The Role Of Chaperone-mediated Autophagy In Degradation Of Huntingtin Disease Protein Htt-552

Aim: To study whether chaperone-mediated autophagy pathway degrades the wild-type and mutant huntingtin disease protein Htt-552, and to study the molecular mechanisms by which CMA recognizes and degrades Htt-552.Methods: In this study, we developed a model of HD by infecting cells with adenoviral vectors encoding the first 552 amino acids of wild-type (18Q) and mutant (100Q) Htt. Double immunofluorescence (IF) was used to measure the levels of Htt-552 expression in cells. MTT and LDH assay were used to detect the viability of cells after viral infection. The effects of macroautophagy specific inhibitor 3-methyadenine (3-MA), lysosomes inhibitor NH4Cl on Htt-552 accumulation were assessed in cells expressing Htt-552 with Western blot analysis. Western Blot analysis and double IF were used to measure the effects of two types of autophagy induced by starvation on Htt-552 degradation. The association of LAMP-2A or Hsc70 with Htt-552 was determined with double IF and co-immunoprecipitation. Western Blot analysis and double IF were used to measure the effect on Htt-552 degradation by changing the activity of CMA through expressing exogenous Hsc70 and LAMP-2A or knock-down them with siRNA. Uptake and degradation of Htt-552 through intact lysosomes was detected by Western Blot analysis. The effects of mutantion of CMA recognition sites in Htt552 mutant Htt-552 on teraction with of Htt552 with Hsc70 and degradation of Htt552 were determined with co-immunoprecipitation and Western blot analysis.Results: Expression of wild-type and mutant Htt-552 in cells was achieved by transfection of adenovirus. Htt552 distributed predominantly in the cytoplasm with relatively low levels in the nucleus. Treatment with the macroautophagy inhibitor 3-MA resulted in the augments of Htt-552 levels in cells. The lysosomal inhibitor, NH4Cl, increased the levels of Htt-552 in cells more dramatically. Serum withdrawal for 6 hours and 12 hours markedly enhanced both macroautophagy and CMA activity. The decrease in Htt-552 levels was observed after starvation for 6 or 12 hours. Co-localization of Htt-552-18Q and Htt552-100Q with LAMP-2A or Hsc70 was observed. Hsc70 was co-immunoprecipitated with Htt-552. The increase in CMA activity by expressing LAMP-2A and Hsc70 decreased the accumulation of Htt-552. Intact lysosomes isolated from the mice liver could could uptake and degrade Htt-552. The efficiency of lysosomal degradation of Htt-552 was impaired by expansion of polyQ (100Q). Mutation of putative KFERQ-like motifs in Htt552 decreased interaction with Hsc70 and degradation through CMA.Conclusion: There is a putative KFERQ recognition motif for CMA. Hsc70 interacts with Htt552 with this motif. Htt552 binds to lysosomal membranes via LAMP-2A and can be degraded by lysosomal enzymes. Expansion of polyQ tract in Htt impairs its clearance by CMA. These studies provide experimental evidence supporting that CMA plays an important role in Htt clearance. These studies also suggest a possible new stratiegy for treatment of HD.