Figure 7. p62-Mediated Clearance of Mutant Htt.

(A) Acetyl-Htt exists as a HMW species and accumulates upon lysosomal inhibition. Neuro2a cells were cotransfected with Htt480-68Q and CBP-HAT, in the presence of bafilomycin A1 (Baf A1, 100 nM) or DMSO. Triton-soluble lysates were analyzed by size-exclusion chromatography (SEC) and western blot using the anti-acetyl K444 antibody (bottom). Neuron-specific enolase (NSE) was used to monitor gel filtration column performance and loading. The horizontal MW marker (kDa) represents the MW obtained by SEC analysis as determined by the elution of globular protein standards. The vertical marker (kDa) represents the MW obtained by SDS-PAGE analysis of fractions. Western blots shown were analyzed by densitometric analysis from at least three independent experiments and normalized to NSE (top). Inset, the amount of monomeric Ac-Htt is shown at a smaller y axis.

(B) Knockdown of p62 in inducible HN10 cell lines expressing Htt573-72Q. Values are the mean ± SEM (n = 5); *p = 0.01 for p62 shRNA compared to control (Ctrl) shRNA, normalized to α-tubulin.

(C) Clearance of Ac-Htt is mediated by p62 in HN10 cells. Mutant Htt half-life was determined in untransfected (Un) or cells transfected with empty vector (Vect), CBP D-Y, CBP-HAT, control shRNA, or p62 shRNA. Half-life values of Htt573-72Q were determined as described in Experimental Procedures. The values represent the mean ± SEM (n = 5). *p < 0.01 compared to Un and Vect; *p < 0.05 compared to CBP D-Y; **p < 0.01 compared to Ctrl-shRNA.