Atg4b-Dependent Autophagic Flux Alleviates Huntington’s Disease Progression
Figure 5
AZD8055, an mTOR inhibitor stimulates neuronal autophagy by inducing Atg4b-dependent autophagy flux in cultured neurons.
A) Schematic representation of the lentiviral backbones used to measure neuronal autophagy flux. tTa was used as a control, mCherryGFPLC3 to measure autophagy flux by monitoring the different fluorescent signal and Atg4b WT or mutant (C74A) were used to block autophagy. B) Left: representative images for the detection of neuronal autophagy flux using mCherry-GFP-LC3 in cultured cortical neurons. Bafilomycin was used as negative control as it blocks acidification of autophagosome, blocking autophagy flux. Right, quantification of mCherry-GFP-LC3 shows how AZD8055 significantly increases autophagy flux (see materials and methods for the quantification of the autophagy flux). C) Representative images of cultured cortical neurons non-infected (NI) or transduced with tTa, Atg4b WT or Atg4b C74A. Note the similar Neurofilament (green) distribution in the different conditions, indicating no toxic impact for the Atg4b WT and Atg4b C74A overexpression (red). D) Left: biochemical analysis of LC3 autophagy markers for the conditions in (C). Both Atg4b WT and Atg4b C74A affected LC3 lipidation (LC3-II form) and induced p62 accumulation. Right: biochemical analysis of the effective mTOR inhibition and p62 degradation upon AZD8055 treatment. Note how the AZD8055-dependent p62 degradation is abolished once Atg4b WT is overexpressed. Far right: quantification of p62 normalized to β-actin shows a significant increase of p62 level upon Atg4b overexpression, as compared with cells transduced with tTa. AZD8055 treatment led to a significant increase in autophagy flux observed by a decrease in p62 level. However it was inefficient in the presence of Atg4b overexpression. E) Left: Representative images of cultured cortical neurons transduced with tTa/mCherry-GFP-LC3 or Atg4b/mCherry-GFP-LC3 with and without AZD8055 treatment. Right: quantitative analysis for neuronal LC3 autophagy. AZD8055 significantly increased LC3 autophagy flux in tTa/mCherry-GFP-LC3 neurons and did not have an effect in Atg4b/mCherry-GFP-LC3 neurons. N = 50 images from 3 independent preparations; median values ± SEM; Student’s t test: *p<0.05, **p<0.01 and ***p<0.001 Bars: (B and E) 10 µm, (C) 30 µm.
