In vitro evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease

A unifying feature of the CAG expansion diseases is the formation ofintracellular aggregates composed of the mutant polyglutamine-expandedprotein. Despite the presence of aggregates in affected patients, theprecise relationship between aggregates and disease pathogenesis isunresolved. Results from in vivo and in vitro studies of mutant huntingtinhave lead to the hypothesis that nuclear localization of aggregates iscritical for the pathology of Huntington's disease (HD). We tested thishypothesis using a 293T cell culture model system that compared thefrequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. Wefirst assessed the mode of nuclear transport of N-terminal fragments ofhuntingtin, and show that the predicted endogenous NLS is not functional,providing data in support of passive nuclear transport. This resultsuggests that proteolysis is a necessary step for nuclear entry ofhuntingtin. Additionally, insertion of nuclear import or export sequencesinto huntingtin fragments containing 548 or 151 amino acids was used toreverse the normal localization of these proteins. Changing the subcellularlocalization of the fragments did not influence their total aggregatefrequency. There were also no significant differences in toxicityassociated with the presence of nuclear compared with cytoplasmicaggregates. The findings of nuclear and cytoplasmic aggregates in affectedbrains, together with these in vitro data, support the nucleus and cytosolas subcellular sites for pathogenesis in HD.