Normal Function Of Huntingtin Research Articles

Allele-Specific Knockdown of Mutant Huntingtin Protein via Editing at Coding Region Single Nucleotide Polymorphism Heterozygosities.

Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

IKKβ slows Huntington’s disease progression in R6/1 mice

Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKβ, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington's disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKβ on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKβ phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKβ to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKβ knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKβ in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKβ knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKβ is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKβ is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKβ may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.

Mechanism suppressing H3K9 trimethylation in pluripotent stem cells and its demise by polyQ-expanded huntingtin mutations

Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.

Potential function for the Huntingtin protein as a scaffold for selective autophagy.

Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51-like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.

Deletion of the huntingtin proline-rich region does not significantly affect normal huntingtin function in mice.

The N-terminus of Huntingtin, the protein encoded by the Huntington's disease gene, contains a stretch of polyglutamine residues that is expanded in Huntington's disease. The polyglutamine stretch is flanked by two conserved protein domains in vertebrates: an N1-17 domain, and a proline-rich region (PRR). The PRR can modulate the structure of the adjacent polyglutamine stretch, and is a binding site for several interacting proteins. To determine the role of the PRR in Huntingtin function, we have generated a knock-in allele of the mouse Huntington's disease gene homolog that expresses full-length normal huntingtin lacking the PRR. Mice that are homozygous for the huntingtin PRR deletion are born at the normal Mendelian frequency, suggesting that the PRR is not required for essential huntingtin functions during embryonic development. Moreover, adult homozygous mutants did not exhibit any significant differences from wild-type controls in general motor function and motor learning. However, 18 month-old male, but not female, homozygous PRR deletion mutants exhibited deficits in the Morris water task, suggesting that age-dependent spatial learning and memory may be affected in a sex-specific fashion by the huntingtin PRR deletion.

Decreased BDNF Levels Are a Major Contributor to the Embryonic Phenotype of Huntingtin Knockdown Zebrafish

Huntington's disease (HD) is an autosomal dominant, neurodegenerative condition caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein huntingtin. Genetic and transgenic studies suggest that the mutation causes disease predominantly via gain-of-function mechanisms. However, loss of normal huntingtin function resulting from the polyglutamine expansion might also contribute to the pathogenesis of HD. Here, we have studied the effects of huntingtin knockdown in zebrafish using morpholino antisense oligonucleotides, as its huntingtin orthologue has 70% amino acid identity with the human protein. Reduced huntingtin levels did not impact on gastrulation and early development, but caused massive apoptosis of neuronal cells by 24 hpf. This was accompanied by impaired neuronal development, resulting in small eyes and heads and enlargement of brain ventricles. Older huntingtin knockdown fish developed lower jaw abnormalities with most branchial arches missing. Molecular analysis revealed that BDNF expression was reduced by approximately 50%. Reduction of BDNF levels by injection of a BDNF morpholino resulted in phenotypes very similar to those seen in huntingtin knockdown zebrafish. The phenotypes of both huntingtin- and BDNF-knockdown zebrafish showed significant rescue when treated with exogenous BDNF protein. This underscores the physiological importance of huntingtin as a regulator of BDNF production and suggests that loss of BDNF is a major cause of the developmental abnormalities seen with huntingtin knockdown in zebrafish. Increasing BDNF expression may represent a useful strategy for Huntington's disease treatment.

Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts

BackgroundThe polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington's disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD.ResultsTo systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO). The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines. We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells. These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in Hdh-KO cells when compared to Hdh-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results.ConclusionFunctional analysis of generated Htt-null MEF cells revealed that Htt plays a direct role in Ca2+ signaling by modulating InsP3R sensitivity to InsP3. The genome-wide transcriptional profiling of Htt-null cells yielded novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD.

Psychopathology in Verified Huntington's Disease Gene Carriers

Psychopathology in Verified Huntington's Disease Gene Carriers

Hypothalamic–endocrine aspects in Huntington's disease

Huntington's disease (HD) is a hereditary and fatal disorder caused by an expanded CAG triplet repeat in the HD gene, resulting in a mutant form of the protein huntingtin. Wild-type and mutant huntingtin are expressed in most tissues of the body but the normal function of huntingtin is not fully known. In HD, the neuropathology is characterized by intranuclear and cytoplasmic inclusions of huntingtin aggregates, and cell death primarily in striatum and cerebral cortex. However, hypothalamic atrophy occurs at early stages of HD with loss of orexin- and somatostatin-containing cell populations. Several symptoms of HD such as sleep disturbances, alterations in circadian rhythm, and weight loss may be due to hypothalamic dysfunction. Endocrine changes including increased cortisol levels, reduced testosterone levels and increased prevalence of diabetes are found in HD patients. In HD mice, alterations in the hypothalamic-pituitary-adrenal axis occurs as well as pancreatic beta-cell and adipocyte dysfunction. Increasing evidence points towards important pathology of the hypothalamus and the endocrine system in HD. As many neuroendocrine factors are secreted into the cerebrospinal fluid, blood and urine, it is possible that their levels may reflect the disease state in the central nervous system. Investigating neuroendocrine changes in HD opens up the possibility of finding biomarkers to evaluate future therapies for HD, as well as of identifying novel targets for therapeutic interventions.

Wild‐type huntingtin protects neurons from excitotoxicity

Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase-mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor-mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over-expressing full-length wild-type huntingtin were dramatically protected from apoptosis and caspase-3 activation compared with cultured striatal neurons from non-transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase-3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full-length huntingtin, and the generation of a specific N-terminal caspase cleavage product of huntingtin. In vivo, over-expression of wild-type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor-mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtin's anti-apoptotic function will contribute to our understanding of the pathogenesis of Huntingdon's disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.

Normal huntingtin function: an alternative approach to Huntington's disease

Several neurological diseases are characterized by the altered activity of one or a few ubiquitously expressed cell proteins, but it is not known how these normal proteins turn into harmful executors of selective neuronal cell death. We selected huntingtin in Huntington's disease to explore this question because the dominant inheritance pattern of the disease seems to exclude the possibility that the wild-type protein has a role in the natural history of this condition. However, even in this extreme case, there is considerable evidence that normal huntingtin is important for neuronal function and that the activity of some of its downstream effectors, such as brain-derived neurotrophic factor, is reduced in Huntington's disease.

Huntington's disease: from gene to potential therapy

Huntington's disease (HD) is a progressive, late-onset neurodegenerative illness with autosomal dominant inheritance that affects one in 10 000 individuals in Western Europe. The disease is caused by a polyglutamine repeat expansion located in the N-terminal region of the huntingtin protein. The mutation is likely to act by a gain of function, but the molecular mechanisms by which it leads to neuronal dysfunction and cell death are not yet known. The normal function of huntingtin in cell metabolism is also unclear. There is no therapy for HD. Research on HD should help elucidate the pathogenetic mechanism of this illness in order to develop successful treatments to prevent or slow down symptoms. This article presents new results in HD research focusing on in vivo and in vitro model systems, potential molecular mechanisms of HD, and the development of therapeutic strategies.

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. J. Neurosci. Res. 54:301–308, 1998. © 1998 Wiley-Liss, Inc.

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.

Differential distribution of the normal and mutated forms of huntingtin in the human brain.

Huntington's disease is an inherited disorder caused by expansion of a CAG trinucleotide repeat in the IT15 gene, which leads to expansion of a polyglutamine tract within the protein called huntingtin. Despite the characterization of the IT15 gene and the mutation involved in the disease, the normal function of huntingtin and the effects of the mutation on its function and on its neuronal location remain unknown. To study whether mutated huntingtin has the same neuronal distribution and intracellular location as normal huntingtin, we analyzed immunohistochemically both forms of this protein in the brain of 5 controls and 5 patients with Huntington's disease. We show that the distribution of mutated huntingtin is, like that of the normal form, heterogeneous throughout the brain, but is not limited to vulnerable neurons in Huntington's disease, supporting the hypothesis that the presence of the mutated huntingtin in a neuron is not in itself sufficient to lead to neuronal death. Moreover, whereas normal huntingtin is detected in some neuronal perikarya, nerve fibers, and nerve endings, the mutated form is observed in some neuronal perikarya and proximal nerve processes but is not detectable in nerve endings. Our results suggest that the expression or processing of the mutated huntingtin in perikarya and nerve endings differs quantitatively or qualitatively from the expression of the normal form in the same neuronal compartments.