The Influence of Huntingtin Protein Size on Nuclear Localization and Cellular Toxicity

Huntington disease is an autosomal dominant neurodegenerative disorder caused by the pathological expansion of a polyglutamine tract. In this study we directly assess the influence of protein size on the formation and subcellular localization of huntingtin aggregates. We have created numerous deletion constructs expressing successively smaller fragments of huntingtin and show that these smaller proteins containing 128 glutamines form both intranuclear and perinuclear aggregates. In contrast, larger NH2-terminal fragments of huntingtin proteins with 128 glutamines form exclusively perinuclear aggregates. These aggregates can form in the absence of endogenous huntingtin. Furthermore, expression of mutant huntingtin results in increased susceptibility to apoptotic stress that is greater with decreasing protein length and increasing polyglutamine size. As both intranuclear and perinuclear aggregates are clearly associated with increased cellular toxicity, this supports an important role for toxic polyglutamine-containing fragments forming aggregates and playing a key role in the pathogenesis of Huntington disease

The isolation and characterization of Huntingtin interacting proteins

Huntington Disease (HD) is an autosomal dominant, neurodegenerative disorder with onset normally occurring at around 40 years of age. This devastating disease is the result of the expression of a polyglutamine tract greater than 35 in a protein with unknown function. The underlying mutation in HD places it in a category of neurodegenerative diseases along with seven other diseases, all of which have widespread expression of the protein with an abnormally long polyglutamine tract, but have disease specific neurodegeneration. Yeast two-hybrid screens were used in an attempt to further elucidate the function of the HD gene product, huntingtin. The results help decipher the biochemical signals that may contribute to the neuronal specific cell death seen in HD patients. Three different cDNA fragments spanning greater than 80 % of the HD cDNA were used to screen for Huntingtin Interacting Proteins (HIPs). Only the N-terminal region of huntingtin produced positive interacting proteins. Of the 14 clones isolated 12 were identical and given the name HIP1. HIP2 and H1P3 were isolated as individual positive clones. Assessment of the expression pattern of each of the HIPs reveal them all to be expressed in most tissues, but preferentially expressed in human brain and subcellular regions similar to that of huntingtin. HJP1 is a novel human gene that shares identity with the yeast Sla2p/End4 protein that is involved in the endocytotic pathway and maintenance of the cytoskeleton. The interaction between HIPl and huntingtin appears to be influenced by the size of the polyglutamine tract, in a manner whereby the larger the CAG tract, the lower the affinity the two proteins have for each other. Biochemical and in vitro assessment of huntingtin and HEP1 place the two proteins in the same cell compartments, providing further evidence that these proteins interact in vivo. HIP2 shares complete identity with the previously cloned bovine E2-25K ubiquitin conjugating enzyme. This protein plays an essential role in the ubiquitin proteolytic pathway, suggesting that huntingtin is degraded via this catabolic mechanism. As part of the investigation into this interaction, huntingtin was shown to coimmunoprecipitate with ubiquitin, without preference for the mutant form of huntingtin. This demonstration of the ubiquitination of huntingtin preceded the description of huntingtin-ubiquitin coimmunoreactive intranuclear inclusions. Presently, four of the eight expanded polyglutamine dependent diseases have been shown to have these ubiquitin positive staining intranuclear inclusions. HEP3 is a protein that is highly expressed in the brain, specifically the caudate nucleus and putamen, regions significantly affected in HD patients. The homology of HIP3 with a membrane associated protein in yeast, Akr1p, places it at the membrane with huntingtin. The involvement of Akr1p in receptor mediated endocytosis is consistent with the role of the SLA2/END4 gene in endocytosis. The data presented in this thesis provides clues into the role huntingtin plays within a cell. It supports data that huntingtin is associated with synaptic vesicles and cytoskeletal components of the cellular membrane. The presence of an expanded polyglutamine tract may alter the ability of huntingtin to either bind its normal cellular target.Medicine, Faculty ofMedical Genetics, Department ofGraduat