Role of calcium in nitric oxide-induced cytotoxicity: EGTA protects mouse oligodendrocytes.

Active nitrogen species are overproduced in inflammatory brain lesions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). NO has been shown to mediate the death of oligodendrocytes (OLs), a primary target of damage in MS. To develop strategies to protect OLs, we examined the mechanisms of cytotoxicity of two NO donors, S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) on mature mouse OLs. Nitrosonium ion (NO+) rather than NO. mediates damage with both SNAP and SNP, as shown by significant protection with hemoglobin (HbO2), but not with the NO. scavenger PTIO. SNAP and SNP differ in time course and mechanisms of killing OLs. With SNAP, OL death is delayed for at least 6 hr, but with SNP, OL death is continuous over 18 hr with no delay. Relative to NO release, SNP is more toxic than SNAP, due to synergism of NO with cyanide released by SNP. SNAP elicits a Ca2+ influx in over half of the OLs within min. Further, OL death due to NO release from SNAP is Ca2+-dependent, because the Ca2+ chelator EGTA protects OLs from killing by SNAP, and also from killing by the NONOates NOC-9 and NOC-18, which spontaneously release NO. SNP does not elicit a Ca2+ influx, and EGTA is not protective. In comparison to the N20.1 OL cell line (Boullerne et al., [1999] J. Neurochem. 72:1050-1060), mature OLs are (1) more sensitive to SNAP, (2) much more resistant to SNP, (3) sensitive to cyanide, but not iron, and (4) exhibit a Ca2+ influx and EGTA protection in response to NO generated by SNAP.

Experimental allergic encephalomyelitis animal models for analyzing features of multiple sclerosis.

Various animal models with experimental allergic encephalomyelitis (EAE) have been developed applying immunologic, virologic, toxic and traumatic parameters in order to understand features of multiple sclerosis (MS). The main simulating aspects of the EAE models and the precautions for their interpretation in determining differences and common features between EAE and MS are presented. In view of an early diagnosis of CNS lesions in human, we present with particular interest the application of human-related technologies, such as MR imaging techniques, and the development of new markers to follow the dynamic of CNS lesions in vivo in EAE animal models.

Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1.

Nitric oxide (NO) produced in inflammatory lesions may play a major role in the destruction of oligodendrocytes in multiple sclerosis and experimental allergic encephalomyelitis. The transformed murine oligodendroglial line N20.1 is much more resistant than primary oligodendrocytes to killing by the NO generator S-nitroso-N-acetyl-DL-penicillamine (SNAP). This observation prompted investigation of the mechanisms leading to cell death in the N20.1 cells and comparison of SNAP with another NO donor, sodium nitroprusside (SNP). We observed that N20.1 cells were 30 times more sensitive to SNP than to SNAP. The specific NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) protected against SNP only, not against SNAP. However, dithiothreitol protected against both SNAP and SNP, indicating that S-nitrosylation of cysteines plays a major role in the cytotoxicity of both NO donors. We did not observe any formation of peroxynitrite or increase of Ca2+ concentration with either SNAP or SNP, thus excluding their involvement in the mechanisms leading to N20.1 cell death. Based on two observations, (a) potentiation of the cytotoxic effect of SNP when coincubated with ferricyanide or ferrocyanide, but not sodium cyanide, and (b) protection by deferoxamine, an iron cyanide chelator, we conclude that the greater sensitivity of N20.1 cells to SNP compared with SNAP is due to synergism between NO released and the iron cyanide portion of SNP, with the cyanide accounting for very little of the cytotoxicity. Finally, SNP but not SNAP induces some apoptosis, as shown by DNA laddering and protection by a caspase-3 inhibitor. These results suggest that low levels of NO in combination with increased iron content lead to apoptotic cell death rather than the necrotic cell death seen with higher levels of NO generated by SNAP.

Indirect evidence for nitric oxide involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S-nitrosocysteine.

Converging data suggest that nitric oxide (NO) production by cytokine-induced immune cells in demyelinating lesions is involved in multiple sclerosis (MS). High levels of NO may complex to suitable amino acids, causing an immune response against the formed neo-epitopes. By testing MS sera with chemically defined nitroso-amino acids conjugated to carrier protein in ELISA, we observed a significant antibody reaction against the S-nitroso-cysteine epitope. The MS antibody response was exclusively of IgM isotype with an avidity of 8 x 10(-7) M. Sera of all clinical MS forms showed a significantly elevated antibody titer versus sera from healthy subjects or from patients affected with other neurological and autoimmune diseases. The detection of circulating antibodies to a conjugated S-nitroso-cysteine epitope provides indirect evidence for NO involvement in MS.