The gene defective in Huntington's disease encodes a protein of unknown function: huntingtin. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking (DiFiglia, 1995).
The cause of neurodegeneration in Huntington's disease (HD) is unknown. Patients with HD have an expanded NH2-terminal polyglutamine region in huntingtin. An NH2-terminal fragment of mutant huntingtin was localized to neuronal intranuclear inclusions (NIIs) and dystrophic neurites (DNs) in the HD cortex and striatum, which are affected in HD, and polyglutamine length influenced the extent of huntingtin accumulation in these structures. Ubiquitin was also found in NIIs and DNs, which suggests that abnormal huntingtin is targeted for proteolysis but is resistant to removal. The aggregation of mutant huntingtin may be part of the pathogenic mechanism in HD (DiFiglia, 1997).
Aggregation of N-terminal mutant huntingtin within nuclear inclusions and dystrophic neurites occurs in the cortex and striatum of Huntington disease (HD) patients and may be involved in neurodegeneration. The prevalence of inclusions and dystrophic neurites has been examined in the cortex and striatum of 15 adult onset HD patients who had mild to severe striatal cell loss (grades 1, 2 or 3) using an antibody that detects the N-terminal region of huntingtin. Nuclear inclusions were more frequent in the cortex than the striatum and were sparse or absent in the striatum of patients with low-grade striatal pathology. Dystrophic neurites occurred in both regions. Patients with low-grade striatal pathology had numerous fibers with immunoreactive puncta and large swellings within the striatal neuropil, the subcortical white matter, and the internal and external capsules. In the globus pallidus of 3 grade 1 cases, N-terminal huntingtin markedly accumulated in the perinuclear cytoplasm and in some axons but not in the nucleus. Findings suggest that in the earlier stages of HD, accumulation of N-terminal mutant huntingtin occurs in the cytoplasm and is associated with degeneration of the corticostriatal pathway (Sapp, 1999).
Huntington's disease is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. Mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients (Davies, 1997).
Yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) have been produced in a developmental and tissue-specific manner identical to that observed in Huntington's disease. YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or micro-aggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration (Hodgson, 1999).
Several lines of mice have been developed that are transgenic for exon 1 of the HD gene containing an expanded CAG sequence. These mice exhibit a defined neurological phenotype along with neuronal changes that are pathognomonic for the disease. Neuronal intranuclear inclusions have been demonstrated in these lines, but no evidence has been found for neurodegeneration. All lines of these mice develop a late onset neurodegeneration within the anterior cingulate cortex, dorsal striatum, and of the Purkinje neurons of the cerebellum. Dying neurons characteristically exhibit neuronal intranuclear inclusions, condensation of both the cytoplasm and nucleus, and ruffling of the plasma membrane while maintaining ultrastructural preservation of cellular organelles. These cells do not develop blebbing of the nucleus or cytoplasm, apoptotic bodies, or fragmentation of DNA. Neuronal death occurs over a period of weeks, not hours. Degenerating cells of similar appearance are found within these same regions in brains of patients who had died with HD. It is therefore suggested that the mechanism of neuronal cell death in both HD and a transgenic mouse model of HD is neither by apoptosis nor by necrosis (Turmaine, 2000).
Despite its widespread expression, mutant huntingtin induces selective neuronal loss in striatal neurons. In mutant mice expressing HD repeats, the production and aggregation of N-terminal huntingtin fragments preferentially occur in HD-affected neurons and their processes and axonal terminals. N-terminal fragments of mutant huntingtin form aggregates and induce neuritic degeneration in cultured striatal neurons. N-terminal mutant huntingtin also binds to synaptic vesicles and inhibits their glutamate uptake in vitro. The specific processing and accumulation of toxic fragments of N-terminal huntingtin in HD-affected striatal neurons, especially in their neuronal processes and axonal terminals, may contribute to the selective neuropathology of HD (Li, 2000).
Huntington's disease (HD) is characterized by the selective loss of striatal projection neurons. In early stages of HD, neurodegeneration preferentially occurs in the lateral globus pallidus (LGP) and substantia nigra (SN), two regions in which the axons of striatal neurons terminate. In mice expressing full-length mutant huntingtin and modeling early stages of HD, neuropil aggregates form preferentially in the LGP and SN. The progressive formation of these neuropil aggregates follows intranuclear accumulation of mutant huntingtin and becomes prominent from 11 to 27 months after birth. Neuropil aggregates, but no intranuclear inclusions, were observed in the LGP and SN, suggesting that huntingtin aggregates are formed in the axons of striatal projection neurons. In the LGP and SN, degenerated axons were observed in which huntingtin aggregates were associated with dark, swollen organelles that resemble degenerated mitochondria. Neuritic aggregates also form in cultured striatal neurons expressing mutant huntingtin, block protein transport in neurites, and cause neuritic degeneration before nuclear DNA fragmentation occurs. These findings suggest that the early neuropathology of HD originates from axonal dysfunction and degeneration associated with huntingtin aggregates (Li, 2001).
Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease. Mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. The co-aggregation of mutant and wild-type htt fragments. Mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. These data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis (Busch, 2003).
Cytoplasmic huntingtin aggregates 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, it was 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 (3H)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 (Li, 2004).
Huntingtin, the protein product of the Huntington's disease gene, associates with vesicle membranes and microtubules in neurons. Analysis of axonal transport with a stop-flow, double crush ligation approach in rat sciatic nerve showed that full length huntingtin (350 kDa) and an N-terminal cleavage product (50 kD) were increased within 6-12 h on both the proximal and distal sides of the crush site when compared with normal unligated nerve. The huntingtin associated protein HAP 1 and the retrograde motor protein dynein also accumulated on both sides of the crush, whereas the vesicle docking protein SNAP-25 was elevated only proximally. The cytoskeletal protein alpha-tubulin was unaffected. The rapid anterograde accumulation of huntingtin and HAP 1 is compatible with their axonal transport on vesicular membranes. Retrograde movement of both proteins, as seen by accumulation distal to the nerve crush, may be necessary for their degradation at the soma or for a function in retrograde membrane trafficking (Block-Galarza, 1997).
Huntington's and Kennedy's disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates (Szebenyi, 2003).
The pathological hallmark of HD is the degeneration of subsets of neurons, primarily those in the striatum and neocortex. Specific morphological markers of affected cells have not been identified in patients with HD, although a unique intranuclear inclusion was recently reported in neurons of transgenic animals expressing a construct encoding the N-terminal part (including the glutamine repeat) of huntingtin. In order to understand the importance of this finding, comparable nuclear abnormalities were sought in autopsy material from patients with HD. In all 20 HD cases examined, anti-ubiquitin and N-terminal huntingtin antibodies identified itranuclear inclusions in neurons and the frequency of these lesions correlated with the length of the CAG repeat in IT15. In addition, examination of material from the related HD-like triplet repeat disorder, dentatorubral and pallidoluysian atrophy, also revealed intranuclear neuronal inclusions. These findings suggest that intranuclear inclusions containing protein aggregates may be common feature of the pathogenesis of glutamine repeat neurodegenerative disorders (Becher, 1999).
To distinguish between 'loss of function' and 'gain of function' models of HD, the murine HD homolog Hdh was inactivated by gene targeting. Mice heterozygous for Hdh inactivation were phenotypically normal, whereas homozygosity resulted in embryonic death. Homozygotes displayed abnormal gastrulation at embryonic day 7.5 and were resorbing by day 8.5. Thus, huntingtin is critical early in embryonic development, before the emergence of the nervous system. That Hdh inactivation does not mimic adult HD neuropathology suggests that the human disease involves a gain of function (Duyao, 1995).
Huntington's disease is an incurable neuropsychiatric disease associated with CAG repeat expansion within a widely expressed gene that causes selective neuronal death. To understand its normal function, a targeted disruption in exon 5 of Hdh (Hdhex5), the murine homolog of the HD gene, has been created. Homozygotes die before embryonic day 8.5, initiate gastrulation, but do not proceed to the formation of somites or to organogenesis. Mice heterozygous for the Hdhex5 mutation display increased motor activity and cognitive deficits. Neuropathological assessment of two heterozygous mice shows significant neuronal loss in the subthalamic nucleus. These studies show that the HD gene is essential for postimplantation development and that it may play an important role in normal functioning of the basal ganglia (Nasir, 1995).
Targeted disruption of the mouse huntingtin gene (Hdh), carried out to examine the normal role of huntingtin, shows that this protein is functionally indispensable, since nullizygous embryos become developmentally retarded and disorganized, and die between days 8.5 and 10.5 of gestation. Based on the observation that the level of the regionalized apoptotic cell death in the embryonic ectoderm, a layer expressing the Hdh gene, is much higher than normal in the null mutants, it is proposed that huntingtin is involved in processes counterbalancing the operation of an apoptotic pathway (Zeitlin, 1995).
Inactivation of the mouse homologue of the Huntington disease gene (Hdh) results in early embryonic lethality. To investigate the normal function of Hdh in the adult and to evaluate current models for Huntington disease (HD), the Cre/loxP site-specific recombination strategy was used to inactivate Hdh expression in the forebrain and testis, resulting in a progressive degenerative neuronal phenotype and sterility. On the basis of these results, it is proposed that huntingtin is required for neuronal function and survival in the brain and that a loss-of-function mechanism may contribute to HD pathogenesis (Dragatsis, 2000).
A molecular base of the HD gene transcription has not been elucidated as yet. Two proteins, HDBP1 and HDBP2, which bind to the promoter region for the HD gene have been identified using a yeast one-hybrid system. Amino acid sequence analysis of the proteins deduced the presence of nuclear localization signal, nuclear export signal, zinc finger, serine/proline-rich region, and highly conserved C-terminal region. In vitro DNA binding assay indicates that the C-terminal conserved regions of the proteins are responsible for binding to the unique promoter DNA sequences of the HD gene. The DNA sequence protected from DNase I digestion is a 7-bp consensus sequence (GCCGGCG), which resides in triplicate at intervals of 13 bp within and proximal to the 20-bp direct repeat sequences of the HD promoter region. The mutation of 7-bp consensus sequence abolishes the HD promoter function in a neuronal cell line (IMR32). In human cultured cells, ectopically expressed green fluorescent protein-fused HDBP1 and HDBP2 localized in the cytoplasm, but both proteins totally shift from cytoplasm to nucleus by the treatment with an inhibitor of the nuclear export, leptomycin B, and mutagenesis of the putative nuclear export signals. Taken together, HDBP1 and HDBP2 are novel transcription factors shuttling between nucleus and cytoplasm and bind to the specific GCCGGCG, which is an essential cis-element for HD gene expression in neuronal cells (Tanaka, 2004).
To explore polyQ-mediated neuronal toxicity, the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins have been expressed in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produces touch insensitivity in young adults. Noticeably, only 28 +/- 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction (Parker, 2001).
Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. A brain-specific protein that associates with huntingtin has been designated huntingtin-associated protein (HAP1). Selective neuronal localizations of HAP1 is described. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy (Li, 1996).
A protein HAP1 has been identified that binds to huntingtin in a glutamine repeat length-dependent manner. HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. The interaction of HAP1 with p150 Glued was examined. Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo. Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD (Engelender, 1997).
Huntingtin-associated protein is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, it was found that HAP1 also binds to dynactin P150Glued (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bind to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitate with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 is enriched in rat brain microtubules and comigrates with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalizes with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. It is proposed that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport (Li, 1998).
The Huntington's disease mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt in vitro causes cell death. Other in vitro studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. N-htt fragments consistent with the size produced by caspase 3 cleavage in vitro are found to be resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred in vitro and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD (Kim, 2001).
Huntingtin has several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspase-cleaved htt, it has been demonstrated that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. Caspase-cleaved htt is detected in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments are detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD (Wellington, 2002).
Huntington's disease (HD) mouse models that express N-terminal huntingtin fragments show rapid disease progression and have been used for developing therapeutics. However, light microscopy reveals no significant neurodegeneration in these mice. It remains unclear how mutant huntingtin induces neurodegeneration. Using caspase staining, TUNEL labelling, and electron microscopy, it has been observed that N171-82Q mice, which express the first 171 aa of mutant huntingtin, display more degenerated neurons than do other HD mouse models. The neurodegeneration was evidenced by increased immunostaining for glial fibrillary acidic protein and ultrastructural features of apoptosis. R6/2 mice, which express exon 1 of mutant huntingtin, showed dark, nonapoptotic neurons and degenerated mitochondria associated with mutant huntingtin. In HD repeat knock-in mice (HdhCAG150), which express full-length mutant huntingtin, degenerated cytoplasmic organelles were found in both axons and neuronal cell bodies in association with mutant huntingtin that was not labeled by an antibody to huntingtin amino acids 342-456. Transfection of cultured cells with mutant huntingtin revealed that an N-terminal huntingtin fragment (amino acids 1-208 plus a 120 glutamine repeat) causes a greater increase in caspase activity than does exon 1 huntingtin and longer huntingtin fragments. These results suggest that context-dependent neurodegeneration in HD may be mediated by different N-terminal huntingtin fragments. In addition, this study has identified neurodegenerative markers for the evaluation of therapeutic treatments in HD mouse models (Yu, 2003).
Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. Interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels have been reported. These associations give rise to a tonic G protein inhibition of the channels. The effects on the CSP chaperone system are reported of huntingtin fragments (exon 1) with [huntingtin(exon1/exp)] and without [huntingtin(exon1/nonexp)] the occurance of expanded polyglutamine (polyQ) tracts. In vitro huntingtin(exon1/exp) sequesters CSP and blocks the association of CSP with G proteins. In contrast, huntingtin(exon1/nonexp) does not interact with CSP and does not alter the CSP/G protein association. Similarly, co-expression of huntingtin(exon1/exp) with CSP and N-type calcium channels eliminates CSP's tonic G protein inhibition of the channels, while coexpression of huntingtin(exon1/nonexp) does not alter the robust inhibition promoted by CSP. These results indicate that CSP's modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts (Miller, 2003).
Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, since the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. These data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease (Ravikumar, 2004).
A pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation (Steffan, 2004).
Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogenesis of HD. Supporting this view, administration of histone deacetylase (HDAC) inhibitors has been shown to rescue lethality and photoreceptor neurodegeneration in a Drosophila model of polyglutamine disease. To further explore the therapeutic potential of HDAC inhibitors, preclinical trials were conducted with suberoylanilide hydroxamic acid (SAHA), a potent HDAC inhibitor, in the R6/2 HD mouse model. SAHA crosses the blood-brain barrier and increases histone acetylation in the brain. SAHA can be administered orally in drinking water when complexed with cyclodextrins. SAHA dramatically improves the motor impairment in R6/2 mice, clearly validating the pursuit of this class of compounds as HD therapeutics (Hockly, 2003).
Pathogenesis in HD appears to include the cytoplasmic cleavage of htt and release of an amino-terminal fragment capable of nuclear localization. Potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) have been investigated including aggregation, protein-protein interactions, and transcription. httex1p was found to coaggregate with p53 in inclusions generated in cell culture and to interact with p53 in vitro and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters, p21(WAF1/CIP1) and MDR-1. httex1p was also found to interact in vitro with CREB-binding protein (CBP) and mSin3a, and CBP to localize to neuronal intranuclear inclusions in a transgenic mouse model of HD. These results raise the possibility that expanded repeat htt causes aberrant transcriptional regulation through its interaction with cellular transcription factors which may result in neuronal dysfunction and cell death in HD (Steffan, 2000).
The polyglutamine-containing domain of Htt, Htt exon 1 protein (Httex1p), directly binds the acetyltransferase domains of two distinct proteins: CREB-binding protein (CBP) and p300/CBP-associated factor (P/CAF). In cell-free assays, Httex1p also inhibits the acetyltransferase activity of at least three enzymes: p300, P/CAF and CBP. Expression of Httex1p in cultured cells reduces the level of the acetylated histones H3 and H4, and this reduction can be reversed by administering inhibitors of histone deacetylase (HDAC). In vivo, HDAC inhibitors arrest ongoing progressive neuronal degeneration induced by polyglutamine repeat expansion, and they reduce lethality in two Drosophila models of polyglutamine disease. These findings raise the possibility that therapy with HDAC inhibitors may slow or prevent the progressive neurodegeneration seen in Huntington's disease and other polyglutamine-repeat diseases, even after the onset of symptoms (Steffan, 2001).
The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions. These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP. In vitro and in cells it has been shown that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner. FRET experiments indicate that these toxic forms are generated through a conformational rearrangement in huntingtin. Interaction of toxic huntingtin with the benign polyQ repeat of TBP structurally destabilizes the transcription factor, independent of the formation of insoluble coaggregates. Hsp70/Hsp40 chaperones interfere with the conformational change in mutant huntingtin and inhibit the deactivation of TBP. These results outline a molecular mechanism of cellular toxicity in polyQ disease and can explain the beneficial effects of molecular chaperones (Schaffar, 2004).
Cystamine, a small disulfide-containing chemical, is neuroprotective in a transgenic mouse and a Drosophila model of Huntington's disease (HD) and decreases huntingtin aggregates in an in vitro model of HD. The mechanism of action of cystamine in these models is widely thought to involve inhibition of transglutaminase mediated cross-linking of mutant huntingtin in the process of aggregate formation/stabilization. Cystamine, both in vitro and in a transgenic mouse model of HD (R6/2), increases levels of the cellular antioxidant L-cysteine. Several oxidative stress markers increase in HD brain. Evidence is provided of oxidative stress in mouse HD by demonstrating compensatory responses in R6/2 HD brains. Age-dependent increases in forebrain glutathione (GSH) were found, and increased levels of transcripts coding for proteins involved in GSH synthesis and detoxification pathways, as revealed by quantitative PCR analysis. Given the general importance of oxidative stress as a mediator of neurodegeneration, it is proposed that an increase in brain L-cysteine levels could be protective in HD. Furthermore, cystamine was dramatically protective against 3-nitropropionic acid-induced striatal injury in mice. It is suggested that cystamine's neuroprotective effect in HD transgenic mice results from pleiotropic effects that include transglutaminase inhibition and antioxidant activity (Fox, 2004).
The mutant huntingtin protein is presumed to acquire a toxic gain of function that is detrimental to striatal neurons in the brain. However, loss of a beneficial activity of wild-type huntingtin may also cause the death of striatal neurons. Wild-type huntingtin up-regulates transcription of brain-derived neurotrophic factor (BDNF), a pro-survival factor produced by cortical neurons that is necessary for survival of striatal neurons in the brain. This beneficial activity of huntingtin is lost when the protein becomes mutated, resulting in decreased production of cortical BDNF. This leads to insufficient neurotrophic support for striatal neurons, which then die. Restoring wild-type huntingtin activity and increasing BDNF production may be therapeutic approaches for treating HD (Zuccato, 2001).
Polyglutamine expansion causes Huntington disease (HD) and at least seven other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin with an expanded glutamine tract are able to aggregate and accumulate in the nucleus. Although intranuclear huntingtin affects the expression of numerous genes, the mechanism of this nuclear effect is unknown. This study reports that huntingtin interacts with Sp1, a transcription factor that binds to GC-rich elements in certain promoters and activates transcription of the corresponding genes. In vitro binding and immunoprecipitation assays show that polyglutamine expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth factor receptor and suppresses its transcriptional activity in cultured cells. Overexpression of Sp1 reduces the cellular toxicity and neuritic extension defects caused by intranuclear mutant huntingtin. These findings suggest that the soluble form of mutant huntingtin in the nucleus may cause cellular dysfunction by binding to Sp1 and thus reducing the expression of Sp1-regulated genes (Li, 2002).
Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. This study reports that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms (Dunah, 2002).
Huntington's disease (HD) is a neurodegenerative disease caused by expansion of a polyglutamine tract within the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis; recent evidence suggests a defect in Sp1-mediated transcription. Chromatin immunoprecipitation (ChIP) assays followed by real-time PCR were used to quantify the association of Sp1 with individual genes. Despite normal protein levels and normal to increased overall nuclear binding activity, Sp1 has decreased binding to specific promoters of susceptible genes in transgenic HD mouse brain, in striatal HD cells, and in human HD brain. Genes whose mRNA levels are decreased in HD have abnormal Sp1-DNA binding, whereas genes with unchanged mRNA levels have normal levels of Sp1 association. Moreover, the altered binding seen with Sp1 is not found with another transcription factor, NF-Y. These findings suggest that mutant huntingtin dissociates Sp1 from target promoters, inhibiting transcription of specific genes (Chen-Plotkin, 2006).
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