global modified microcirculation prior to leakage of the blood-brain barrier in the pathophysiology of MS. Perfusion changes might therefore be sensitive to active inflammation apart from lesion development without local blood–brain barrier breakdown, and could be utilized to further assess the metabolic aspect of current inflammation.     

多发性硬化与病毒感染研究进展

多发性硬化发病机制复杂,病毒感染在其发病中可能起着举足轻重的作用,目前多项研究支持他们之间的关联性;分子模拟、免疫调节网络的失调、表位扩展、旁路激活、超抗原激活和直接细胞损伤等是病毒感染导致多发性硬化发病的主要机理。本文就相关研究进行综述。     

Analysis of the Primary Sequence of Human Myelin Basic Protein Peptides 1–44 and 90–170 by Fast Atom Bombardment Mass Spectrometry

The originally described sequence of human myelin basic protein peptide 45-89 has recently been shown to contain two errors which have now been resolved. In the present study fast atom bombardment mass spectrometry was utilized to analyze the primary sequence of the other portions, peptides 1-44 and 90-170 of human myelin basic protein. The results obtained confirm the accuracy of the primary sequence published for both of these terminal peptides.     

Lipid and Protein Alterations of Spinal Cord and Cord Myelin of Multiple Sclerosis

Abstract: A comprehensive study was carried out to clarify the chemical compositions of spinal cord, cord myelin, and myelin subfractions of multiple sclerosis (MS). The protein compositions of normal-appearing cerebral white matter and cerebral plaque and periplaque tissues were also analyzed for comparison. MS whole cord samples were found to contain higher amounts of water compared with normal samples. The total lipid contents were below normal. Among the individual lipids, cholesterol content remained unchanged, whereas cholesteryl esters appeared increased in MS cords. The acidic phospholipid concentrations were found to be lower than normal. Glycolipids, such as cerebrosides G_M4, G_M1, and G_D1b, which are abundant in myelin, were all decreased. However, the concentrations of G_M3 and G_D3, which are more characteristic of reactive astrocytes, were highly elevated. The total protein content of MS cord samples was decreased, and the decrease was attributable to the loss of myelin proteins as evidenced by the low recovery of myelin. The concentrations of myelin-specific proteins, such as proteolipid protein and myelin basic protein, were significantly reduced. Other changes in the protein compositions included the accretion of two low molecular weight proteins of approximately 11,000 and 12,000, and the appearance of a periodic acid-Schiff-positive protein with the same electrophoretic mobility as the P₀ protein. Analysis of the isolated myelin indicated that it had a grossly normal protein composition. However, the two low molecular weight proteins and the P₀ protein appeared to be enriched in an upper-phase cord subtraction. We attribute the appearance of the two low molecular weight proteins to the breakdown of proteolipid protein and/or myelin basic protein as a result of demyelination, and the appearance of P₀ to the involvement of PNS myelin. The latter finding provides the first biochemical evidence that in MS cord, remyelination can be achieved in part by invading Schwann cells and/or by the small number of Schwann cells that may be present in the cord.     

Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

Quantitative Electroimmunoblotting Study of the Calcium-Activated Neutral Protease in Human Myelin

Degradation of myelin basic protein (MBP) in human man myelin was monitored by electroimmunoblotting. Problems of variation between, as well as within, electroimmunoblots were overcome by the introduction of an internal standard in each sample, thus allowing reproducible quantification of MBP. The Ca2+-dependent protease acting on MBP was active at endogenous levels of Ca2+ (congruent to 300 micrograms/g myelin) and was inhibited in the presence of Ca2+ chelators. Extensive degradation of MBP occurred rapidly in the presence of added Ca2+, reaching a plateau after a 1 h incubation (80-85% degradation). The proteolytic activity was not enhanced in the presence of 2-mercaptoethanol. It was most active at neutral pH and at temperatures approaching physiological conditions. No difference was observed between proteolytic activities of control and multiple sclerotic myelin. It is suggested that fluctuations in the accessibility of free Ca2+ to the protease may lead to the regulation of Ca2+-activated myelinolysis.     

Association between Change in Normal Appearing White Matter Metabolites and Intrathecal Inflammation in Natalizumab-Treated Multiple Sclerosis

Background

Multiple sclerosis (MS) is associated not only with focal inflammatory lesions but also diffuse pathology in the central nervous system (CNS). Since there is no firm association between the amount of focal inflammatory lesions and disease severity, diffuse pathology in normal appearing white matter (NAWM) may be crucial for disease progression. Immunomodulating treatments for MS reduce the number of focal lesions, but possible effects on diffuse white matter pathology are less studied. Furthermore, it is not known whether intrathecal levels of inflammatory or neurodegenerative markers are associated with development of pathology in NAWM.

Methods

Quantitative proton magnetic resonance spectroscopy (¹H-MRS) was used to investigate NAWM in 27 patients with relapsing MS before and after one year of treatment with natalizumab as well as NAWM in 20 healthy controls at baseline. Changes in ¹H-MRS metabolite concentrations during treatment were also correlated with a panel of intrathecal markers of inflammation and neurodegeneration in 24 of these 27 patients.

Results

The group levels of ¹H-MRS metabolite concentrations were unchanged pre-to posttreatment, but a pattern of high magnitude correlation coefficients (r = 0.43–0.67, p<0.0005–0.03) were found between changes in individual metabolite concentrations (total creatine and total choline) and levels of pro-inflammatory markers (IL-1β and CXCL8).

Conclusions

Despite a clinical improvement and a global decrease in levels of inflammatory markers in cerebrospinal fluid during treatment, high levels of pro-inflammatory CXCL8 and IL-1β were associated with an increase in ¹H-MRS metabolites indicative of continued gliosis development and membrane turnover in NAWM.