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PubMed2006

Aggregate-centered redistribution of proteins by mutant huntingtin.

Huntingtin is a widely expressed 350-kDa cytosolic multidomain of unknown function. Aberrant expansion of the polyglutamine tract located in the N-terminal region of huntingtin results in Huntington's disease. The presence of insoluble huntingtin inclusions in the brains of patients is one of the hallmarks of Huntington's disease. Experimentally, both full-length huntingtin and N-terminal fragments of huntingtin with expanded polyglutamine tracts trigger aggregate formation. Here, we report that upon the formation of huntingtin aggregates; endogenous cytosolic huntingtin, Hsc70/Hsp70 (heat shock protein and cognate protein of 70kDa) and syntaxin 1A become aggregate-centered. This redistribution suggests that these proteins are eventually depleted and become unavailable for normal cellular function. These results indicate that the cellular targeting of several key proteins are altered in the presence of mutant huntingtin and suggest that aggregate depletion of these proteins may underlie, in part, the sequence of disease progression.

Animals↗
PubMed2004

The serum- and glucocorticoid-induced kinase SGK inhibits mutant huntingtin-induced toxicity by phosphorylating serine 421 of huntingtin.

Huntington's disease (HD) is caused by abnormal polyglutamine (polyQ) expansion in the protein huntingtin. We have previously demonstrated the importance of the insulin-like growth factor I (IGF-1)/Akt pathway in HD. Indeed, upon IGF-1 activation, Akt phosphorylates polyQ-huntingtin at serine 421 and abrogates its toxicity. In addition, we have demonstrated that Akt is altered in the brain of HD patients. Here, we investigate the role of the serum- and glucocorticoid-induced kinase (SGK) in HD. We show that SGK phosphorylates huntingtin at serine 421 and that phosphorylation can protect striatal neurons against polyQ-huntingtin-induced toxicity. We find that SGK levels are increased in the brain of HD patients. Using a cellular model of HD, we demonstrate that the SGK dysregulation induced by polyQ-huntingtin occurs via the p38/MAPK pathway. Collectively, our results strongly suggest the involvement of SGK in HD and further imply that IGF-1 downstream signalling is a key transduction pathway that regulates the toxicity of huntingtin.

Animals↗
PubMedDate not supplied

The early cellular pathology of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder that affects about one in 10,000 individuals in North America. The genetic defect responsible for the disease is an expansion of a CAG repeat that encodes a polyglutamine tract in the expressed protein, huntingtin. The disease is characterized by involuntary movements, cognitive impairment, and emotional disturbance. Despite the widespread expression of huntingtin, the brains of HD patients show selective neuronal loss in the striatum and the deep layers of the cerebral cortex. Recent studies have shown that polyglutamine expansion causes huntingtin to aggregate, to accumulate in the nucleus, and to interact abnormally with other proteins. Several cellular and animal models for HD have revealed that intranuclear accumulation of mutant huntingtin and the formation of neuropil aggregates precede neurological symptoms and neurodegeneration. Intranuclear huntingtin may affect nuclear function and the expression of genes important for neuronal function, whereas neuropil aggregates may interfere with neuritic transport and function. These early pathological events, which occur in the absence of neurodegeneration, may contribute to the neurological symptoms of HD and ultimately lead to neuronal cell death.

Animals↗
PubMed2006

Is Huntington's a glutamine storage disease?

Huntington's disease is a neurological disorder caused by the expansion of a polyglutamine tract in the protein huntingtin. Several other neurological diseases also result from the expansion of polyglutamine regions in different proteins. Despite intense efforts, no definitive biochemical or physiological role for huntingtin has been described, nor has a function been assigned to the polyglutamine region in unaffected individuals. This article presents the hypothesis that polyglutamine expansions within huntingtin and other polyglutamine proteins provide a function in and of themselves. Incorporating multiple glutamine residues into a protein during synthesis, and releasing them during protein turnover, may represent a means of minimizing interruptions in brain levels of glutamine and glutamate during periods of malnutrition. The number and variety of different proteins containing polyglutamine expansions can be interpreted as a series of evolutionary "experiments" toward a nontoxic form for glutamine storage.

Animals↗
PubMed2003

Inclusion formation in Huntington's disease R6/2 mouse muscle cultures.

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion in the number of glutamine repeats in the N-terminal region of the huntingtin protein. Nuclear and cytoplasmic aggregates of the N-terminal portion of huntingtin have been found in the brains of HD patients and the brains and non-neuronal tissues of the R6/2 HD transgenic mouse. We have cultured myoblasts and myotubes from transgenic R6/2 mice and littermate controls to investigate the formation of these inclusions in post mitotic cells. Huntingtin immunoreactivity was intense in differentiating, desmin positive myoblasts and myotubes from both control and R6/2 mice suggesting that it may play a role in myotube differentiation. Following differentiation huntingtin and ubiquitin positive aggregates were observed in R6/2 but not control cultures. After 3 weeks in differentiation medium cytoplasmic huntingtin and ubiquitin immunoreactive aggregates were observed in non-myotube cells, while nuclear huntingtin aggregates were seen in a proportion of myotubes after 6 weeks. Growth in the absence of serum resulted in a marked increase in the number of R6/2 myotubes containing nuclear inclusions after 6 weeks demonstrating that environmental factors influenced huntingtin aggregate formation in these cells. Consequently, cultured myotubes from R6/2 mice may be a useful post mitotic cell culture model to study both the biochemical consequences of huntingtin aggregates and the factors that may influence aggregate formation.

Animals↗
PubMed2003

Decreased cAMP response element-mediated transcription: an early event in exon 1 and full-length cell models of Huntington's disease that contributes to polyglutamine pathogenesis.

Huntington's disease (HD) is one of nine neurodegenerative diseases caused by an expanded polyglutamine (polyQ) tract within the disease protein. To characterize pathways induced early in HD, we have developed stable inducible PC12 cell lines expressing wild-type or mutant forms of huntingtin exon 1 fragments or the full-length huntingtin protein. Three cAMP response element-binding protein (CREB)-binding protein-dependent transcriptional pathways, regulated by cAMP response element (CRE), retinoic acid response element, and nuclear factor kappaB, show abnormalities in our exon 1 cell model. Of these, the CRE pathway shows the earliest disruption and is significantly down-regulated as early as 12 h following mutant htt transgene induction. This pathway is also the only one of the three that is similarly perturbed in our full-length HD model, where it is also down-regulated at an early time point, compatible with observations in HD brains. Reduced CRE-dependent transcription may contribute to polyQ disease pathogenesis because overexpression of transcriptionally active CREB, but not an inactive form of the protein, is able to protect against polyQ-induced cell death and reduce aggregation.

Animals↗
PubMed2004

SUMO modification of Huntingtin and Huntington's disease pathology.

Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that 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.

Animals↗
PubMed2002

Protein aggregation in Huntington's disease.

The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.

Animals↗
PubMed2004

Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.

Huntington's disease is caused by an abnormal polyglutamine expansion within the protein huntingtin and is characterized by microscopic inclusion bodies of aggregated huntingtin and by the death of selected types of neuron. Whether inclusion bodies are pathogenic, incidental or a beneficial coping response is controversial. To resolve this issue we have developed an automated microscope that returns to precisely the same neuron after arbitrary intervals, even after cells have been removed from the microscope stage. Here we show, by survival analysis, that neurons die in a time-independent fashion but one that is dependent on mutant huntingtin dose and polyglutamine expansion; many neurons die without forming an inclusion body. Rather, the amount of diffuse intracellular huntingtin predicts whether and when inclusion body formation or death will occur. Surprisingly, inclusion body formation predicts improved survival and leads to decreased levels of mutant huntingtin elsewhere in a neuron. Thus, inclusion body formation can function as a coping response to toxic mutant huntingtin.

Animals↗
PubMed2006

Huntingtin inhibits caspase-3 activation.

Huntington's disease results from a mutation in the HD gene encoding for the protein huntingtin. The function of huntingtin, although beginning to be elucidated, remains largely unclear. To probe the prosurvival function of huntingtin, we modulate levels of wild-type huntingtin in a number of cellular and in vivo models. Huntingtin depletion resulted in caspase-3 activation, and overexpression of huntingtin resulted in caspase-3 inhibition. Additionally, we demonstrate that huntingtin physically interacts with active caspase-3. Interestingly, mutant huntingtin binds active caspase-3 with a lower affinity and lower inhibitory effect on active caspase-3 than does wild-type huntingtin. Although reduction of huntingtin levels resulted in caspase-3 activation in all conditions examined, the cellular response was cell-type specific. Depletion of huntingtin resulted in either overt cell death, or in increased vulnerability to cell death. These data demonstrate that huntingtin inhibits caspase-3 activity, suggesting a mechanism whereby caspase-mediated huntingtin depletion results in a detrimental amplification cascade leading to further caspase-3 activation, resulting in cell dysfunction and cell death.

Animals↗
PubMed1993

Characterization and localization of the Huntington disease gene product.

The recent identification of the Huntington's disease (HD) gene, enabled us to synthesize oligopeptides corresponding with the carboxy-terminal end of the predicted HD-gene (IT15) product. Immunobiochemcial studies with polyclonal antibodies directed against this synthetic peptide (position 3114-3141) on lymphoblastoid cells from normal individuals and patients with Huntington disease, revealed the presence of a protein (huntingtin) with a molecular mass of approximately 330 kDa. Immunocytochemical studies showed a cytoplasmic localization of huntingtin in various cell types including neurons. In most of the neuronal cells the protein was also present in the nucleus. No difference in molecular mass or intracellular localization was found between normal and mutant cells.

Amino Acid Sequence↗
PubMed2005

Neuronal intranuclear inclusions and neuropil aggregates in HdhCAG(150) knockin mice.

We studied the development of neuronal intranuclear inclusions (NIIs), neuropil aggregates (NAs), and expression of expanded repeat polyglutamine protein in the HdhCAG(150) knockin mouse model of Huntington's disease (HD). Diffuse nuclear localization of huntingtin protein (htt) was noted initially within striatal neurons at approximately 28 weeks, followed by the development of striatal htt immunoreactive NIIs by approximately 40 weeks. Striatal NIIs were observed initially in clusters within the matrix compartment but subsequently became diffusely distributed throughout the striatum. In the oldest animals (107 weeks), NIIs were enlarged and diffuse nuclear htt immunoreactivity reduced. Expression of ubiquitin immunoreactive NIIs paralleled but lagged behind the expression of htt immunoreactive NIIs. Abundant NIIs were found by approximately 75 weeks in layers 3 and 4 of somatosensory cortex and in layer 2 of piriform cortex. In the oldest animals, greater than 100 weeks, some NIIs were found in many brain regions. NAs were found mainly within the globus pallidus and substantia nigra, perhaps reflecting expression in striatal terminals. Cyclic AMP response element binding protein (CBP) was not localized to NIIs, arguing against gross sequestration of this transcriptionally active protein. Comparison of the relative levels of a common polyglutamine epitope in HdhCAG(150) knockin and hprtCAG(146) knockin mice shows greater expression of the polyglutamine epitope in the phenotypically less aggressive HdhCAG(150) knockin line. HdhCAG(150) knockin mice may be a model of early pathologic changes in HD.

Aging↗
PubMed2001

Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila.

Proteins with expanded polyglutamine repeats cause Huntington's disease and other neurodegenerative diseases. Transcriptional dysregulation and loss of function of transcriptional co-activator proteins have been implicated in the pathogenesis of these diseases. Huntington's disease is caused by expansion of a repeated sequence of the amino acid glutamine in the abnormal protein huntingtin (Htt). Here we show that 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.

Acetylation↗
PubMed1999

Life without huntingtin: normal differentiation into functional neurons.

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmitter-gated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.

Animals↗
PubMed2005

Unraveling a role for dopamine in Huntington's disease: the dual role of reactive oxygen species and D2 receptor stimulation.

Huntington's disease (HD), an inherited neurodegenerative disorder, results from an abnormal polyglutamine extension in the N-terminal region of the huntingtin protein. This mutation leads to protein aggregation and neurotoxicity. Despite its widespread expression in the brain and body, mutated huntingtin causes selective degeneration of striatal projection neurons. In the present study, we investigate the role of dopamine (DA) in this preferential vulnerability. Using primary cultures of striatal neurons transiently expressing GFP-tagged-exon 1 of mutated huntingtin, we show that low doses of DA (100 microM) act synergistically with mutated huntingtin to activate the proapoptotic transcription factor c-Jun. Surprisingly, DA also increases aggregate formation of mutated huntingtin in all cellular compartments, including neurites, soma, and nuclei. DA-dependent potentiation of c-Jun activation was reversed by ascorbate, a reactive oxygen species (ROS) scavenger, and SP-600125, a selective inhibitor of the c-Jun N-terminal kinase (JNK) pathway. By contrast, DA effects on aggregate formation were reversed by a selective D2 receptor antagonist and reproduced by a D2 agonist. Similarly, striatal neurons from D2 knockout mice showed no effect of DA on aggregate formation. Blocking ROS production, JNK activation, or D2 receptor stimulation significantly reversed DA aggravation of mutated huntingtin-induced striatal death. The combined treatment with the ROS scavenger and D2 antagonist totally reversed DA's effects on mutated huntingtin-induced striatal death. Thus, the present results provide insights into the cellular mechanisms that govern striatal vulnerability in HD and strongly support a dual role of JNK activation and D2 receptor signaling in this process.

Analysis of Variance↗
PubMed2005

Compounds blocking mutant huntingtin toxicity identified using a Huntington's disease neuronal cell model.

Neuronal cell death in HD is believed to be largely a dominant cell-autonomous effect of the mutant huntingtin protein. We previously developed an inducible PC12 cell model which expresses an N-terminal huntingtin fragment with an expanded poly Q repeat (N63-148Q) under the control of the tet-off system. In order to evaluate the ability of compounds to protect against mutant huntingtin toxicity in our model, we measured LDH released by dead cells into the medium. We have now screened the library of 1040 compounds from the NINDS Custom Collection as part of a National Institute of Neurological Disorders and Stroke (NINDS) collaborative project. Each positive compound was tested at 3-8 concentrations. Five compounds significantly attenuated mutant huntingtin (htt)-induced LDH release without affecting the expression level of huntingtin and independent of effect on aggregates. We also tested a broad spectrum caspase inhibitor Z-VAD-fmk and previously proposed candidate compounds. This cell model can provide a method to screen potential therapeutic compounds for treating Huntington's disease.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.

Amino Acid Sequence↗
PubMed2003

Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes.

Huntingtin protein is mutated in Huntington disease. We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild-type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE. In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE-containing genes involved in the maintenance of the neuronal phenotype. Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes.

Animals↗