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Motor neurons rapidly accumulate DNA single‐strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy
Author(s) -
Liu Zhiping,
Martin Lee J.
Publication year - 2001
Publication title -
journal of comparative neurology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.855
H-Index - 209
eISSN - 1096-9861
pISSN - 0021-9967
DOI - 10.1002/cne.1087
Subject(s) - motor neuron , biology , axotomy , neuron , dna damage , comet assay , neuroscience , microbiology and biotechnology , spinal cord , biochemistry , central nervous system , dna
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 ∼84% neurons, with ∼86% of these neurons being motor neurons; ∼72% of these motor neurons are α‐motor neurons. Motor neuron viability in suspension is ∼100% immediately after isolation and ∼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), H 2 O 2 , or NO donor plus H 2 O 2 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 H 2 O 2 (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 H 2 O 2 , 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. J. Comp. Neurol. 432:35–60, 2001. © 2001 Wiley‐Liss, Inc.