Figure 6.

ER accumulation of polyubiquitin, and ER caspase activation with α-synucleinopathy in the A53TαS Tg mice. A, Immunoblot analysis of total SpC lysates from the end-stage A53TαS Tg mice and nTg littermates. Neuropathology in the A53TαS Tg mice is clearly associated with cleavage of ER-dependent caspase-12 and its downstream effectors, caspase- 9 and -3 (pro, pro-peptide; Δ, cleaved). Both pro-peptide and cleaved peptide were detected on the same blot but cropped to save space. The bar graph is the quantitative analysis of blots shown. The amount of cleaved products was normalized to the full-length protein and represents mean ± SEM (n = 4). ***p < 0.001, **p < 0.01, Student's t test, Tg versus nTg littermates. B, C, Caspase-12 activation is selective for α-synucleinopathy. (B) Caspase-12 immunoblot showing cleavage (Δ) of caspase-12 propeptide (pro) in end-stage (End) Tg mice compared with presymptomatic (PreS) mice. (C) Caspase-12 analysis of affected areas (SpC) compared with disease-free regions (Ctx) from end-stage Tg and nTg mice. Note that the cleaved caspase is associated with the presence of end-stage disease. D, Ubiquitin immunoblot of total SDS-soluble fraction (Tot), Triton X-100-soluble (S-Tx), and Triton X-100-insoluble pellets (P-Tx) from SpC of end-stage A53TαS Tg mice and nTg littermates. The graph shows quantitative analysis. Mean ± SEM (n = 6), *p < 0.05, Student's t test. E, Ubiquitin immunoblot of microsome fractions from nTg and A53TαS Tg mice at various stages: 6 months old, 10 months old, presymptomatic stage (PreS), and symptomatic stages (Early, End). Densitometry analysis of immunoblots reveals the dramatic ER accumulation (30- to 40-fold increase) of polyubiquitin with α-synucleinopathy. In contrast, total tissue increase in polyubiquitin is only 2- to 3-fold (D). The plots are mean ± SEM of percentage of nTg average (n = 6). ***p < 0.001, **p < 0.01, one-way ANOVA. Both Early- and End-stage Tg mice are also different from PreS (p < 0.05 and p <0.001).