Cell-Specific Deletion of PGC-1α from Medium Spiny Neurons Causes Transcriptional Alterations and Age-Related Motor Impairment.

Multiple lines of evidence indicate that a reduction in the expression and function of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is associated with neurodegeneration in diseases such as Huntington's disease (HD). Polymorphisms in the PGC-1α gene modify HD progression and PGC-1α expression is reduced in striatal medium spiny neurons (MSNs) of HD patients and mouse models. However, neither the MSN-specific function of PGC-1α nor the contribution of PGC-1α deficiency to motor dysfunction is known. We identified novel, PGC-1α-dependent transcripts involved in RNA processing, signal transduction, and neuronal morphology and confirmed reductions in these transcripts in male and female mice lacking PGC-1α specifically in MSNs, indicating a cell-autonomous effect in this population. MSN-specific PGC-1α deletion caused reductions in previously identified neuronal and metabolic PGC-1α-dependent genes without causing striatal vacuolizations. Interestingly, these mice exhibited a hypoactivity with age, similar to several HD animal models. However, these newly identified PGC-1α-dependent genes were upregulated with disease severity and age in knock-in HD mouse models independent of changes in PGC-1α transcript, contrary to what would be predicted from a loss-of-function etiological mechanism. These data indicate that PGC-1α is necessary for MSN transcriptional homeostasis and function with age and that, whereas PGC-1α loss in MSNs does not replicate an HD-like phenocopy, its downstream genes are altered in a repeat-length and age-dependent fashion. Understanding the additive effects of PGC-1α gene functional variation and mutant huntingtin on transcription in this cell type may provide insight into the selective vulnerability of MSNs in HD.SIGNIFICANCE STATEMENT Reductions in peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α)-mediated transcription have been implicated in the pathogenesis of Huntington's disease (HD). We show that, although PGC-1α-dependent transcription is necessary to maintain medium spiny neuron (MSN) function with age, its loss is insufficient to cause striatal atrophy in mice. We also highlight a set of genes that can serve as proxies for PGC-1α functional activity in the striatum for target engagement studies. Furthermore, we demonstrate that PGC-1α-dependent genes are upregulated in a dose- and age-dependent fashion in HD mouse models, contrary to what would be predicted from a loss-of-function etiological mechanism. However, given this role for PGC-1α in MSN transcriptional homeostasis, it is important to consider how genetic variation in PGC-1α could contribute to mutant-huntingtin-induced cell death and disease progression.

Characterisation of progressive motor deficits in whisker movements in R6/2, Q175 and Hdh knock-in mouse models of Huntington's disease.

BACKGROUND: Motor dysfunction is a major component of the Huntington's disease (HD) phenotype, both in patients and animal models. Motor function in mice is usually measured using tests that involve a novel environment, or require a degree of learning, which creates potential confounds in animals, such as anxiety and/or learning. NEW METHOD: We propose that studying whisker control provides a more naturalistic way to measure motor function in HD mice. To this end we tested three strains of HD mice; R6/2 (CAG250), zQ175 and Hdh (CAG50, 150 and 250) mice. RESULTS: We discovered a clear and progressive whisking deficit in the most severe model, the R6/2 CAG250 mouse. At 10 weeks, R6/2 mice showed an increase in whisking movements, which may be a correlate of the hyperkinesia seen in HD patients. By 18 weeks the R6/2 mice showed a reduction in whisking movements. Hdh Q250 mice showed a hyperkinetic profile at 10 weeks, approximately 4 months before other motor deficits have previously been reported in these mice. Q175 mice showed very little change in whisking behaviour, apart from a transient increase in retraction velocity at 10 weeks. COMPARISONS WITH EXISTING METHODS: Our findings suggest that whisking may be a more sensitive test of motor function in HD mice than more commonly used methods, such as the rotarod. CONCLUSIONS: Our data suggest that whisking deficits represent a novel way of assessing the progression of the motor phenotype, and are early indicators for reversal of phenotype studies, such as drug trials.

Sex-dependent behavioral impairments in the HdhQ350/+ mouse line.

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease characterized by gradual deterioration of motor and cognitive functions and development of psychiatric deficits. Animal models provide powerful means to study the pathological processes, molecular dysfunctions and symptoms associated with HD. We performed a longitudinal behavioral study of the newly developed HdhQ350/+ mouse line, a knock-in model that expresses a repeat of 350 glutamines. We found remarkable sex-dependent differences on symptom onset and severity. While both sexes lose weight and grip strength, only HdhQ350/+ males have impaired motor coordination as measured by the rotarod and alterations in gait as measured by the catwalk assay. While HdhQ350/+ females do not exhibit impairment in motor coordination, we found a reduction in dark phase locomotor activity. Male and female HdhQ350/+ mice do not show anxiety as measured by the elevated plus maze or changes in exploration as measured by the open field test. To investigate these sex-dependent differences, we performed western blot analyses of striatal tissue. We measured equal mutant huntingtin protein expression in both sexes and found evidence of aggregation. We found the expected decrease of DARPP-32 expression only in female HdhQ350/+ mice. Remarkably, we found no evidence of reduction in synaptophysin or CB1 receptors in HdhQ350/+ tissue of either sex. Our study indicates that male and female HdhQ350/+ mice differentially recapitulate select behavioral impairments commonly measured in other HD mouse models with limited sex-dependent changes in recognized histopathological markers. We conclude that expanded polyglutamine repeats influence HD pathogenesis in a sex-dependent manner.

Allelic series of Huntington's disease knock-in mice reveals expression discorrelates.

Identifying molecular drivers of pathology provides potential therapeutic targets. Differentiating between drivers and coincidental molecular alterations presents a major challenge. Variation unrelated to pathology further complicates transcriptomic, proteomic and metabolomic studies which measure large numbers of individual molecules. To overcome these challenges towards the goal of determining drivers of Huntington's disease (HD), we generated an allelic series of HD knock-in mice with graded levels of phenotypic severity for comparison with molecular alterations. RNA-sequencing analysis of this series reveals high numbers of transcripts with level alterations that do not correlate with phenotypic severity. These discorrelated molecular changes are unlikely to be drivers of pathology allowing an exclusion-based strategy to provide a short list of driver candidates. Further analysis of the data shows that a majority of transcript level changes in HD knock-in mice involve alteration of the rate of mRNA processing and/or degradation rather than solely being due to alteration of transcription rate. The overall strategy described can be applied to assess the influence of any molecular change on pathology for diseases where different mutations cause graded phenotypic severity.

Progression of behavioural despair in R6/2 and Hdh knock-in mouse models recapitulates depression in Huntington's disease.

In Huntington's disease (HD) depression is observed before the disease is diagnosed, and is likely to be a component of the disease, rather than a consequence. Depression in HD patients does not progress in parallel with other symptoms; rather it peaks at early- to mid-stages of the disease and declines thereafter. In mice, depressive-like behaviours can be measured as an increase in behavioural despair (floating) observed in the forced swim test (FST). Floating in the FST is modulated differently by antidepressants with different mechanisms of action. Drugs that increase levels of serotonin inhibit floating by promoting horizontal swimming, whereas drugs that increase levels of noradrenaline inhibit floating by enhancing vertical swimming (climbing). We compared the FST behavioural profiles of two different allelic series of HD mice, a fragment model (R6/2 mice carrying 120, 250, or 350 CAG repeats), and a knock-in model (Hdh mice carrying 50, 150, or 250 CAG repeats). The FST behavioural profile was similar in both lines. It was characterized by an early-stage increase in floating, and then, as the mice aged, floating decreased, whereas active behaviours of swimming and climbing increased. Our results show that, as with depression in HD patients, floating in HD mice does not progress linearly, suggesting that, at the late stages of the disease, an increase in serotonergic and noradrenergic activity might contribute to lower floating levels in HD mice. If similar compensatory changes occur in humans, this should be taken into account when considering the treatment of depression in HD patients.

Early white matter abnormalities, progressive brain pathology and motor deficits in a novel knock-in mouse model of Huntington's disease.

White matter abnormalities have been reported in premanifest Huntington's disease (HD) subjects before overt striatal neuronal loss, but whether the white matter changes represent a necessary step towards further pathology and the underlying mechanism of these changes remains unknown. Here, we characterized a novel knock-in mouse model that expresses mouse HD gene homolog (Hdh) with extended CAG repeat- HdhQ250, which was derived from the selective breeding of HdhQ150 mice. HdhQ250 mice manifest an accelerated and robust phenotype compared with its parent line. HdhQ250 mice exhibit progressive motor deficits, reduction in striatal and cortical volume, accumulation of mutant huntingtin aggregation, decreased levels of DARPP32 and BDNF and altered striatal metabolites. The abnormalities detected in this mouse model are reminiscent of several aspects of human HD. In addition, disturbed myelination was evident in postnatal Day 14 HdhQ250 mouse brain, including reduced levels of myelin regulatory factor and myelin basic protein, and decreased numbers of myelinated axons in the corpus callosum. Thinner myelin sheaths, indicated by increased G-ratio of myelin, were also detected in the corpus callosum of adult HdhQ250 mice. Moreover, proliferation of oligodendrocyte precursor cells is altered by mutant huntingtin both in vitro and in vivo. Our data indicate that this model is suitable for understanding comprehensive pathogenesis of HD in white matter and gray matter as well as developing therapeutics for HD.

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

Huntington disease (HD) is a devastating, late-onset, inherited neurodegenerative disorder that manifests with personality changes, movement disorders, and cognitive decline. It is caused by a CAG repeat expansion in exon 1 of the HTT gene that translates to a polyglutamine tract in the huntingtin protein (HTT). The formation of HTT fragments has been implicated as an essential step in the molecular pathogenesis of HD and several proteases that cleave HTT have been identified. However, the importance of smaller N-terminal fragments has been highlighted by their presence in HD postmortem brains and by the fact that nuclear inclusions are only detected by antibodies to the N terminus of HTT. Despite an intense research effort, the precise length of these fragments and the mechanism by which they are generated remains unknown. Here we show that CAG repeat length-dependent aberrant splicing of exon 1 HTT results in a short polyadenylated mRNA that is translated into an exon 1 HTT protein. Given that mutant exon 1 HTT proteins have consistently been shown to be highly pathogenic in HD mouse models, the aberrant splicing of HTT mRNA provides a mechanistic basis for the molecular pathogenesis of HD. RNA-targeted therapeutic strategies designed to lower the levels of HTT are under development. Many of these approaches would not prevent the production of exon 1 HTT and should be reviewed in light of our findings.

Tissue transglutaminase overexpression does not modify the disease phenotype of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a devastating autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine in the huntingtin protein. Determining the contribution of specific factors to the pathogenesis of HD should provide rational targets for therapeutic intervention. One suggested contributor is the type 2 transglutaminase (TG2), a multifunctional calcium dependent enzyme. A role for TG2 in HD has been suggested because a polypeptide-bound glutamine is a rate-limiting factor for a TG2-catalyzed reaction, and TG2 can cross-link mutant huntingtin in vitro. Further, TG2 is up regulated in brain areas affected in HD. The objective of this study was to further examine the contribution of TG2 as a potential modifier of HD pathogenesis and its validity as a therapeutic target in HD. In particular our goal was to determine whether an increase in TG2 level, as documented in human HD brains, modulates the well-characterized phenotype of the R6/2 HD mouse model. To accomplish this objective a genetic cross was performed between R6/2 mice and an established transgenic mouse line that constitutively expresses human TG2 (hTG2) under control of the prion promoter. Constitutive expression of hTG2 did not affect the onset and progression of the behavioral and neuropathological HD phenotype of R6/2 mice. We found no alterations in body weight changes, rotarod performances, grip strength, overall activity, and no significant effect on the neuropathological features of R6/2 mice. Overall the results of this study suggest that an increase in hTG2 expression does not significantly modify the pathology of HD.

Early alterations of autophagy in Huntington disease-like mice.

In a recent study, we reported in vivo evidence of early and sustained alterations of autophagy markers in a novel knock-in mouse model of Huntington disease (HD). The novel model is derived from selective breeding of HdhQ150 knock-in mice to generate mice with ~200 CAG/polyglutamine repeats (HdhQ200). HdhQ200 knockin mice exhibit an accelerated and more robust motor phenotype than the parent line with detectable abnormalities at 50 weeks and substantial impairments at 80 weeks. Heterozygous HdhQ200 knock-in mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age followed by striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Increased LC3-II protein expression is noted at 9 weeks and sustained throughout the disease course, and is paralleled by increased expression of p62. Early and sustained expression of: autophagy-related proteins in this genetically precise mouse model of HD suggests that alteration of autophagic flux is an important and early component of neuronal response to polyglutamine expanded huntingtin.

Early autophagic response in a novel knock-in model of Huntington disease.

The aggregation of mutant polyglutamine (polyQ) proteins has sparked interest in the role of protein quality-control pathways in Huntington's disease (HD) and related polyQ disorders. Employing a novel knock-in HD mouse model, we provide in vivo evidence of early, sustained alterations of autophagy in response to mutant huntingtin (mhtt). The HdhQ200 knock-in model, derived from the selective breeding of HdhQ150 knock-in mice, manifests an accelerated and more robust phenotype than the parent line. Heterozygous HdhQ200 mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age and striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Striatal NIIs accumulate earlier in HdhQ200 mice than in HdhQ150 mice. The earlier appearance of aggregate pathology in HdhQ200 mice is paralleled by earlier and more rapidly progressive motor deficits: progressive imbalance and decreased motor coordination by 50 weeks, gait deficits by 60 weeks and gross motor impairment by 80 weeks of age. At 80 weeks, heterozygous HdhQ200 mice exhibit striatal and cortical astrogliosis and a approximately 50% reduction in striatal dopamine receptor binding. Increased LC3-II protein expression, which is noted early and sustained throughout the disease course, is paralleled by increased expression of the autophagy-related protein, p62. Early and sustained expression of autophagy-related proteins in this genetically precise mouse model of HD suggests that the alteration of autophagic flux is an important and early component of the neuronal response to mhtt.

Mitochondrial calcium uptake capacity as a therapeutic target in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is an incurable autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine domain in the huntingtin protein. It is proposed that abnormal mitochondrial Ca2+ capacity results in an increased susceptibility to mitochondrial permeability transition (MPT) induction that may contribute significantly to HD pathogenesis. The in vivo contribution of these hypothesized defects remains to be elucidated. In this proof-of-principle study, we examined whether increasing mitochondrial Ca2+ capacity could ameliorate the well-characterized phenotype of the R6/2 transgenic mouse model. Mouse models lacking cyclophilin D demonstrate convincingly that cyclophilin D is an essential component and a key regulator of MPT induction. Mitochondria of cyclophilin D knockout mice are particularly resistant to Ca2+ overload. We generated R6/2 mice with normal, reduced or absent cyclophilin D expression and examined the effect of increasing mitochondrial Ca2+ capacity on the behavioral and neuropathological features of the R6/2 model. A predicted outcome of this approach was the finding that cyclophilin D deletion enhanced the R6/2 brain mitochondria Ca2+ capacity significantly. Increased neuronal mitochondrial Ca2+ capacity failed to ameliorate either the behavioral and neuropathological features of R6/2 mice. We found no alterations in body weight changes, lifespan, RotaRod performances, grip strength, overall activity and no significant effect on the neuropathological features of R6/2 mice. The results of this study demonstrate that increasing neuronal mitochondrial Ca2+-buffering capacity is not beneficial in the R6/2 mouse model of HD.

In vivo evidence for NMDA receptor-mediated excitotoxicity in a murine genetic model of Huntington disease.

N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity is implicated as a proximate cause of neurodegeneration in Huntington Disease (HD). This hypothesis has not been tested rigorously in vivo. NMDAR-NR2B subunits are a major NR2 subunit expressed by striatal medium spiny neurons that degenerate in HD. To test the excitotoxic hypothesis, we crossed a well validated murine genetic model of HD (Hdh((CAG)150)) with a transgenic line overexpressing NMDAR-NR2B subunits. In the resulting double-mutant line, we show exacerbation of selective striatal neuron degeneration. This is the first direct in vivo evidence of NR2B-NMDAR-mediated excitotoxicity in the context of HD. Our results are consistent with previous suggestions that direct and/or indirect interactions of mutant huntingtin with NMDARs are a proximate cause of neurodegeneration in HD.

Rodent genetic models of Huntington disease.

Huntington disease (HD) is a dominantly inherited human neurodegenerative disorder characterized by motor deficits, cognitive impairment, and psychiatric symptoms leading to inexorable decline and death. Since the identification of the huntingtin gene and the characteristic expanded CAG repeat/polyglutamine mutation, multiple murine genetic models and one rat genetic model have been generated. These models fall into two general categories: transgenic models with ectopic expression of the characteristic expanded CAG codon mutation, and knock-in models with expression of mutant huntingtin under control of endogenous regulatory elements. Rodent genetic models are valuable tools for studying mechanisms of pathogenesis in HD and for preclinical evaluation of possible therapies. In this mini-review, we provide a concise comparative summary of rodent genetic models of HD.

DNA instability in postmitotic neurons.

Huntington's disease (HD) is caused by a CAG repeat expansion that is unstable upon germ-line transmission and exhibits mosaicism in somatic tissues. We show that region-specific CAG repeat mosaicism profiles are conserved between several mouse models of HD and therefore develop in a predetermined manner. Furthermore, we demonstrate that these synchronous, radical changes in CAG repeat size occur in terminally differentiated neurons. In HD this ongoing mutation of the repeat continuously generates genetically distinct neuronal populations in the adult brain of mouse models and HD patients. The neuronal population of the striatum is particularly distinguished by a high rate of CAG repeat allele instability and expression driving the repeat upwards and would be expected to enhance its toxicity. In both mice and humans, neurons are distinguished from nonneuronal cells by expression of MSH3, which provides a permissive environment for genetic instability independent of pathology. The neuronal mutations described here accumulate to generate genetically discrete populations of cells in the absence of selection. This is in contrast to the traditional view in which genetically discrete cellular populations are generated by the sequence of random variation, selection, and clonal proliferation. We are unaware of any previous demonstration that mutations can occur in terminally differentiated neurons and provide a proof of principle that, dependent on a specific set of conditions, functional DNA polymorphisms can be produced in adult neurons.

Longitudinal evaluation of the Hdh(CAG)150 knock-in murine model of Huntington's disease.

Several murine genetic models of Huntington's disease (HD) have been developed. Murine genetic models are crucial for identifying mechanisms of neurodegeneration in HD and for preclinical evaluation of possible therapies for HD. Longitudinal analysis of mutant phenotypes is necessary to validate models and to identify appropriate periods for analysis of early events in the pathogenesis of neurodegeneration. Here we report longitudinal characterization of the murine Hdh(CAG)150 knock-in model of HD. A series of behavioral tests at five different time points (20, 40, 50, 70, and 100 weeks) demonstrates an age-dependent, late-onset behavioral phenotype with significant motor abnormalities at 70 and 100 weeks of age. Pathological analysis demonstrated loss of striatal dopamine D1 and D2 receptor binding sites at 70 and 100 weeks of age, and stereological analysis showed significant loss of striatal neuron number at 100 weeks. Late-onset behavioral abnormalities, decrease in striatal dopamine receptors, and diminished striatal neuron number observed in this mouse model recapitulate key features of HD. The Hdh(CAG)150 knock-in mouse is a valid model to evaluate early events in the pathogenesis of neurodegeneration in HD.

Intraflagellar transport is essential for endochondral bone formation.

While cilia are present on most cells in the mammalian body, their functional importance has only recently been discovered. Cilia formation requires intraflagellar transport (IFT), and mutations disrupting the IFT process result in loss of cilia and mid-gestation lethality with developmental defects that include polydactyly and abnormal neural tube patterning. The early lethality in IFT mutants has hindered research efforts to study the role of this organelle at later developmental stages. Thus, to investigate the role of cilia during limb development, we generated a conditional allele of the IFT protein Ift88 (polaris). Using the Cre-lox system, we disrupted cilia on different cell populations within the developing limb. While deleting cilia in regions of the limb ectoderm had no overt effect on patterning, disruption in the mesenchyme resulted in extensive polydactyly with loss of anteroposterior digit patterning and shortening of the proximodistal axis. The digit patterning abnormalities were associated with aberrant Shh pathway activity, whereas defects in limb outgrowth were due in part to disruption of Ihh signaling during endochondral bone formation. In addition, the limbs of mesenchymal cilia mutants have ectopic domains of cells that resemble chondrocytes derived from the perichondrium, which is not typical of Indian hedgehog mutants. Overall these data provide evidence that IFT is essential for normal formation of the appendicular skeleton through disruption of multiple signaling pathways.

Allele-specific conditional destabilization of glutamine repeat mRNAs.

Several late-onset neurological diseases are caused by the inheritance of an expanded CAG repeat coding for polyglutamine. To date there is no effective means of halting the progression of these diseases, and their underlying molecular mechanisms remain a mystery. Strategies designed to specifically reduce the levels of long repeat mRNA might provide an effective therapy for these diseases. An emphasis on allele specificity is necessary to avoid the potential toxicities associated with reduction of expression. The experiments described here are based on the relationship between translation and mRNA stability and the idea that translation of a repeated codon might be extremely sensitive to reductions in levels of cognate aminoacylated tRNA. Consistent with this hypothesis, we have discovered that reduced glutamine concentration destabilizes mRNAs coding for long glutamine repeats while sparing short repeat versions of the same mRNAs. These results suggest therapy might be attained with existing compounds or environmental conditions known to decrease free glutamine levels.

Verification of somatic CAG repeat expansion by pre-PCR fractionation.

The inheritance of a long CAG repeat causes several late onset neurological disorders including Huntington's disease (HD). Longer CAG repeats correlate with earlier onset of HD suggesting an increased toxicity for the products of long repeat alleles. PCR based data has been used to show that HD CAG repeat expansion beyond the inherited length occurs in affected tissues indicating a possible role for somatic instability in the disease process. PCR, however, is prone to artifacts resulting from expansion of repeat sequences during amplification. We describe a method to distinguish between CAG repeat expansions that exist in vivo and those that potentially occur during PCR. The method involves size fractionation of genomic restriction fragments containing the expanded repeats followed by PCR amplification. The application of this method confirms the presence of somatic expansions in the brains of a knock-in mouse model of HD.

Mouse Huntington's disease homolog mRNA levels: variation and allele effects.

Huntington's disease homolog (Hdh) mRNA levels in mice with different Hdh alleles were measured. Brain Hdh mRNA levels varied up to threefold in genetically identical wild-type mice, indicating nongenetic factors influence Hdh expression. Striatal Hdh mRNA levels from an allele with a repeat expanded to 150 CAGs were diminished compared with wild-type and showed variation that might contribute to phenotypic variability in the Hdh(CAG)150 knock-in mouse model. To determine whether Hdh mRNA levels are tightly regulated, we assessed these levels in mice heterozygous for a deletion of the Hdh promoter. The loss of one allele reduced Hdh mRNA levels in most tissues, suggesting mechanisms to maintain Hdh mRNA levels are not in effect and should not impede therapies designed to destroy mutant huntingtin mRNA. Finally, we found a correlation between tissue mRNA levels and the susceptibility of the Hdh locus to Cre-mediated deletion. The two tissues with the highest levels of Hdh mRNA, testes and brain, were the only tissues susceptible to Cre-mediated recombination between loxP sites at Hdh locus. In contrast, the same Cre-expressing line caused recombination in every tissue for loxP sites at another genomic location. The pattern of Cre susceptibility at Hdh suggests a correlation between chromatin accessibility and high levels of Hdh expression in testes and brain.

Embryonic stem cells expressing expanded CAG repeats undergo aberrant neuronal differentiation and have persistent Oct-4 and REST/NRSF expression.

Nine neurodegenerative disorders are caused by CAG/polyglutamine (polyQ) repeat expansions. The molecular mechanisms responsible for disease-specific neurodegeneration remain elusive. We developed an embryonic stem (ES) cell-based model to probe the role of polyQ tract expansion in neuronal degeneration. ES cells containing expanded CAG repeats in the hypoxanthine phosphoribosyltransferase (Hprt) gene develop features typical of CAG-mediated neuropathology, exhibit length-dependent decrease in survival, undergo aberrant neuronal differentiation as well as persistent Oct-4 and Repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF) expression. This novel model will allow analysis of the molecular pathogenesis of neuronal degeneration and can be used to rapidly screen therapeutic interventions for these fatal diseases.