Figure 7

Co-expression of the PolgA D257A mutation and APP/Ld transgene causes increased levels of neurodegeneration markers, cleaved caspase-3 and p25. 30 μg of brain homogenates from D257A; APP/Ld, D257A, APP/Ld, and wild type mice were randomly loaded and subjected to immunoblot analysis for cleaved caspase-3 fragment and p25. Immunoblot signals were normalized to Ponceau S staining intensities in a given lane. The numbers on immunoblot correspond to a mouse with a specific genotype as denoted in the legend key (box). (A) Full-length (f.l.) and activated cleaved caspase-3 run at ~35 kDa and ~17 kDa respectively, as previously reported [33]. (B) Ratio of immunosignal intensities of cleaved to f.l. caspase-3. Note that the cleaved:f.l. caspase-3 ratio is significantly increased in APP/Ld mice, although D257A; APP/Ld mice have an even greater cleaved:f.l. caspase-3 ratio, suggesting synergism between the D257A mutation and APP/Ld transgene. (C) DAB staining using cleaved caspase-3 neoepitope antibody on coronal sections (30 μm) demonstrate cleaved caspase-3-positive puncta in cell bodies of cortical neurons (cortical layers V and VI) of APP/Ld and D257A; APP/Ld mice (black arrows; scale bar: 100 μm). No immunoreactivity for cleaved caspase-3 is observed in wild-type and D257A mice. (D) Immunoblots using an antibody that recognizes both p35 and the neurodegeneration marker, calpain-cleaved p25 fragment. (E) Ratio of immunosignal intensities of p25 to p35. Note that the p25:p35 ratio is significantly elevated in APP/Ld and D257A; APP/Ld mice compared to wild-type mice. Increased p25:p35 ratio is seen in D257A; APP/Ld compared to APP/Ld mice suggesting synergism, although this trend did not reach statistical significance. D257A mice do not have an elevation of p25:p35 ratio (mean ± SEM; n.s. = not significant; *p < 0.05; **p < 0.01; One-way ANOVA followed by Newman–Keuls multiple-comparisons post hoc test).