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 of intrac ellular aggregates composed of the mutant polyglutamine-expanded protein. D espite the presence of aggregates in affected patients, the precise relatio nship between aggregates and disease pathogenesis is unresolved. Results fr om in vivo and in vitro studies of mutant huntingtin have lead to the hypot hesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD), We tested this hypothesis using a 293T cell culture model system that compared the frequency and toxicity of cytoplasmi c and nuclear huntingtin aggregates. We first assessed the mode of nuclear transport of N-terminal fragments of huntingtin, and show that the predicte d endogenous NLS is not functional, providing data in support of passive nu clear transport, This result suggests that proteolysis is a necessary step for nuclear entry of huntingtin, Additionally, insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino a cids was used to reverse the normal localization of these proteins. Changin g the subcellular localization of the fragments did not influence their tot al aggregate frequency. There were also no significant differences in toxic ity associated with the presence of nuclear compared with cytoplasmic aggre gates. The findings of nuclear and cytoplasmic aggregates in affected brain s, together with these in vitro data, support the nucleus and cytosol as su bcellular sites for pathogenesis in HD.