Activation of autophagy via mTOR inhibition rescues HD phenotype in CStS from R6/2 mice.

<p>A) Effective mTOR inhibition and autophagy induction in CStS upon AZD8055 treatment at DIV21. Left: biochemical detection of downstream signaling molecules in the mTOR pathway show how phospho-S6 (P-S6) and phospho-Akt (P-Akt) are efficiently blocked upon AZD8055 treatment. Biochemical detection of LC3 and p62 show how AZD8055 is inducing autophagy in brain slices by increasing the LC3 conversion and by decreasing p62 levels. Right: quantification of LC3II/LC3I conversion ratios and p62 levels in the AZD8055-treated and non-treated condition. Note how AZD8055 is efficiently inducing autophagy in both WT and R6/2 slices. B) Electron microscopy (EM) of representative WT and R6/2 brain slices treated with AZD8055 from DIV14 to DIV16. Up: autophagosomes (white arrows) and autolysosomes (white arrowheads) in AZD8055 R6/2 untreated and treated slices. Note the zoom of one autophagosome containing different structures (yellow arrow). Down: representative autophagosomes containing cytosolic contents (yellow arrows) in WT and R6/2 slices with and without AZD8055 treatment. Right: quantification of the number of autophagosome/100 µm² indicates an increase upon AZD8055 treatment. C) Representative images of R6/2 and WT slices that were stained with mHtt (green), DARPP-32 (red) and NeuF (green). AZD8055 treatment led to obvious decrease in mHtt staining as well as amelioration in DARPP-32 distribution. D) Quantification of mHtt aggregation from C). AZD8055 treatment led to a reduction of striatal mHtt aggregate number as well as size. E) Quantification of mHtt levels in total lysates of DIV21 R6/2 slices treated with AZD8055 or with vehicle by AGERA. F) Quantification of C) shows that AZD8055 leads to a rescue of DARPP-32 and NeuF levels. G) Total protein levels were not changed by mTOR inhibition with AZD8055 in DIV21 slices. N = 10 images from 5 independent slices; median values ± SEM; Student’s t test: *p<0.05, **p<0.01 and ***p<0.001 Bar: (B) 1 µm and 0.3 µm, (C) 30 µm.