Cytokines induce nitric oxide-mediated mtDNA damage and apoptosis in oligodendrocytes. Protective role of targeting 8-oxoguanine glycosylase to mitochondria.
Nitric oxide (NO) that is produced by inducible NO synthase (iNOS) in glial cells is thought to contribute significantly to the pathogenesis of multiple sclerosis. Oligodendrocytes can be stimulated to express iNOS by inflammatory cytokines, which are known to accumulate in the multiple sclerotic brain. The potentially pathological levels of NO produced under these circumstances can target a wide spectrum of intracellular components. We hypothesized that one of the critical targets for damage that leads to disease is mtDNA. In this study, we found that cytokines, in particular a combination of tumor necrosis factor-alpha (50 ng/ml) and IFNgamma (25 ng/ml), cause elevated NO production in primary cultures of rat oligodendrocytes. Western blot analysis revealed a strong enhancement of iNOS expression 48 h after cytokine treatment. Within the same time period, NO-mediated mtDNA damage was shown by Southern blot analysis and by ligation-mediated PCR. Targeting the DNA repair enzyme human 8-oxoguanine DNA glycosylase (hOGG1) to the mitochondria of oligodendrocytes had a protective effect against this cytokine-mediated mtDNA damage. Moreover, it was shown that mitochondrial transport sequence hOGG1-transfected oligodendrocytes had fewer apoptotic cells compared with cells containing vector only following treatment with the cytokines. Subsequent experiments revealed that targeting hOGG1 to mitochondria reduces the activation of caspase-9, showing that this recombinant protein works to reduce apoptosis that is occurring through a mitochondria-based pathway.
Druzhyna
Nadiya M
NM
Department of Cell Biology and Neuroscience, College of Medicine, University of South Alabama, Mobile, Alabama 36688, USA.
Musiyenko
Sergyi I
SI
Wilson
Glenn L
GL
LeDoux
Susan P
SP
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry*; D004272:DNA, Mitochondrial / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007249:Inflammation; D007371:Interferon-gamma / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009103:Multiple Sclerosis / Q000378:metabolism / Q000473:pathology; D009457:Neuroglia / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009836:Oligodendroglia / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D000077278:RNA, Mitochondrial; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D004272:DNA, Mitochondrial; D000077278:RNA, Mitochondrial; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D012313:RNA; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D045647:DNA Glycosylases; C483741:OGG1 protein, rat; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases