Figure 1.
Bax deletion increases lifespan and delays disease onset of SOD1 G93A mutant mice without altering expression of SOD1 or interacting with SOD1. A, Bax KO/SOD1 mutants live longer (higher SOD1 copy number: mean = 137 ± 7 d, n = 43 vs 157 ± 9 d, n = 17, p < 0.001; lower SOD1 copy number: mean = 261 ± 26 d, n = 73, vs 283 ± 30 d, n = 19, p < 0.01) and (B) exhibit a decline in motor performance later (119 ± 11 d, n = 10 vs 136 ± 13 d, n = 10; p < 0.001) than Bax WT/SOD1 mutants. C, Mutant SOD1 mRNA is expressed by normal (asterisk) and developmentally rescued, small MNs (arrowheads) that express VAchT mRNA in adjacent sections. Lateral is to the left, medial to the right. Scale bar, 100 μm. D, Expression of Bax, pan-caspase-3 (arrow), and human mutant SOD1 protein in the spinal cord of Bax WT/SOD1 and Bax KO/SOD1 mutants (asterisk denotes a nonspecific band). Immunoblots are representative samples from each genotype (Bax, main effect of genotype: F(1,14) = 68.5, p < 0.0001; of age: F(2,14) = 4.05, p < 0.05, n = 3; Caspase-3, main effect of genotype: F(1,14) = 16.0, p < 0.005; of age: F(2,14) = 50.6, p < 0.0001, n = 3; SOD1, no main effects on genotype or age, n = 3). E, Antibodies against human/mouse SOD1 (lane 1 or 2, 4) failed to immunoprecipitate Bax protein, which is present in P100 spinal extract (arrow, lane 3). The bottom arrowhead in lane 2 is the IgG light chain, as confirmed by the result in lane 4, after omission of the primary antibody. The top arrowhead is the IgG heavy chain. Image is a representative example from P100 (n = 4), although similar results were also obtained at P60 and at end stage. F, Bcl-2 protein is expressed in spinal cord (arrow in first four lanes) but is not observed in P25 spinal cord-derived SOD1 immunoprecipitates (second 4 lanes; arrowhead shows rabbit cross-reactivity to sheep IgG heavy chain, as in E). Image is a representative example from P25 and P45 (n = 4). G, SOD1 is present in Bcl-2 and IgG IPs. Both sheep anti-human/mouse SOD1 (arrow, lanes 1–3) and rabbit anti-human SOD1 (arrow, lanes 4–5) are detected in spinal cord-derived Bcl-2 or IgG IPs at every age examined (n = 3). Arrowheads denote heavy- and light-chain IgG. IP, Immunoprecipitate.