Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease

Psychopathology in Verified Huntington's Disease Gene Carriers

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown.The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model.Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis.Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

<h3>Background</h3> Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by expansion of CAG repeats in the Huntingtin (HTT) gene. <h3>Aim</h3> There is human evidence of increased complement biosynthesis and activation in HD, with increased immunoreactivity for C1q, C4, C3 and C3b observed in striatal neurons, astrocyte, and microglia cells. However, the role of the classical pathway in HD pathogenesis is unclear. <h3>Methods</h3> Using the R6/2 mouse model of HD with ~ 120 CAG expansion, we investigated alteration of classical complement cascade in plasma and cerebral spinal fluid (CSF), we evaluated correlation of classical complement C1q levels with the neurodegenerative biomarker Neurofilament Light Chain (NFL) levels and assessed efficacy of anti C1q mAb therapy in preventing neurodegeneration and ameliorating disease progression. <h3>Results</h3> We found evidence of perturbations in the complement pathway, such as significant increase in plasma levels of C1q, C1s, C4, C3 and C3d during disease progression. Positive correlation of plasma C1q with CSF NFL levels was observed, suggesting a role of the classical complement cascade in neurodegeneration and disease progression. To test this hypothesis, we pharmacologically inhibited the classical pathway via systemic administration of anti-C1q mAb (ANX-M1, Annexon Biosciences). We observed that reduction of free C1q by anti C1q mAb therapy resulted in normalized complement pathway activity, reduced levels of CSF NFL, improved motor behavior measured with the open field assay and improved R6/2 mouse survival. <h3>Conclusions</h3> Altogether this study suggests that inhibiting C1q restores synaptic and neuronal health in mice with expanded CAG repeats in the HTT gene and C1q is a potential pharmacological target in HD. A Phase 2 study of ANX005 anti-C1q therapy in HD patients (clinical trials.gov NCT04514367) is ongoing.

Huntington's disease (HD) is a late-onset progressive neurodegenerative disorder characterised by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The proteolytic cleavage of huntingtin (HTT) to generate N-terminal fragments has been proposed to be a key aspect of HD pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 (CASP6) and that prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 as a therapeutic strategy should be approached with caution in the context of this complex disease. Abstract Huntington's disease (HD) is a late-onset progressive neurodegenerative disorder characterised by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The proteolytic cleavage of huntingtin (HTT) to generate N-terminal fragments has been proposed to be a key aspect of HD pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 (CASP6) and that prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 as a therapeutic strategy should be approached with caution in the context of this complex disease. Abstract Huntington's disease (HD) is a late-onset progressive neurodegenerative disorder characterised by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The proteolytic cleavage of huntingtin (HTT) to generate N-terminal fragments has been proposed to be a key aspect of HD pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 (CASP6) and that prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 as a therapeutic strategy should be approached with caution in the context of this complex disease.

<h3></h3> Huntington disease (HD) is an autosomal dominant neurodegenerative disorder, characterised by motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the first exon of the HTT gene, resulting in an expanded polyglutamine tract at the N-terminus of the huntingtin protein. Typical disease onset is around mid-life (adult-onset HD) whereas onset below 21 years is classified as juvenile HD. While much research has been done on the underlying HD disease mechanisms, little is known about regulation and expression levels of huntingtin RNA and protein. In this study we used a unique collection of human post-mortem HD brain tissue and fibroblast cells to investigate huntingtin mRNA and protein expression, as well as huntingtin antisense isoforms. In adult-onset HD brain samples, there was only a small but significant lower expression of mutant huntingtin mRNA compared to wild-type huntingtin mRNA, while protein expression levels were equal. Juvenile HD subjects did show a lower protein expression of mutant huntingtin compared to wild-type huntingtin protein. Additionally, in brain tissue we did not find any evidence for a reduced expression of huntingtin antisense, as we showed HTTAS_v1 expression in a homozygous HD patient. Finally, we have identified a novel huntingtin antisense isoform and named it HTTAS_v2.2. Our study shows an intricate mechanism of huntingtin RNA and protein regulation of expression with slightly less mutant huntingtin mRNA, but equal wild-type and mutant huntingtin protein levels in adult-onset HD, indicating subtle differences in huntingtin protein expression between adult-onset and juvenile HD.

Huntington Disease (HD) is a dominantly inherited, relentlessly progressive neurodegenerative disease. Caused by a polyglutamine expansion in the huntingtin protein (mutant huntingtin, mhtt), HD pathogenesis impairs function in the cerebral cortex and in medium spiny neurons of the striatum. HD is also characterized by the transcriptional dysregulation of a number of genes. Of these genes, the silencing of genes related to mitochondrial function is believed to explain metabolic dysfunction in rodent models of HD. Here we show that transglutaminase 2 (TG2), which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes. In a cellular model of HD, TG2 inhibition by RNA knockdown, genetic deletion, or administration of a novel, peptide-based irreversible TG2 inhibitor (ZDON), de-repressed two established regulators of mitochondrial function, PGC-1α and cytochrome c. We showed that TG2 must localize to non-coding or coding regions of these mitochondrial metabolic genes to silence their transcription. As expected, TG2 inhibition reversed the increased susceptibility of HD mouse cells and human HD myoblasts to the mitochondrial toxin, 3-nitroproprionic acid (3-NP); however, protection mediated by TG2 inhibition was not associated with improved mitochondrial bioenergetics. Indeed, an unbiased array analysis indicated that TG2 inhibition leads to normalization of not only mitochondrial genes but of nearly 40% of genes that are dysregulated in HD mouse striatal neurons, including chaperone and histone genes. Indeed, TG2 interacts directly with Histone H3 in the nucleus. Moreover, TG2 inhibition significantly attenuated photoreceptor degeneration in a Drosophila model of HD and protected mouse HD striatal neurons (YAC128) from NMDA-induced toxicity. Altogether these findings demonstrate that TG2 mediates its deleterious effects in HD by contributing to broad transcriptional dysregulation of genes representing many cellular functions. These studies define a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

Chapter 6 - Huntington Disease and the Huntingtin Protein

Huntington’s disease (HD) is a neurodegenerative disease that results in motor and cognitive dysfunction, leading to early death. HD is caused by an expansion of CAG repeats in the huntingtin gene (HTT). Here, we review the mouse models of HD. They have been used extensively to better understand the molecular and cellular basis of disease pathogenesis as well as to provide non-human subjects to test the efficacy of potential therapeutics. The first and best-studied in vivo rodent model of HD is the R6/2 mouse, in which a transgene containing the promoter and exon 1 fragment of human HTT with 150 CAG repeats was inserted into the mouse genome. R6/2 mice express rapid, robust behavioral pathologies and display a number of degenerative abnormalities in neuronal populations most vulnerable in HD. The first conditional full-length mutant huntingtin (mHTT) mouse model of HD was the bacterial artificial chromosome (BAC) transgenic mouse model of HD (BACHD), which expresses human full-length mHTT with a mixture of 97 CAG-CAA repeats under the control of endogenous HTT regulatory machinery. It has been useful in identifying the role of mHTT in specific neuronal populations in degenerative processes. In the knock-in (KI) model of HD, the expanded human CAG repeats and human exon 1 are inserted into the mouse Htt locus, so a chimera of the full-length mouse protein with the N-terminal human portion is expressed. Many of aspects of the pathology and behavioral deficits in the KI model better mimic disease characteristics found in HD patients than other models. Accordingly, some have proposed that these mice may be preferable models of the disease over others. Indeed, as our understanding of HD advances, so will the design of animal models to test and develop HD therapies.

Lack of huntingtin promotes neural stem cells differentiation into glial cells while neurons expressing huntingtin with expanded polyglutamine tracts undergo cell death

Longitudinal Evaluation of "Presymptomatic" Carriers of Huntington's Disease

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full‐length HTT or an N‐terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP‐positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50–61

​TheThe gene for Huntington's disease (HD) was discovered in 1993 and ever since has been puzzling researchers intent on understanding its effects. The mutation, an expanded CAG repeat, is translated into an extended polyglutamine tract in the huntingtin protein (HTT), which leads to protein misfolding, accumulation of sticky protein aggregates in both cytoplasm and nucleus, and degeneration of neurons, first in the brain's striatum and later in the cortex and elsewhere. There are no disease-altering treatments and no single hypothesis of disease pathogenesis. In a new study, Michal Mielcarek, Gillian Bates, and colleagues elucidate a key role for the transcription regulator histone deactylase 4 (HDAC4)—not within the nucleus as you might expect, but instead in the cytoplasm. HDAC4 acts in the cytoplasm of brain cells to exacerbate the Huntington's disease (HD) pathogenic process. Reduction of HDAC4 levels ameliorates cytoplasmic-related HD phenotypes and improves survival, but does not change gene transcription. One prominent feature of HD molecular pathology is a global transcriptional dysregulation, likely driven in part by reduced histone acetylation. To counterbalance that effect, researchers have investigated whether inhibiting HDACs might be therapeutic, and one HDAC inhibitor has produced promising results in preclinical trials in models of HD. There are 11 different mammalian HDACs, and in order to better characterize their individual contributions to HD, the authors examined the effects of partially or completely knocking out each one. They found that reducing HDAC4 by 50% in a mouse model of HD reduced neuronal dysfunction of striatal neurons and delayed loss of motor function of the mice, allowing them to perform a balancing task at 12 weeks of age as well as untreated mice who were a month younger. Treatment also extended lifespan by about 20%, a significant improvement in this aggressive disease model of HD. Antibodies against HDAC4 and either mutant or wild-type HTT indicated that HDAC4 bound to mutant HTT in either soluble or aggregated form, but not to normal protein. Like HTT, HDAC4 includes a polyglutamine region, and it is likely that its interaction with mutant HTT is through the reciprocal attractions between these regions. Reducing HDAC4 reduced the total burden of HTT aggregates throughout the brain while increasing the amount of soluble HTT, indicating that treatment delayed the aggregation process. The work thereby finds a novel route to modulating the toxicity of HTT. HDAC4 remains sequestered in the cytoplasm until it is called upon to shuttle into the nucleus to take part in regulating transcription. Co-labeling HDAC4 and mutant HTT indicated that they localized together in the cytoplasm, but not the nucleus, and that reducing HDAC4 reduced aggregation in the cytoplasm while leaving the number of nuclear aggregates unchanged. This ability of the work to separate cytoplasmic pathologies from nuclear ones is an important advance in this field. Surprisingly, reducing HDAC4 had no effect on the widespread transcriptional dysregulation that led HD researchers to consider HDAC inhibitors in the first place. It did, however, have one potentially important effect on gene expression: it largely restored the levels of brain-derived neurotrophic factor, a growth factor that neurons need for survival and that is known to be lost in HD. This effect is likely mediated through a cytoplasmic, not nuclear, mechanism, based on previous work on this pathway in HD; further work will be needed to understand it in detail. These results have several implications for understanding and treating HD. There is no shortage of hypotheses of pathogenic mechanisms in HD, and this new mechanism is unlikely to be the sole cause of neuronal damage. But the discovery of a strictly cytoplasmic pathogenic effect of mutant HTT is new and will likely bring more attention to the cytoplasm as a site for further research. The demonstration that shifting the balance of mutant protein from aggregated to soluble forms has a beneficial effect further informs a long-standing debate in disorders of protein misfolding, about whether aggregates are toxic or protective, and will likely accelerate exploration of therapies to promote disaggregation. As mentioned, there are currently no disease-modifying therapeutics available for HD. The discovery that reducing HDAC4 has therapeutic effects in the HD model tested here—which happens to be a gold standard model in which most therapeutics fail when tested—will spur efforts to mimic this effect with small molecules to obstruct the interaction of HDAC4 and mutant HTT, or antisense therapies designed to prevent production of HDAC4 protein. Mielcarek M, Landles C, Weiss A, Bradaia A, Seredenina T, et al (2013). HDAC4 Reduction: A Novel Therapeutic Strategy to Target Cytoplasmic Huntingtin and Ameliorate Neurodegeneration. doi:10.1371/journal.pbio.1001717

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.

Huntington disease (HD) is an inherited neurodegenerative disease characterized by a clinical triad of motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Homozygosity for the HD-causing mutation is extremely rare; thus, the majority of HD patients express the mutant huntingtin protein in addition to reduced levels of the non-pathogenic huntingtin protein. Deficits in synaptic plasticity, including hippocampal long-term potentiation (LTP), have been identified in various mouse models of HD and are thought to contribute to the debilitating cognitive symptoms associated with the disease. However, the bulk of these studies used N-terminal fragment or homozygous knock-in mouse models of HD at symptomatic ages, and our understanding of the onset and progression of synaptic plasticity deficits in the HD brain is lacking. To better understand the time-course of synaptic plasticity deficits in HD, as well as the impact of heterozygous and homozygous huntingtin mutations, we quantified basal synaptic connectivity, presynaptic release probability, presynaptically mediated post-tetanic potentiation (PTP) and postsynaptically mediated LTP at presymptomatic, early symptomatic and late symptomatic ages in heterozygous and homozygous Q175FDN knock-in HD mice. Our results demonstrate clear age-dependent effects of the HD-causing mutation on both short and long-term plasticity that generally emerge earlier in homozygous mice. Interestingly, deficits in presynaptic short-term plasticity were more closely linked to disease progression than deficits in postsynaptic LTP, and heterozygous mice were more susceptible to an LTP deficit when induced by high frequency stimulation compared to theta burst stimulation. To the best of our knowledge, the present study represents the most thorough characterization to date of the onset and progression of hippocampal synaptic plasticity deficits in a mouse model of HD, and should prove valuable to future studies exploring cellular mechanisms underlying the debilitating cognitive decline in HD.