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Huntingtin is a cytoplasmic protein of unknown function that associates with vesicle membranes and microtubules. Its protein interactions suggest that huntingtin has a role in endocytosis and organelle transport. In this study we sought to identify factors that regulate the transport of huntingtin in striatal neurons, which are the cells most affected in Huntington's disease. In clonal striatal cells derived from fusions of neuroblastoma and embryonic striatal neurons, huntingtin localization is diffuse and slightly punctate in the cytoplasm. When these neurons were differentiated by treatment with forskolin, huntingtin redistributed to perinuclear regions, discrete puncta along plasma membranes, and branch points and terminal growth cones in neurites. Huntingtin staining overlapped with clathrin, a coat protein involved in endocytosis. Immunoblot analysis of subcellular membrane fractions separated by differential centrifugation confirmed that huntingtin immunoreactivity in differentiated neurons markedly increased in membrane fractions enriched with clathrin and with huntingtin-interacting protein 1. Dopamine treatment altered the subcellular localization of huntingtin and increased its expression in clathrin-enriched membrane fractions. The dopamine-induced changes were blocked by the D1 antagonist SCH 23390 and were absent in a clonal cell line lacking D1 receptors. Results suggest that the transport of huntingtin and its co-expression in clathrin and huntingtin-interacting protein 1-enriched membranes is influenced by activation of adenylyl cyclase and stimulation of dopamine D1 receptors.

Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin. Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae. The huntingtin-HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin-HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain.

Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, -4 and -5 enzymes of Saccharomyces cerevisiae. This protein is highly expressed in brain and a slightly larger protein recognized by an anti-E2-25K polyclonal antibody is selectively expressed in brain regions affected in HD. The huntingtin-E2-25K interaction is not obviously modulated by CAG length. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD.

Apoptosis has recently been recognized as a mode of cell death in Huntington disease (HD). Apopain, a human counterpart of the nematode cysteine protease death-gene product, CED-3, has a key role in proteolytic events leading to apoptosis. Here we show that apoptotic extracts and apopain itself specifically cleave the HD gene product, huntingtin. The rate of cleavage increases with the length of the huntingtin polyglutamine tract, providing an explanation for the gain-of-function associated with CAG expansion. Our results show that huntingtin is cleaved by cysteine proteases and suggest that HD might be a disorder of inappropriate apoptosis.

The mutation underlying Huntington disease (HD) is CAG expansion in the first exon of the HD gene. In order to investigate the role of CAG expansion in the pathogenesis of HD, we have produced transgenic mice containing the full length human HD cDNA with 44 CAG repeats. By 1 year, these mice have no behavioral abnormalities and morphometric analysis at 6 (one animal) and 9 (two animals) months age revealed no changes. Despite high levels of mRNA expression, there was no evidence of the HD gene product in any of these transgenic mice. In vitro transfection studies indicated that the inclusion of 120 bp of the 5' UTR in the cDNA construct and the presence of a frameshift mutation at nucleotide 2349 prevented expression of the HD cDNA. These findings suggest that the pathogenesis of HD is not mediated through DNA-protein interaction and that presence of the RNA transcript with an expanded CAG repeat is insufficient to cause the disease. Rather, translation of the CAG is crucial for the pathogenesis of HD. In contrast to that seen in humans, the CAG repeat in these mice was remarkably stable in 97 meioses. This suggests that genomic sequences may play a critical role in influencing repeat instability.

We have previously cloned and characterized the murine homologue of the Huntington disease (HD) gene and shown that it maps to mouse chromosome 5 within a region of conserved synteny with human chromosome 4p16.3. Here we present a detailed comparison of the sequence of the putative promoter and the organization of the 5' genomic region of the murine (Hdh) and human HD genes encompassing the first five exons. We show that in this region these two genes share identical exon boundaries, but have different-size introns. Two dinucleotide (CT) and one trinucleotide intronic polymorphism in Hdh and an intronic CA polymorphism in the HD gene were identified. Comparison of 940-bp sequence 5' to the putative translation start site reveals a highly conserved region (78.8% nucleotide identity) between Hdh and the HD gene from nucleotide -56 to -206 (of Hdh). Neither Hdh nor the HD gene have typical TATA or CCAAT elements, but both show one putative AP2 binding site and numerous potential Sp1 binding sites. The high sequence identity between Hdh and the HD gene for approximately 200 bp 5' to the putative translation start site indicates that these sequences may play a role in regulating expression of the Huntington disease gene.

Huntington's disease (HD) is associated with the expansion of a CAG trinucleotide repeat in a novel gene. We have assessed 360 HD individuals from 259 unrelated families and found a highly significant correlation (r = 0.70, p = 10(-7)) between the age of onset and the repeat length, which accounts for approximately 50% of the variation in the age of onset. Significant associations were also found between repeat length and age of death and onset of other clinical features. Sib pair and parent-child analysis revealed that the CAG repeat demonstrates only mild instability. Affected HD siblings had significant correlations for trinucleotide expansion (r = 0.66, p < 0.001) which was not apparent for affected parent-child pairs.