Mutation of superoxide dismutase elevates reactive species: comparison of nitration and oxidation of proteins in different brain regions of transgenic mice with amyotrophic lateral sclerosis.
As part of our effort to study the role of reactive species in amyotrophic lateral sclerosis (ALS), the goal of this work is to explore the correlation between nitration and oxidation of proteins and mutation of Cu, Zn-superoxide dismutase (SOD1) in ALS. Transgenic mice overexpressing the mutant Cu, Zn-superoxide dismutase (mSOD1) gene from humans with familial ALS, wild-type mice overexpressing the normal human SOD1 gene and normal mice without gene overexpression were used. Brain sections from different regions of three groups of mice were double immunohistochemically stained with anti-neurofilament plus anti-nitrotyrosine or treated with 2,4-dinitrophenylhydrazine to label protein carbonyls, then double stained with anti-neurofilament plus anti-2,4-dinitrophenyl (anti-DNP). Neurons containing nitrated and oxidized proteins were visualized only in mSOD1 mice in the motor cortex, the cerebellar cortex and nucleus of hypoglossal nerves (regions related with movement). This correlates mutation of SOD1 to nitration and oxidation of neurons in the movement regions. By counting double-stained neurons, we demonstrated that the number of nitrotyrosine- and DNP-positive neurons was significantly higher in the brain sections of both motor and sensory cortex in mSOD1 mice than in the corresponding regions of control mice (P=0.005 to <0.001), further correlating nitration and oxidation of proteins to SOD1 mutation. Neurons underwent significantly more nitration and oxidation in the motor cortex than in the sensory cortex in mSOD1 mice (P=0.002 and 0.02 respectively), indicating enhanced susceptibility of the motor cortex to nitration and oxidation of proteins and thereby targeting oxidation and nitration of proteins in neurons of the motor cortex in ALS. Significantly elevated protein nitration and nitric oxide synthesis were also demonstrated biochemically in the brain tissues and in cerebrospinal fluid of mutant SOD1 mice. Our in vivo evidence correlates mutation of the SOD1 gene to increased nitric oxide, nitration and oxidation of proteins in ALS.
Liu
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D
Department of Neurology, University of Texas Medical Branch, 301 University Boulevard, Route 0881, Galveston, TX 77555-0881, USA. dliu@utmb.edu
Bao
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F
Wen
J
J
Liu
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J
D000704:Analysis of Variance; D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000201:enzymology*; D002452:Cell Count / Q000379:methods; D002956:Citrulline / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D017551:Microdialysis / Q000379:methods; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D006834:Hydrazines; D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; C446799:dinitrophenylhydrazine; D002956:Citrulline; C002744:3-nitrotyrosine; D014443:Tyrosine; D013482:Superoxide Dismutase