Branislava Mitrovic

Nitric oxide as a potential pathological mechanism in demyelination: Its differential effects on primary glial cellsin vitro

Because we believe that macrophage-derived nitric oxide contributes to pathology of demyelinating diseases, we have determined the differential effects of nitric oxide on primary rat glial cells in vitro. Enriched cultures of microglia, astrocytes and oligodendrocytes were treated with S-nitroso,N-acetyl-DL-penicillamine, a nitric oxide-releasing chemical. There was a significantly decreased function of one of the ferrosulfur-containing mitochondrial enzymes after S-nitroso,N-acetyl-DL-penicillamine/nitric oxide treatment in oligodendrocytes and astrocytes compared to microglia, which were much less sensitive to S-nitroso,N-acetyl-DL-penicillamine/nitric oxide at all concentrations. At 0.5 mM S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, astrocytes and oligodendrocytes suffered a 40% loss in succinate dehydrogenase activity, while microglia were unaffected. A control non-ferrosulfur-containing mitochondrial enzyme, isocitrate dehydrogenase, was not affected in any glial cell type. Although the per cent of mitochondrial damage in oligodendrocytes and astrocytes was the same for all concentrations of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, significant cell death occurred in oligodendrocytes at 1.0 mM; at this concentration there was no significant killing of microglia or astrocytes. Furthermore, at a 0.5 mM concentration of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, which inhibited mitochondrial respiration but did not kill oligodendrocytes, significant changes in oligodendrocyte morphology (e.g. retraction of processes) occurred. Morphological changes were not seen in microglia and astrocytes at any concentration of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide. In addition, oligodendrocytes were more sensitive to S-nitroso,N-acetyl-DL-penicillamine/nitric oxide-induced single stranded DNA breaks than microglia or astrocytes. The mitochondrial damage was attributable to nitric oxide since N-acetyl-DL-penicillamine had no effect. Oxyhemoglobin, which competitively inhibits toxic effects of nitric oxide, protected these glial cells from mitochondrial damage, single stranded breaks in DNA and cell death in a time- and dose-dependent manner. Once again, oligodendrocytes were less easily rescued from nitric oxide effects by oxyhemoglobin than were astrocytes, suggesting greater vulnerability of the myelin-producing cell to nitric oxide. These findings suggest that there is differential sensitivity of glial cells to nitric oxide. Although oligodendrocytes and astrocytes are equally susceptible to nitric oxide-induced mitochondrial damage, oligodendrocytes are more sensitive to nitric oxide-induced single stranded DNA breaks, morphological changes and cell death. Compared to both oligodendrocytes and astrocytes, microglia, nitric oxide-producing cells, are resistant to nitric oxide-induced damage.

The role of nitric oxide in multiple sclerosis.

Abstract During the past decade nitric oxide has emerged as an important mediator of physiological and pathophysiological processes. Elevated nitric oxide biosynthesis has been associated with nonspecific immune-mediated cellular cytotoxicity and the pathogenesis of chronic, inflammatory autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, and mutiple sclerosis. Recent evidence suggests, however, that nitric oxide is also immunoregulatory and suppresses the function of activated proinflammatory macrophages and T lymphocytes involved in these diseases. This article reviews the role of nitric oxide in the biology of central nervous system glial cells (astrocytes and microglia) as it pertains to the pathogenesis of multiple sclerosis in humans and experimental allergic encephalitis, the animal model of this disease. Although nitric oxide has been clearly implicated as a potential mediator of microglia-dependent primary demyelination, a hallmark of multiple sclerosis, studies with nitric oxide synthase inhibitors in the encephalitis model have been equivocal. These data are critically reviewed in the context of what is know from clinical research on the nitric oxide pathway in multiple sclerosis. Specific recommendations for future preclinical animal model research and clinical research on the nitric oxide pathway in patients are suggested. These studies are necessary to further define the role of nitric oxide in the pathology of multiple sclerosis and to fully explore the potential for nitric oxide synthase inhibitors as novel therapeutics for this disease.

Inducible nitric oxide synthase and nitric oxide production by oligodendrocytes

It has been previously demonstrated that microglia and astrocytes produce micromolar amounts of nitric oxide in vitro. In this study, we demonstrate that primary rat oligodendrocytes can be stimulated to produce iNOS mRNA as detected by Northern blot and in situ hybridization analysis and a 131‐kDa iNOS protein by Western blot analysis; protein was also detected in cells by single‐ and double‐label immunohistochemistry for iNOS and the oligodendrocyte‐specific marker CNPase. NO/NOS are produced as a consequence of activation of the gene encoding the inducible nitric oxide synthase as determined by inhibition with actinomycin D and cyclohexamide. The iNOS is functional, leading to calcium/calmodulin‐independent NO production in these in vitro cultures. J. Neurosci. Res. 48:372–384, 1997.

Oxidative stress in a clonal cell line of neuronal origin: effects of antioxidant enzyme modulation.

Abstract: The effects of intracellularly generated H2O2 on cell viability, morphology, and biochemical markers of injury have been investigated in a clonal cell line of neuronal origin (140‐3, mouse neuroblastoma X rat glioma) as a cell culture model for the role of oxidative stress in the longterm loss of neurons in the brain. The H2O2 was generated from the redox cycling of menadione, or by the oxidation of serotonin catalyzed by monoamine oxidase, to simulate the effect of amine neurotransmitter turnover. Incubation with menadione at concentrations as low as 10 γM for several hours resulted in significant losses of cell viability and altered morphology. Similar effects were evident in the presence of serotonin only after incubation overnight with concentrations > 1 mM. The cytotoxicity of either agent was potentiated by preincubation with specific inhibitors of two enzymes important to cellular antioxidant defenses, 3‐amino‐1,2,4‐trazole for catalase and 1,3‐bis(chloromethyl)‐1‐nitrosourea for glutathione reductase. Activity of another antioxidant enzyme of particular importance to antioxidant defenses in brain, the selenoprotein glutathione peroxidase, was stimulated fourfold by growth of cultures in the presence of sodium selenite as a source of active‐site Se for the enzyme. The only effect of the selenite on other functionally coupled antioxidant enzymes was a decrease in activity of superoxide dismutase at concentrations >200 nM. The selenite substantially protected cells against oxidative stress induced by combinations of menadione, 3‐amino‐1,2,4‐trazole, and 1,3‐bis(chloromethyl)‐1‐nitrosourea, but was only marginally effective with serotonin as a source of oxidative stress. The monoamine oxidase inhibitor pargyline increased cell survival in the presence of serotonin, demonstrating the role of this enzyme in its cytotoxicity. DNA damage (single strand breaks), but not lipid peroxidation, correlated with the cytotoxic effects of menadione.

Differential sensitivity to nitric oxide in immortalized, cloned murine oligodendrocyte cell lines.

Using five different immortalized, cloned murine oligodendrocyte cells lines, we have assessed their sensitivity to the nitric oxide donor chemical S-nitroso, N-acetyl-DL-penicillamine (SNAP). The five lines were quite different in their sensitivity to SNAP as determined by assessment of viability, mitochondrial function, single-stranded DNA breaks, DNA/RNA/protein synthesis, morphology, and myelin gene expression. Single-stranded DNA breaks as well as mitochondrial and DNA damage occurred in viable cells. Thus, cell damage was independent of cell death. In the most mature oligodendrocyte cell line, cell morphology was altered and the expression of myelin basic protein mRNA was inhibited by nitric oxide in a time- and dose-dependent fashion. These findings suggest two things. First, oligodendrocytes at different states of differentiation are differentially sensitive to nitric oxide. Second, while not all oligodendrocytes may be destroyed in multiple sclerosis plaques, many could be significantly damaged and thereby nonfunctional in repair processes triggered during the pathology of this disease.

Chapter 26 Neurotransmitters and cytokines in CNS pathology

Publisher Summary This chapter demonstrates an in vitro model for oligodendrocyte cell death that may be relevant to events in formation of lesions in multiple sclerosis (MS). It involves cell contact to oligodendrocytes with activated, viable microglia, surface tumor necrosis factor (TNF)-α, surface adhesion molecules, and production of NO. MS is a demyelinating disease of the central nervous system, in which myelin sheaths and the myelin-producing cells, oligodendrocytes, are destroyed. There is an accumulation of activated microglia and inflammatory macrophages as well as presence of proinflammatory cytokines such as TNF-α and interferon gamma (IFN-γ) at the lesion edge. The asymmetry of the pathology of these lesions with myelin loss and oligodendrocyte death in the wake or trailing edge of the growing lesion, but not at the leading edge of the lesion, suggests that the plaque formation is cell-mediated. Indeed, proliferation of oligodendrocytes is seen just beyond the plaque margin. Precise mechanisms of TNF-α and intercellular adhesion molecule-l/ leukocyte functional antigen-l participation and the nature of the susceptibility of the oligodendrocyte are presently being studied.

Molekulare Aspekte der Behandlung der Multiplen Sklerose

Die Multiple Sklerose (MS) ist die haufigste Autoimmunerkrankung des Nervensystems und mit die Hauptursache einer neurologischen Behinderung junger Erwachsener. Pathologie, Pathophysiologie und Pathogenese der MS, die als Prototyp einer Entmarkungskrankheit angesehen wird, werden zunehmend besser verstanden.