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PubMed1998

Molecular aspects of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disease striking principally medium spiny GABAergic neurons of the caudate nucleus of the basal ganglia. It affects about one in 10,000 individuals and is transmitted in an autosomal dominant fashion. The molecular basis of the disease is expansion of the trinucleotide CAG in the first exon of a gene on chromosome four. The CAG repeats are translated to polyglutamine repeats in the expressed protein, huntingtin. The normal function of huntingtin remains incompletely characterized, but based upon recently defined protein-protein interactions, it appears to be associated with the cytoskeleton and required for neurogenesis. Huntingtin has been demonstrated to interact with such proteins as HAP1, HIP1, microtubules, GADPH, calmodulin, and an ubiquitin-conjugating enzyme. Polyglutamine expansion alters many of these interactions and leads to huntingtin aggregation and the formation of neuronal nuclear inclusions, ultimately culminating in cell death. In this review, we discuss the molecular aspects of HD, including the present understanding of huntingtin-protein interactions, studies with transgenic mice, and postulated mechanisms of huntingtin aggregation.

Animals↗
PubMed2003

Abnormal association of mutant huntingtin with synaptic vesicles inhibits glutamate release.

In Huntington disease (HD), polyglutamine expansion causes the disease protein huntingtin to aggregate and accumulate in the nucleus and cytoplasm. The cytoplasmic huntingtin aggregates are found in axonal terminals and electrophysiological studies show that mutant huntingtin affects synaptic neurotransmission. However, the biochemical basis for huntingtin-mediated synaptic dysfunction is unclear. Using electron microscopy on sections of HD mouse brains, we found that axonal terminals containing huntingtin aggregates often had fewer synaptic vesicles than did normal axonal terminals. Subcellular fractionation and electron microscopy revealed that mutant huntingtin is co-localized with huntingtin-associated protein-1 (HAP1) in axonal terminals in the brains of HD transgenic mice. Mutant huntingtin binds more tightly to synaptic vesicles than does normal huntingtin, and it decreases the association of HAP1 with synaptic vesicles in HD mouse brains. Brain slices from HD transgenic mice that had axonal aggregates showed a significant decrease in [(3)H]glutamate release, suggesting that neurotransmitter release from synaptic vesicles was impaired. Taken together, these findings suggest that mutant huntingtin has an abnormal association with synaptic vesicles and this association impairs synaptic function.

Animals↗
PubMed2001

Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease.

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein-protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.

Amino Acid Sequence↗
PubMed1999

A putative Drosophila homolog of the Huntington's disease gene.

The Huntington's disease (HD) gene encodes a protein, huntingtin, with no known function and no detectable sequence similarity to other proteins in current databases. To gain insight into the normal biological role of huntingtin, we isolated and sequenced a cDNA encoding a protein that is a likely homolog of the HD gene product in Drosophila melanogaster. We also determined the complete sequence of 43 125 contiguous base pairs of genomic DNA that encompass the Drosophila HD gene, allowing the intron-exon structure and 5'- and 3'-flanking regions to be delineated. The predicted Drosophila huntingtin protein has 3583 amino acids, which is several hundred amino acids larger than any other previously characterized member of the HD family. Analysis of the genomic and cDNA sequences indicates that Drosophila HD has 29 exons, compared with the 67 exons present in vertebrate HD genes, and that Drosophila huntingtin lacks the polyglutamine and polyproline stretches present in its mammalian counterparts. The Drosophila HD mRNA is expressed in a broad range of developmental stages and in the adult, a temporal pattern of expression similar to that observed for mammalian HD transcripts. We can discern five regions of high similarity from multiple sequence alignments between Drosophila and vertebrate huntingtins. These regions may define functionally important domains within the protein.

Amino Acid Sequence↗
PubMed1998

A human HAP1 homologue. Cloning, expression, and interaction with huntingtin.

Huntington's disease (HD) is caused by the expansion of a glutamine repeat in the protein huntingtin. The expanded glutamine repeat is thought to mediate a gain of function by causing huntingtin to abnormally interact with other proteins. We previously identified a rat huntingtin-associated protein (HAP1) that binds to huntingtin; HAP1 binds more tightly to huntingtin with an expanded glutamine repeat than to wild type huntingtin. Identification of the human homologue of HAP1 is necessary for investigation of the potential role of HAP1 in HD pathology. Here, we report the cloning of a human HAP1 homologue (hHAP) that shares 62% identity with rat HAP1 over its entire sequence and 82% amino acid identity in the putative huntingtin-binding region. The hHAP gene encodes a 4.1-kilobase transcript and a 75-kDa protein which are specifically expressed in human brain tissues. Its expression in Huntington's disease brains is reduced in parallel with a decreased expression of huntingtin. While two isoforms of rat HAP1 are expressed at similar levels in rat brain, only a single major form of hHAP is found in primate brains. In vitro binding, immunoprecipitation, and coexpression studies confirm the interaction of hHAP with huntingtin. The in vitro binding of hHAP to huntingtin is enhanced by lengthening the glutamine repeat. Despite similar binding properties of rat HAP1 and hHAP, differences in the sequences and expression of hHAP may contribute to a specific role for its interaction with huntingtin in humans.

Amino Acid Sequence↗
PubMed2005

Synaptic dysfunction in Huntington's disease: a new perspective.

Huntington's disease (HD) is caused by a polyglutamine expansion in the protein huntingtin and is characterized by intraneuronal inclusions and widespread neuronal death at the late stage of the disease. In research, most of the emphasis has been on understanding the cell death and its mechanisms. Until recently, it was believed that the vast majority, if not all, of the symptoms in HD are a direct consequence of neurodegeneration. However, increasing evidence shows that subtle alterations in synaptic function could underlie the early symptoms. It is of particular interest to understand the nature of this neuronal dysfunction. Normal huntingtin interacts with various cytoskeletal and synaptic vesicle proteins that are essential for exocytosis and endocytosis. Altered interactions of mutant huntingtin with its associated partners could contribute to abnormal synaptic transmission in HD. This review describes recent advances in understanding synaptic dysfunction in HD.

Animals↗
PubMed2006

Expression of an expanded polyglutamine domain in yeast causes death with apoptotic markers.

Huntington's disease is caused by specific mutations in huntingtin protein. Expansion of a polyglutamine (polyQ) repeat of huntingtin leads to protein aggregation in neurons followed by cell death with apoptotic markers. The connection between the aggregation and the degeneration of neurons is poorly understood. Here, we show that the physiological consequences of expanded polyQ domain expression in yeast are similar to those in neurons. In particular, expression of expanded polyQ in yeast causes apoptotic changes in mitochondria, caspase activation, nuclear DNA fragmentation and death. Similar to neurons, at the late stages of expression the expanded polyQ accumulates in the nuclei and seems to affect the cell cycle of yeast. Interestingly, nuclear localization of the aggregates is dependent on functional caspase Yca1. We speculate that the aggregates in the nuclei disturb the cell cycle and thus contribute to the development of the cell death process in both systems. Our data show that expression of the polyQ construct in yeast can be used to model patho-physiological effects of polyQ expansion in neurons.

Antioxidants↗
PubMed2001

Tissue transglutaminase selectively modifies proteins associated with truncated mutant huntingtin in intact cells.

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the N-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. However, how the expanded polyglutamine repeats of mutant huntingtin cause HD is not known. Because in vitro expanded polyglutamine repeats are excellent glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. However, an association between huntingtin and tTG or modification of huntingtin by tTG has not been demonstrated in cells. To examine the interactions between tTG and huntingtin human neuroblastoma SH-SY5Y cells were stably transfected with full-length huntingtin containing 23 (FL-Q23) (wild type) or 82 (FL-Q82) (mutant) glutamine repeats or a truncated N-terminal huntingtin construct containing 23 (Q23) (wild type) or 62 (Q62) (mutant) glutamine repeats. Aggregates were rarely observed in the cells expressing full-length mutant huntingtin, and no specific colocalization of full-length huntingtin and tTG was observed. In contrast, in cells expressing truncated mutant huntingtin (Q62) there were numerous complexes of truncated mutant huntingtin and many of these complexes co-localized with tTG. However, the complexes were not insoluble structures. Further, truncated huntingtin coimmunoprecipitated with tTG, and this association increased when tTG was activated. Activation of tTG did not result in the modification of either truncated or full-length huntingtin, however proteins that were associated with truncated mutant huntingtin were selectively modified by tTG. This study is the first to demonstrate that tTG specifically interacts with a truncated form of huntingtin, and that activated tTG selectively modifies mutant huntingtin-associated proteins. These data suggest that proteolysis of full-length mutant huntingtin likely precedes its interaction with tTG and this process may facilitate the modification of huntingtin-associated proteins and thus contribute to the etiology of HD.

Antineoplastic Agents↗
PubMed2001

Loss of cortical and thalamic neuronal tenascin-C expression in a transgenic mouse expressing exon 1 of the human Huntington disease gene.

A transgenic mouse containing the first exon of the human Huntington's disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntington's disease (HD). The present study has found that expression of the extracellular matrix glycoprotein tenascin-C appears to be unaffected in astroglial cells in wild-type and R6/2 transgenic mice that express the mutant huntingtin protein but that it is conspicuously absent in two neuronal populations within the cerebral cortex and thalamus of the R6/2 mice. Loss of tenascin-C expression begins between the fourth and eighth postnatal weeks, coincidental with the onset of abnormal behavioral phenotype and the appearance of intranuclear inclusion bodies and neuropil aggregates. By 12 weeks, R6/2 mice exhibit a complete absence of tenascin-C neuronal immunolabeling, a disappearance of cRNA probe-positive neurons across discrete cytoarchitectonic regions of the dorsal thalamus (e.g., the ventromedial, parafascicular, lateral posterior, and posterior thalamic groups) and frontal cortex, and an accompanying thalamic astrogliosis. The loss of neuronal tenascin-C expression includes structures that are known to send converging excitatory axonal projections to the caudate-putamen, the structure that is most at risk for neurodegeneration in HD. Altered neuronal expression of tenascin-C in R6/2 mice implicates altered transcriptional activities of the mutant huntingtin protein. The abnormal biochemistry and possibly abnormal activity of thalamostriate and corticostriate projection neurons may also affect abnormal neuronal activities in their primary connectional target, the neostriatum, which is severely compromised in HD.

Animals↗
PubMed2004

Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules.

Polyglutamine expansion (polyQ) in the protein huntingtin is pathogenic and responsible for the neuronal toxicity associated with Huntington's disease (HD). Although wild-type huntingtin possesses antiapoptotic properties, the relationship between the neuroprotective functions of huntingtin and pathogenesis of HD remains unclear. Here, we show that huntingtin specifically enhances vesicular transport of brain-derived neurotrophic factor (BDNF) along microtubules. Huntingtin-mediated transport involves huntingtin-associated protein-1 (HAP1) and the p150(Glued) subunit of dynactin, an essential component of molecular motors. BDNF transport is attenuated both in the disease context and by reducing the levels of wild-type huntingtin. The alteration of the huntingtin/HAP1/p150(Glued) complex correlates with reduced association of motor proteins with microtubules. Finally, we find that the polyQ-huntingtin-induced transport deficit results in the loss of neurotrophic support and neuronal toxicity. Our findings indicate that a key role of huntingtin is to promote BDNF transport and suggest that loss of this function might contribute to pathogenesis.

Animals↗
PubMed2005

HAP1 and intracellular trafficking.

Huntington's disease is caused by a polyglutamine expansion in the protein huntingtin. Several studies suggest that huntingtin and its associated protein HAP1 participate in intracellular trafficking and that polyglutamine expansion affects vesicular transport. A study now provides new evidence that HAP1 is also involved in the endocytosis of membrane receptors. These studies offer insight into the normal function of HAP1 and its involvement in Huntington's disease.

Animals↗
PubMed1997

Huntington disease: advances in molecular and cell biology.

Huntington disease is an inherited neurodegeneration, for which the associated mutation was isolated in 1993. The mutation is an expansion of a CAG trinucleotide repeat, which translates to give a polyglutamine tract at the N-terminus of a large protein, huntingtin. Neither the normal nor the pathogenic functions of this protein have been identified, but it is clear that pathogenesis is mediated through the expanded polyglutamine tract within the protein, and that polyglutamine is toxic to cells. A number of proteins which interact with the N-terminal region of huntingtin have been isolated, but this has not, so far, yielded a rationale for pathogenesis. Huntingtin is found in areas of the brain that degenerate in this disease but is also associated with pathogenic inclusions in Alzheimer disease and Pick disease. It is possible that Huntington disease has pathogenic mechanisms in common with these other neurodegenerative diseases, and that the mechanism may relate to the formation of abnormal, cytoskeletal-associated, inclusions within cells.

Animals↗
PubMed2000

Transplanted fetal striatum in Huntington's disease: phenotypic development and lack of pathology.

Neural and stem cell transplantation is emerging as a potential treatment for neurodegenerative diseases. Transplantation of specific committed neuroblasts (fetal neurons) to the adult brain provides such scientific exploration of these new potential therapies. Huntington's disease (HD) is a fatal, incurable autosomal dominant (CAG repeat expansion of huntingtin protein) neurodegenerative disorder with primary neuronal pathology within the caudate-putamen (striatum). In a clinical trial of human fetal striatal tissue transplantation, one patient died 18 months after transplantation from cardiovascular disease, and postmortem histological analysis demonstrated surviving transplanted cells with typical morphology of the developing striatum. Selective markers of both striatal projection and interneurons such as dopamine and c-AMP-related phosphoprotein, calretinin, acetylcholinesterase, choline acetyltransferase, tyrosine hydroxylase, calbindin, enkephalin, and substance P showed positive transplant regions clearly innervated by host tyrosine hydroxylase fibers. There was no histological evidence of immune rejection including microglia and macrophages. Notably, neuronal protein aggregates of mutated huntingtin, which is typical HD neuropathology, were not found within the transplanted fetal tissue. Thus, although there is a genetically predetermined process causing neuronal death within the HD striatum, implanted fetal neural cells lacking the mutant HD gene may be able to replace damaged host neurons and reconstitute damaged neuronal connections. This study demonstrates that grafts derived from human fetal striatal tissue can survive, develop, and are unaffected by the disease process, at least for 18 months, after transplantation into a patient with HD.

Corpus Striatum↗
PubMed2004

Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo.

Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of clathrin light chains.

Actins↗
PubMed2001

The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably, transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an imperfect (Gln-Ala)(38) tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full-length htt from lymphoblastoid cells. The expression of CA150, measured immunohistochemically, was markedly increased in human HD brain tissue compared with normal age-matched human brain tissue, and CA150 showed aggregate formation with partial colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small, but statistically significant, amount of the variability in the onset age. Our data suggest that abnormal expression of CA150, mediated by interaction with polyglutamine-expanded htt, may alter transcription and have a role in HD pathogenesis.

Age of Onset↗
PubMed2004

Expanded huntingtin activates the c-Jun terminal kinase/c-Jun pathway prior to aggregate formation in striatal neurons in culture.

Huntington's disease (HD) is an autosomal neurodegenerative disorder, caused by expansion of a glutamine repeat in the Huntingtin protein. Pathogenesis in HD includes the cytoplasmic cleavage of Huntingtin and release of an amino-terminal fragment capable of nuclear localization, where expanded-Huntingtin (Exp-Htt) might lead to aberrant transcriptional regulation, neuronal dysfunction and degeneration. Recent evidence, from hippocampal cell lines, also implicates altered interaction of Exp-Htt with components of the c-Jun N-terminal kinase (JNK) cascade. However, there is yet no proven implication of the JNK/c-Jun module in degeneration of striatal neurons, the more vulnerable cell population, in HD. In the present study, we used primary striatal neurons in culture to analyze c-Jun activation by Exp-Htt. Green fluorescent protein (GFP)-tagged exon 1 of human Huntingtin either in its normal (25Q, normal-Htt) or expanded (103Q, Exp-Htt) version was transiently transfected in these cells. We first set out, in our conditions, the time course of striatal degeneration produced by Exp-Htt, and found it occurred rapidly. At 48 h post-transfection, 60% of striatal neurons expressing Exp-Htt had apoptotic characteristics including DNA fragmentation and neuritic retraction. Most of these neurons also showed nuclear aggregates of GFP-Exp Htt. Kinetics of c-Jun activation were tested in transfected cells using immunocytochemical detection of phospho-c-Jun. We found a significant activation and induction of c-Jun in Exp-Htt but not normal-Htt-transfected neurons. Of interest, these events occurred prior to nuclear translocation of Exp-Htt. Finally, overexpression of a dominant negative version of c-Jun, as well as pharmacological inhibition of JNK strongly protected against DNA fragmentation and neuritic retraction induced by Exp-Htt. Thus our data suggest that c-Jun activation and induction, is an early event in the pathogenesis of HD, occurring prior to formation of nuclear aggregates of Exp-Htt.

Animals↗
PubMed2000

Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by polyglutamine expansion in the disease protein, huntingtin. In HD patients and transgenic mice, the affected neurons form characteristic ubiquitin-positive nuclear inclusions (NIs). We have established ecdysone-inducible stable mouse Neuro2a cell lines that express truncated N-terminal huntingtin (tNhtt) with different polyglutamine lengths which form both cytoplasmic and nuclear aggregates in a polyglutamine length- and inducer dose-dependent manner. Here we demonstrate that newly synthesized polyglutamine-expanded truncated huntingtin interacts with members of Hsp40 and Hsp70 families of chaperones in a polyglutamine length-dependent manner. Of these interacting chaperones, only Hdj-2 and Hsc70 frequently (Hdj-2 > Hsc70) co-localize with both the aggregates in the cellular model and with the NIs in the brains of HD exon 1 transgenic mice. However, Hdj-2 and Hsc70 do not co-localize with cytoplasmic aggregates in the brains of transgenic mice despite these chaperones being primarily localized in the cytoplasmic compartment. This strongly suggests that the chaperone interaction and their redistribution to the aggregates are two completely different phenomena of the cellular unfolded protein response. This unfolded protein response is also evident from the dramatic induction of Hsp70 on expression of polyglutamine-expanded protein in the cellular model. Transient overexpression of either Hdj-1 or Hsc70 suppresses the aggregate formation; however, suppression efficiency is much higher in Hdj-1 compared with Hsc70. Overexpression of Hdj-1 and Hsc70 is also able to protect cell death caused by polyglutamine-expanded tNhtt and their combination proved to be most effective.

Animals↗