Construction, Expression Of GFP-Huntingtin CDNA In Mammalian Cells And Effects Of Huntingtin Expression On Lysosomal Enzymes

Objective: To construct plasmids expressing GFP-huntingtin and to observe expression of constructed plasmids in mammalian cells as well as to study the effects of exogenous Htt on cellular lososomal enzymes.Methods: Plasmids expressing truncated huntingtin were constructed, denoted as pcDNA3-GFP/Htt969-18 (expressing wild type truncated htt N terminal amino acids with 18 glutamine repeat) and pcDNA3-GFP/Htt969-100 (expressing mutant type truncated htt N terminal amino acids with 100 glutamine repeat). Plasmids were transfected by method of lipofection. Western blot analysis and immunofluorescence were used to confirm the expression of huntingtin. Immunofluorescence was used to study effects of over-expressed exogenous Htt on cytoplasmic lysosomal enzyme cathepsin B and D .Results: Plasmids were successfully constructed, which were confirmed by polymerase chain reaction, restrictive cleavage and DNA sequencing. Over-expression of exogenous Htt in cells significantly increased levels of the lysosomal enzymes cathepsin B and D.Conclusions: Exogenous Htt significantly increased levels of the lysosomal enzymes cathepsin B and D. Autophagy/lososome pathway may play a critical role in the processing of Htt.