Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons resulting in progressive paralysis. To date, more than 140 different mutations in the gene encoding CuZn-superoxide dismutase (SOD1) have been associated with ALS. Several transgenic murine models exist in which various mutant SOD1s are expressed. We used DIGE to analyze the changes in the spinal cord proteome induced by expression of the unstable SOD1 truncation mutant G127insTGGG (G127X) in mice. Unlike mutants used in most other models, G127X lacks SOD activity and is present at low levels, thus reducing the risk of overexpression artifacts. The mice were analyzed at their peak body weights just before onset of symptoms. Variable importance plot analysis showed that 420 of 1,800 detected protein spots contributed significantly to the differences between the groups. By MALDI-TOF MS analysis, 54 differentially regulated proteins were identified. One spot was found to be a covalently linked mutant SOD1 dimer, apparently analogous to SOD1-immunoreactive bands migrating at double the molecular weight of SOD1 monomers previously detected in humans and mice carrying mutant SOD1s and in sporadic ALS cases. Analyses of affected functional pathways and the subcellular representation of alterations suggest that the toxicity exerted by mutant SODs induces oxidative stress and affects mitochondria, cellular assembly/organization, and protein degradation.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons resulting in progressive paralysis
The DIGE analysis of the gels covering pI 3–11 found about 1,800 potential protein spots that could be matched between the different gels and that could be subjected to statistical analysis
In our final analysis of alterations caused by expression of the G127X mutant SOD1, we only considered proteins that showed good correlations (p(corr)p Ͼ 0.70) by the multivariate OPLS-DA and that differed from controls with significances better than p Ͻ 0.01 using the parametric t test
Summary
Transgenic Mice—The G127X hSOD1 mice [7] were developed in our laboratory and backcrossed with C57BL/6JBomTac mice for Ͼ25 generations. Immunohistochemistry—Brain and spinal cords from 200-day-old G127X and G85R transgenic mice and C57BL/6JBomTac control mice were fixed with 4% paraformaldehyde in phosphate-buffered solution (10 mM potassium phosphate, pH 7.4, 0.15 M NaCl) and embedded in paraffin. An iterative threshold approach, a modified version of the approach presented by Karp et al [17], was applied to identify the protein spots that contributed most to the calculated OPLS-DA model (VIP analysis). This was done by successively removing the 20 most significant protein spots, based on p(corr)p, and recalculating the model until the cross-validated score vector (tcv) revealed the first overlap between the two classes. The cross-validated predictive ability (VIP-Q2) and the p value were calculated
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