A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity (original) (raw)
Figure 4
sCAG neurotoxic effect is dependent on Dicer and Ago proteins.
A. Dicer knockdown inhibits the generation of sCAGs produced by the expression of 80*CAG HTT-e1. sCAG levels were normalized to RNU66 levels. GFP blots indicate the expression of the HTT-constructs (n = 3; interaction p-value = 0.000138; F = 46.220). B. In the same experiments, cell viability and caspase 9 cleavage analysis show that Dicer depletion mitigates cell death induced by expanded HTT (n = 5; interaction p-value = 0.000135; F = 18.263). C. Ago2 depletion mitigates the toxicity of sRNA obtained from mutant HTT expressing cells (n = 3; interaction p-value = 0.011; F = 10.821). D. sCAG efficiently associate to Ago2 in vivo. _HTT_-expressing constructs were transfected on cells stably expressing Flag-Ago2. Flag IP demonstrate that sCAG binds to Ago2 complex. No significant binding was detected in control IP experiments (α-V5). The plot shows the mean ratio of sCAG levels in FLAG IP vs. control V5 IP (n = 3; *p<0.05). E. The expression of Flag-Ago2 in cells depleted for endogenous Ago2 partially, but significantly, rescued CAG-expanded HTT toxic effect (n = 3; *p<0,05). Values represent the mean of the ratio expanded-HTT sRNA toxicity vs non-expanded-HTT sRNA toxicity ± SEM in each experimental condition. Toxicity levels are referred to the control cells lacking HTT expression. In A. B. and C., values represent the mean fold change with respect to the control, non-transfected cells ± SEM. Cells were processed 24 hours after double transfection in all the experiments.