Multiple sclerosis (MS) is a common autoimmune neurodegenerative disease of unknown cause, which results in inflammation and plaques of demyelination in brain and eventual axonal degeneration. We report the novel presence of oxidized phosphatidylcholine [1-palmitoyl-2-(5'-oxo)valeryl-sn-glycero-3-phosphorylcholine (POVPC)], a lipid associated with inflammatory diseases such as atherosclerosis and lung disease, in the brain of MS patients. The OxPC epitope was detected by Western blotting with the E06 monoclonal antibody. E06-positive lipid was present in the highest amounts in MS plaques, which also showed evidence of low-molecular-weight (15-kDa) OxPC-modified protein. E06 reactivity did not change with post-mortem interval, and E06-positive lipids were largely absent from control tissue. We then used a second monoclonal antibody (AB1-2, which recognizes the E06/T15 idiotype and therefore detects the presence of antibody to OxPC) to show that MS brain samples were strongly positive for the 50-kDa antibody heavy chain. We also showed that isoelectric focussing of the oligoclonal IgG characteristic of MS revealed some immunoglobulin bands that Western blotted with the AB1-2 antibody. Spinal cords from mice induced to undergo experimental allergic encephalomyelitis (EAE) also showed strong AB1-2 reactivity by both immunocytochemistry and Western blot analysis. We therefore conclude that we can detect both OxPC and 15-kDa protein modified by OxPC and the antibody to the antibody to OxPC (antiidiotype) in pathological tissue and suggest that this could play a role in the progression of MS.

Background:Fibroblasts from the fro/fro mouse contained reduced amounts of ceramides and elevated amounts of hyaluronan (HA). Results: Increased HA secretion was associated with increased activity of the Akt pathway and enhanced expression of the HAS2 gene. Conclusion: HA synthesis is regulated by NSMase2/ceramide through ceramide-activated phosphatase PP2A and Akt signaling pathway. Significance: There is a direct link between sphingolipid and glycosaminoglycan metabolism.

Both cultured neonatal rat hippocampal neurons and differentiated oligodendrocytes rapidly metabolized exogenous C 2 -and C 6 -ceramides to sphingosine (Sph) and sphingosine 1-phosphate (S1P) but only minimally to C 16 -24 -ceramides. Dihydrosphinolipids were unaffected but were increased by exogenous C 6 -dihydroceramide. Conversely, quantitative liquid chromatography-tandem mass spectrometry technology showed that exogenous S1P (0.25-10 M) was rapidly metabolized to both Sph (a >200-fold increase) and predominantly C 18 -ceramide (a >2-fold increase). Longer treatments with either C 2 -ceramide (>2.5 M) or S1P (10 M) led to apoptotic cell death. Thus, there is an active sphingolipid salvage pathway in both neurons and oligodendrocytes. Staurosporine-induced cell death was shown to be associated with decreased S1P and increased Sph and C 16/18 -ceramide levels. The physiological significance of this observation was confirmed by the analysis of affected white matter and plaques from brains of multiple sclerosis patients in which reduced S1P and increased Sph and C 16/18 -ceramides were observed.

Demyelination is a common result of oxidative stress in the nervous system, and we report here that the response of oligodendrocytes to oxidative stress involves the receptor for advanced glycation end products (RAGE). RAGE has not previously been reported in neonatal rat oligodendrocytes (NRO), but, by using primers specific for rat RAGE, we were able to show expression of messenger RNA (mRNA) for RAGE in NRO, and a 55-kDa protein was detected by Western blotting with antibodies to RAGE. Neonatal rat oligodendrocytes stained strongly for RAGE, suggesting membrane localization of RAGE. Addition of low concentrations of hydrogen peroxide (100 μM) initiated 55-kDa RAGE shedding from the cell membrane and the appearance of "soluble" 45-kDa RAGE in the culture medium, followed by restoration of RAGE expression to normal levels. Increasing hydrogen peroxide concentration (>200 μM) resulted in no restoration of RAGE, and the cells underwent apoptosis and necrosis. We further confirmed the observation in a human oligodendroglioma-derived (HOG) cell line. Both the antioxidant N-acetyl-L-cysteine and the broad-spectrum metalloproteases inhibitor TAPI0 were able partially to inhibit shedding of RAGE, suggesting involvement of metalloproteases in cleavage to produce soluble RAGE. The level of 55-kDa RAGE in autopsy brain of patients undergoing neurodegeneration with accompanying inflammation [multiple sclerosis and neuronal ceroid-lipofuscinosis (Batten's disease)] was much lower than that in age-matched controls, suggesting that shedding of RAGE might occur as reactive oxygen species accumulate in brain cells and be part of the process of neurodegeneration. Keywordsoligodendrocytes; reactive oxygen species; hydrogen peroxide; RAGE; shedding proteolysis Oligodendrocytes have been implicated in the pathogenesis of demyelinating diseases such as multiple sclerosis (MS) in which the active agents are reactive oxygen species (ROS) and the products of inflammation (LeVine, 1992;Smith et al., 1999). Evidence of ROS damage has been reported in demyelinating lesions in the brains of MS patients (Langemann et al., 1992;LeVine and Wetzel, 1998) and in the demyelinating experimental allergic encephalitis (EAE) mouse spinal cord (MacMicking et al., 1992). Lipid peroxidation products and ROS have been identified in cerebrospinal fluid (CSF), plasma, and brain from MS patients (Naidoo and Knapp, 1992;LeVine, 1992;Qin et al., 2007), and this, together with evidence for peroxynitrite modification of proteins (Liu et al., 2001) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript 1998), strongly supports the view that oxidative damage is important in demyelination. Protein carbonyl content, mainly glutamic semialdehyde (from arginine and proline) and aminoadipic semialde-hyde (from lysine oxidation), is a measure of ROS-mediated protein oxidation (Berlett and Stadtman, 1997) and is increased in MS brain (Bizzozero et al., 2005).RAGE is a 45−55-kDa member of the immunoglobulin superfamily (Rong et al., 2004a,b;...

The immunomodulator drug Gilenya (FTY720), marketed as the first oral sphingosine 1-phosphate receptor (S1P-R) modulator for treatment of Multiple Sclerosis (MS) also inhibits lysosomal acid sphingomyelinase (ASMase). Treatment of cultured cells for 24h with FTY720 (up to 10μM) inhibited ASMase by >80% and this could be reversed by pre-treatment with the cathepsin protease inhibitor leupeptin (5μM). In contrast, neutral sphingomyelinase activity was unaffected and sphingosine-1-phosphate treatment had no effect on ASMase. RT-PCR revealed no inhibition of ASMase mRNA and there was no direct (in vitro) inhibition of ASMase by either FTY720 or FTY720-phosphate. This suggests that its mechanism of inhibition is similar to that of tricyclic anti-depressants such as desipramine, which are also amphiphilic cationic drugs. Both Desipramine and FTY720 treatment reduced ASMase without significant inhibition of other lysosomal hydrolases but most hydrolases showed increased secretion (up to a 50% increase) providing more evidence of lysosomal disruption by these drugs.