The Expression And Catabolism Of Caspase 6-cleaved Huntingtin-586 And Its Influence On Cell Functions

Objective: To study the activation of autophagy by over-expressing of N-terminal Htt-586 and the contribution of the autophagy/lysosomal pathway and ubiquitin-proteasomal pathway on the metabolism of the wild-type and mutant N-terminal Htt-586; and to investigate the effects of Htt-586 on the function of cellular organelles.Methods: In this study, we have established the stable PC12 cellular models of Huntington's disease using lentivirus to express Caspase 6 cleavage products Htt-586 with 18Q (wild-type) or 100Q (mutant). Proteins related to autophagy, LC3 and Beclin1 were determined with Western Blot analysis and double immunofluorescence. The effects of autophagy specific inhibitor 3-methyadenine (3-MA), autophagy activator rapamycin, and the proteasome inhibitors lactacystin and MG132 on Htt accumulation were assessed in PC12 cells expressing Htt-586 with Western Blot analysis. The Golgi complex morphology in PC12 cells were measured in the presence of wild-type Htt-586 or mutant Htt-586 with double immunofluorescence. The expression level of brain-derived neurotrophic factor (BDNF) was detected in PC12 cells expressing Htt-586 with ELISA analysis, Western Blot analysis and double immunofluorescence.Results: Wild-type and mutant Htt-586 were stably expressed in highly differentiated PC12 cells by lentivirus transfection. Treatment with proteasome inhibitors (lactacystin and MG132) resulted in the augments of Htt-586 levels in cells, especially in those expressing mutant Htt-586(100Q). The Golgi complex morphology was shrinked in PC12 cells expressing mutant Htt-586(100Q). The levels of pro-BDNF was increased in the PC12 cells expressing mutant Htt-586(100Q), while mature BDNF was decreased in culture medium of PC12 cells expressing mutant Htt-586(100Q).Conclusion: In this study, we have established stable PC12 cellular models of Huntington's disease using lentivirus-mediated expression of Caspase-6 cleavage fragment Htt-586 with 18Q (wild type) or 100Q (mutant). The ubiquitin-proteasome pathway may be the major pathway in the catabolism of Htt-586. Over-expressing mutant Htt-586 in PC12 cells can reduce the size of Golgi complex and inhibit the maturation of BDNF. These studies will open a new avenue to study the physiological and pathological functions of N-terminal Htt fragments endogenously produced in the cells.