Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy.
The mechanisms of neuronal degeneration in motor neuron disease are not fully understood. We tested the hypothesis that oxidative stress in vitro and axotomy in vivo induce single-strand breaks (SSB) in DNA, a form of early DNA damage, in adult motor neurons early during their degeneration. We developed and characterized a novel cell suspension system enriched in motor neurons from adult rat spinal cord ventral horn. This cell system is approximately 84% neurons, with approximately 86% of these neurons being motor neurons; approximately 72% of these motor neurons are alpha-motor neurons. Motor neuron viability in suspension is approximately 100% immediately after isolation and approximately 61% after 12 hours of incubation. During incubation, isolated motor neurons generate high levels of superoxide. We used single-cell gel electrophoresis (comet assay) to detect DNA-SSB in motor neurons. Exposure of motor neurons to nitric oxide (NO) donors (sodium nitroprusside or NONOate), H2O2, or NO donor plus H2O2 rapidly induces DNA-SSB and causes motor neuron degeneration, the occurrence of which is dose and time related, as represented by comet formation and cell loss. Motor neuron toxicity is potentiated by cotreatment with NO donor and H2O2 (at nontoxic concentrations alone). Peroxynitrite causes DNA-SSB in motor neurons. The DNA damage profiles (shown by the comet morphology and moment) of NO donors, NO donor plus H2O2, and peroxynitrite are similar. In an in vivo model of motor neuron apoptosis, DNA-SSB accumulate slowly in avulsed motor neurons before apoptotic nuclear features emerge, and the comet fingerprint is similar to NO toxicity. We conclude that motor neurons challenged by oxidative stress and axotomy accumulate DNA-SSB early in their degeneration and that the formation of peroxynitrite is involved in the mechanisms.
Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205-2196, USA.
Z
Z
Liu
L J
LJ
Martin
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D020552:Comet Assay; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D007839:Functional Laterality; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008959:Models, Neurological; D009046:Motor Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009566:Nitrates / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D012584:Sciatic Nerve / Q000502:physiology; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology; D013481:Superoxides / Q000378:metabolism
D009566:Nitrates; D020030:Nitric Oxide Donors; D013481:Superoxides; D009599:Nitroprusside; C039642:peroxynitric acid; D004247:DNA; D006861:Hydrogen Peroxide