Shang-Zhi Xu and Chun- Juan Shan
The present study was to explore whether overload o f cholesterol elicited by Mutant huntingtin (mhtt) may directly induce transfected PC12 cell death. PC12 cells were transfected with huntingtin flanked with normal range of poly-Q (26Q) or mhtt ( 82Q). Expression of mhtt was turned on by addition of doxycycline (Dox). PC12 + Dox, Q26 + Do x and Q82 + Dox were continuously cultured for 60 days to monitor cell survival rate. The amount o f cholesterol induced by mhtt was quantitatively analyzed by fluorescent intensities from filipin sta ining under confocal microscopy. Several key protein levels of huntingtin, caveolin1, and sterol regulatory element-binding proteins (SREBP-2) were determined by western blotting. Our results in dicated that PC12 cells with Q82 were induced death around 25 days. Total numbers of survival cel ls were decreased to 40-50% by the end of 60 days in comparison with PC12 parental cells and Q26 cells. Cholesterol within Q82 cells started to accumulate from Day 18, with cholesterol overloaded around plasma membrane on Day 39, and eventually plasma membrane was collapsed and induce d Q82 cells death on Day 60. Huntingtin and caveolin1 proteins in Q82 cells were greatly increa sed on 60 days; SREBP2 in Q82 cells was significantly cleaved and activated. Our results su ggest that mhtt leads to cholesterol accumulation by over activated SREBP2, which stimulates choleste rol synthesis. The overload of cholesterol causes plasma membrane fragility and eventually ind uces transfected PC12 cell death.
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