Figure 1.

Genetic reduction in SNAP-25 levels accelerates mortality of CSPα KO mice and decreases SNARE-complex assembly. (A) Images of representative WT mice, CSPα KO mice (CSP^−/−), heterozygous CSPα KO/heterozygous SNAP-25 KO mice (CSP^+/−SNAP-25^+/−), and homozygous CSPα KO/heterozygous SNAP-25 KO mice (CSP^−/−SNAP-25^+/−) at 30 days of age (P30). (B) Paralysis, as a neuromuscular sign of motor neuron degeneration, was recorded in mice of the indicated genotypes as a function of age (shown as the percentage of mice that uprighted in <5 s when placed on the side). CSP^−/−SNAP-25^+/− mice were paralyzed significantly earlier than CSP^−/− mice containing two WT SNAP-25 alleles (n=9–13 mice; ^***P<0.001 by Mantel–Cox test). (C) Survival of littermate mice with the indicated genotypes as a function of age. In comparison, CSP^−/−SNAP-25^+/− mice perish significantly earlier than CSP^−/− mice (n=9–13 mice; ^***P<0.001 by Mantel–Cox test). For a survival study including non-littermate mice, see Supplementary Figure S1E. (D, E) Protein (D) and SNARE-complex levels (E) in the brains from six different genotypes of mice at P20: WT, SNAP-25^+/−, CSP^−/−, CSP^−/−SNAP-25^+/−, transgenic α-synuclein (tSyn), and tSyn/CSP^−/−. (D) Representative immunoblots (left) and protein quantitations (right), obtained using ¹²⁵I-labelled secondary antibodies and normalized to GDI as internal standard. (E) SNARE-complex measurements in the same samples using co-immunoprecipitation of SNARE proteins (top, representative immunoblots; bottom, quantitations). Recovered protein (relative to the input) was normalized to the immunoprecipitated protein and then to WT levels (n=4 mice). Protein levels were measured by quantitative immunoblotting using ¹²⁵I-labelled secondary antibodies. Data shown are mean values±s.e.m.; ^*P<0.05; ^**P<0.01; ^***P<0.001 (assessed using Mantel–Cox (B, C) or Student’s t-test (D, E), comparing each test condition to the control analysed in the same experiment). Figure source data can be found in Supplementary data.