Oxidative stress has been implicated in the pathophysiology of multiple sclerosis (MS). Increased levels of reactive oxygen species (ROS) derived from infiltrating macrophages and microglial cells have been shown to reduce the levels of endogenous antioxidants and to cause the oxidation of various substrates within the MS plaque. To determine whether oxidative damage takes place beyond visible MS plaques, the occurrence of total carbonyls (TCOs) and protein carbonyls (PCOs) in the normal-appearing white matter (NAWM) and gray matter (NAGM) of eight MS brains was assessed and compared with those of four control brains. The data show that most (7/8) of the MS-WM samples contain increased amounts of PCOs as determined by reaction with 2,4-dinitrophenylhydrazine and Western blot analysis. These samples also have high levels of glial fibrilary acidic protein (GFAP), suggesting that oxidative damage is related to the presence of small lesions. In contrast, we detected no evidence of protein thiolation (glutathionylation and cysteinylation) in the diseased tissue. To our surprise, MS-NAGM specimens with high GFAP content also showed three times the concentration of TCOs and PCOs as the controls. The increase in PCOs is likely to be a consequence of reduced levels of antioxidants, in that the concentration of nonprotein thiols in both MS-WM and -GM decreased by 30%. Overall, our data support the current view that both NAWM and -GM from MS brains contain considerable biochemical alterations. The involvement of GM in MS was also supported by the decrease in the levels of neurofilament light protein in all the specimens analyzed. To the best of our knowledge, this is the first study demonstrating the presence of increased protein carbonylation in post-mortem WM and GM tissue of MS patients.

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.

This study investigates the effect of nitric oxide (NO) on both the chemical modifications of CNS proteins and the architecture of the myelinated internode. Incubation of rat optic nerves for 2 h with 1 mM concentration of the NO-donors S-nitroso-N-acetyl-penicillamine (SNAP), ethyl-2-[hydroxyimino]-5-nitro-3-hexeneamide (NOR-3), and 4-phenyl-3-furoxan carbonitrile (PFC) led to decompaction of myelin at the level of the intraperiod line (IPL). In contrast, incubation with 1 mM sodium nitroprusside, which slowly releases NO, sodium nitrite, and N-nitrosopyrrolidine failed to cause myelin disassembly. This suggests that free NO and/or some of its direct oxidation products (e.g., N2O3) are the active molecular species. NO-induced alterations in myelin architecture could not be assigned to protein or lipid degradation, lipid peroxidation, ATP depletion, calcium uptake, protein nitration, protein carbonylation, and nerve depolarization. NO-treatment, however, resulted in the S-nitrosation of a number of proteins. In myelin, one of the major S-nitrosated substrates was identified as proteolipid protein (PLP), an abundant cysteine-rich protein that is responsible for IPL stabilization. Peripheral nervous system myelin, whose stability depends on proteins other than PLP, was not decompacted upon incubation of sciatic nerves with SNAP. It is proposed that NO-mediated nitrosation of sulfhydryl groups is likely to interfere with the normal function of PLP and other important CNS myelin proteins leading to the structural demise of this membrane. These findings are relevant to multiple sclerosis and other inflammatory demyelinating disorders where both excessive NO production and myelin instability are known to occur.

We have investigated the effect of documented protein palmitoylation inhibitors on the fatty acylation and intracellular transport of myelin proteolipid protein (PLP). To this end, brain slices from 20-day-old rats were incubated with either [3H]palmitate or [3H]leucine in the presence or absence of various concentrations of 2-fluoropalmitate (FP), cerulenin (CER), or tunicamycin (TM). FP (> or = 10 microM) decreased the cellular uptake of [3H]palmitate and consequently reduced the labeling of palmitoyl-CoA, glycerolipids and PLP. CER (> or = 1 mM) reduced the palmitoylation of PLP with a concomitant decline in protein thiols. Consistent with being a fatty acyl-CoA analogue, TM (> or = 200 microM) diminished the palmitoylation of PLP and lipids while increasing the amount of [3H]palmitoyl-CoA. Although both CER and TM decreased protein palmitoylation, only the latter affected the appearance of newly synthesized PLP into myelin. Because TM, but not CER, also reduced the formation of lipids, it is concluded that palmitoylation is not required for intracellular transport. Finally, comparison of the effect of TM in brain slices and in a cell-free system suggests that palmitoylation of PLP in whole cells may be an enzymatic process.

We have investigated the structure of the native PLP, DM‐20 and several low molecular weight proteolipids (LMWPs) by mass spectrometry (MS). The various proteolipid species were isolated from bovine spinal cord by size‐exclusion and ion‐exchange chromatography. Matrix‐assisted laser desorption ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) of PLP and DM‐20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester‐bound fatty acids. Electrospray ionization‐MS analysis of the deacylated proteins produced the predicted molecular masses of the apoproteins, demonstrating that palmitoylation is the major modification of PLP, and that the majority of PLP and DM‐20 molecules in the CNS are fully acylated. A series of proteolipids with molecular masses from 12 to 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N‐ and C‐terminal sequencing, tryptic digestion and peptide mapping by MALDI‐TOF‐MS. The results showed that these polypeptides correspond to the N‐terminal region (residues 1‐105/112) and C‐terminal region (residues 113/131‐276) of PLP, and they appear to be generated by natural proteolytic cleavage within the 50 amino acid‐long cytoplasmic domain. These LMWPs are derived from myelin, and are not generated during their isolation or during the postmortem period. Moreover, they are present in other species and are unevenly distributed across the CNS. It is tempting to speculate that these minor proteolipids may play a role in myelin formation. Acknowledgements:  Supported by PHHS grant NS 38325.