Fig. 3

CYP46A1 overexpression promotes the activation of autophagy, independently of the UPS system. a N2a cells were transfected with pEGFP-HTTQ74, co-transfected with pEGFP-HTTQ74 and CYP46A1. Western blots were probed with rabbit anti-LC3B, rabbit anti-p62/SQSTM1, and mouse anti-actin. b The densitometric analysis showed that CYP46A1 expression leads to a significant increase in the LC3B-II levels and c to a significant reduction in the SQSTM1/p62 levels, both compared to the experimental control. The results with chloroquine inhibition of autophagy follow the same trend, suggesting a robust autophagy activation upon CYP46A1 overexpression. d This activation is also supported by a significant increase in the autophagic net flux. e Representative western blot probed for GFP of protein lysates from N2a cells of the different experimental conditions, with and without the proteasome inhibitor MG132 (5 M). The western blot was also probed for tubulin, and poly-ubiquitin to highlight the UPS inhibition. f The densitometric analysis showed that CYP46A1 expression decreases GFP levels (HTT-MUT), with and without ubiquitin–proteasome system inhibition. (n = 4 independent experiments, Unpaired Student’s t-test; one-way ANOVA with Bonferroni’s multiple comparisons test, *P < 0.05; **P < 0.001; ***P < 0.0001)