Figure 5: Loss of endogenous C9ORF72 inhibits autophagy accompanied by increased accumulation of polydipeptides produced by AUG-independent translation in C9^450C transgenic mice.

(a) Repeat-containing C9orf72 RNA expression in the cortex of C9orf72 transgenic mice with normal, reduced, or absence of endogenous C9ORF72 at 6 months of age. Error bars represent SEM (n = 4 Non-Tg, n = 5 C9^450C], n = 4 C9^450C,C9orf72^+/−, n = 5 C9^450C,C9orf72^−/−).

(b-c) Levels of poly(GP) (b) and poly(GA) (c) by immunoassay in the cortical extracts of C9ORF72 transgenic mice with normal, reduced, or absence of C9ORF72 at 6 months of age. Error bars represent SEM (n = 4 Non-Tg, n = 5 C9^450C, n = 4 C9^450C,C9orf72^+/−, n = 5 C9^450C,C9orf72^−/−). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.

(d-e) Representative images (d) and quantification (e) of poly(GA) aggregates in the retrosplenial granular cortex of C9orf72 transgenic mice with normal, reduced or absence of C9ORF72 at 6 months of age. White arrowheads indicate poly(GA) aggregates. Error bars represent SEM (n = 3 Non-Tg, n = 4 C9^450C, n = 3 C9^450C,C9orf72^+/−, n = 3 C9^450C,C9orf72^−/−). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.

(f-g) Immunoblotting analyses of cortical extracts from 6-month-old C9orf72 transgenic mice with reduced or loss of C9ORF72 visualized with antibodies to the autophagic marker LC3 (f). Actin was used as a loading control. Quantification of LC3B-II accumulated levels were normalized to actin (g). Error bars represent SEM (n = 6 per group). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.