Effects Of Mutant Huntingtin-552 On Glutamate Transporter GLT-1 In Astrocytes Are Reversed By Rapamycin

Object: Study the effect of N-terminal Htt-552 on the expression and functions of glutamate transporters in primary cultured rat astrocyteMethod: In this study, we have established an astrocytes model of Huntingtin's disease using adenoviral expression of caspase2/3 cleavage products Htt-552 with 18Q (wild type) or 100Q (mutant). Using western blotting or realtime PCR, we detected the protein and mRNA levels of glutamate transporters (GLT-1 and GLAST). In addition, we detected the glutamate uptake of astrocytes by liquid scintillation spectrometer. The effects of autophagy activator rapamycin and autophagy specific inhibitor 3-methyadenine (3-MA) on mutant Htt's accumulation were assessed in astrocytes expressing Htt-552 by western blotting. At the same time, we detected the expression of GLT-1 and the glutamate uptake of astrocytes with or without the treatment of rapamycin or 3-MA.Results: Wild type and mutant Htt-552 were sucessfully expressed mediated adenoviral expression system in primary cultured rat astrocytes. Western blotting and realtime PCR analysis revealed decreased expression of GLT-1, but not GLAST. The decreased GLT-1 contributed to decreased glutamate uptake of astrocytes. After the clearance of mutant Htt by rapamycin, the autophagy stimulator, the expression of GLT-1 and the glutamate uptake of astrocytes were both recovered. Conclusion:Mutant Htt-552 fragments could decrease the expression of glutamate transporter GLT-1, which contributed to the decline in the glutamate uptake by astrocytes. The expression of GLT-1 and the glutamamte uptake of astrocytes both recovered after mutant Htt cleared by autophagy.