Figure 1.

Rilmenidine enhances autophagy in wild-type mice. (A) Endogenous LC3-II levels were measured in stable inducible PC12 cells 48 h after switching on expression of either huntingtin exon 1 with 23 polyglutamine repeats (htt-23Q) or 74 polyglutamine repeats (htt-74Q) in the presence or absence of rilmenidine (for the final 24 h). Actin was used as a loading control. (B) LC3-II levels were measured using fluorescent intensity of the bands by Li-Cor Odyssey. Results are shown as a percentage of control in each individual cell line (n = 5, *P < 0.05 by t-test). The increase in LC3-II levels in rilmenidine treated, htt-74Q is not significantly greater than in htt-23Q treated cells, and possible differences may be due to variations between the clonal cell lines used. (C) LC3-II levels in muscle lysates from rilmenidine-treated and placebo-treated wild-type mice after 24 weeks of treatment. Western blots were also probed for tubulin as a loading control (D) Densitometric analysis of LC3-II-levels relative to tubulin. Control condition is set to 100%. Error bars show SEM (*P = 0.036, t-test, n = 4 for rilmenidine, n = 5 for control). (E) In cultured primary cortical neurons, LC3-II levels were assessed by western blot. Two exposures are shown to allow comparison of weaker bands in non-bafilomycin A1-treated lanes (−Baf A1) and stronger bands in bafilomycin A1-treated lanes without saturation. (F) Densitometric quantification of LC3-II levels relative to actin in triplicate experiments. (*P < 0.05 by t-test). Effect of rilmenidine treatment on phosphorylation of downstream mTOR targets was investigated by western blotting, (G) phosphorylated p70 S6 kinase levels, (H) phosphorylated S6 ribosomal protein and (I) phosphorylated 4EBP1. In these experiments, rapamycin treatment was used as a control for the inactivation of mTOR where the effects of treatment can be clearly seen.