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 intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have lead to the hypothesis 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 cytoplasmic and nuclear huntingtin aggregates. We first assessed the mode of nuclear transport of N-terminal fragments of huntingtin, and show that the predicted endogenous NLS is not functional, providing data in support of passive nuclear 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 acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. The findings of nuclear and cytoplasmic aggregates in affected brains, together with these in vitro data, support the nucleus and cytosol as subcellular sites for pathogenesis in HD.
Hackam
A S
AS
Centre for Molecular Medicine and Therapeutics, 980 West 28th Avenue, Vancouver, BC, Canada V5Z 4H4 and Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
Singaraja
R
R
Zhang
T
T
Gan
L
L
Hayden
M R
MR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000209:etiology* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008954:Models, Biological; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; C086055:HTT protein, human; D000071058:Huntingtin Protein; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010446:Peptide Fragments