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1.  ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice 
PLoS Genetics  2012;8(8):e1002920.
Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissue-specific effects, which may be partially alleviated by the induction of FBXW8.
Author Summary
Frequent age-associated neurodegenerative disorders like Alzheimer's, Parkinson's, and Lou Gehrig's disease are being elucidated molecularly by studying rare heritable variants. Various hereditary neurodegenerative disorders are caused by polyglutamine expansions in different proteins. In spite of this common pathogenesis and the pathological aggregation of most affected proteins, investigators were puzzled that the pattern of affected neuron population varies and that molecular mechanisms seem different between such disorders. The polyglutamine expansions in the Ataxin-2 (ATXN2) protein are exceptional in view of the lack of aggregate clumps in nuclei of affected Purkinje neurons and well documented alterations of RNA processing in the resulting disorders SCA2 and ALS. Here, as a faithful disease model and to overcome the unavailability of autopsied patient brain tissues, we generated and characterized an ATXN2-CAG42-knock-in mouse mutant. Our data show that the unspecific, chronically present mutation leads to progressive insolubility and to reduced soluble levels of the disease protein and of an interactor protein, which modulates RNA processing. Compensatory efforts are particularly weak in vulnerable tissue. They appear to include the increased degradation of the toxic disease protein by FBXW8. Thus the link between protein and RNA pathology becomes clear, and crucial molecular targets for preventive therapy are identified.
doi:10.1371/journal.pgen.1002920
PMCID: PMC3431311  PMID: 22956915
2.  Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis 
PLoS Genetics  2010;6(7):e1001021.
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a CAG repeat that encodes a polyglutamine tract in ATAXIN1 (ATXN1). Molecular and genetic data indicate that SCA1 is mainly caused by a gain-of-function mechanism. However, deletion of wild-type ATXN1 enhances SCA1 pathogenesis, whereas increased levels of an evolutionarily conserved paralog of ATXN1, Ataxin 1-Like, ameliorate it. These data suggest that a partial loss of ATXN1 function contributes to SCA1. To address this possibility, we set out to determine if the SCA1 disease model (Atxn1154Q/+ mice) and the loss of Atxn1 function model (Atxn1−/− mice) share molecular changes that could potentially contribute to SCA1 pathogenesis. To identify transcriptional changes that might result from loss of function of ATXN1 in SCA1, we performed gene expression microarray studies on cerebellar RNA from Atxn1−/− and Atxn1154Q/+ cerebella and uncovered shared gene expression changes. We further show that mild overexpression of Ataxin-1-Like rescues several of the molecular and behavioral defects in Atxn1−/− mice. These results support a model in which Ataxin 1-Like overexpression represses SCA1 pathogenesis by compensating for a partial loss of function of Atxn1. Altogether, these data provide evidence that partial loss of Atxn1 function contributes to SCA1 pathogenesis and raise the possibility that loss-of-function mechanisms contribute to other dominantly inherited neurodegenerative diseases.
Author Summary
Spinocerebellar Ataxia type 1 (SCA1) is one of nine neurodegenerative diseases caused by an increase in the number of the amino acid glutamine in their respective proteins. Genetic studies have pointed to the fact that the glutamine expansion in Ataxin-1 causes SCA1 by causing Ataxin-1 to gain some function(s). Here, we demonstrate that in addition to the toxic gain-of-function mechanism, partial loss of the normal functions of Ataxin-1 contributes to SCA1. Ataxin-1 forms protein complexes with Capicua, a protein that silences expression of other genes, and we found that in SCA1 mouse models the levels of these complexes are reduced, resulting in increased expression of some genes. We also demonstrate that increased levels of Ataxin-1-Like, a protein that is similar to Ataxin-1 and protects against mutant Ataxin-1 in mice, rescues molecular and behavioral defects in mice deficient in Ataxin-1. These results show that Ataxin-1-Like compensates for loss of Ataxin-1 and that Ataxin-1 and Ataxin-1-Like share some normal functions. Together, these findings suggest that rescue of SCA1 symptoms by Ataxin-1-Like could be partly due to restoration of lost normal functions of Ataxin-1 in mice that express the mutant polyglutamine-expanded Ataxin-1.
doi:10.1371/journal.pgen.1001021
PMCID: PMC2900305  PMID: 20628574
3.  Lithium Therapy Improves Neurological Function and Hippocampal Dendritic Arborization in a Spinocerebellar Ataxia Type 1 Mouse Model 
PLoS Medicine  2007;4(5):e182.
Background
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder characterized by progressive motor and cognitive dysfunction. Caused by an expanded polyglutamine tract in ataxin 1 (ATXN1), SCA1 pathogenesis involves a multifactorial process that likely begins with misfolding of ATXN1, which has functional consequences on its interactions, leading to transcriptional dysregulation. Because lithium has been shown to exert neuroprotective effects in a variety of conditions, possibly by affecting gene expression, we tested the efficacy of lithium treatment in a knock-in mouse model of SCA1 (Sca1154Q/2Q mice) that replicates many features of the human disease.
Methods and Findings
Sca1154Q/2Q mice and their wild-type littermates were fed either regular chow or chow that contained 0.2% lithium carbonate. Dietary lithium carbonate supplementation resulted in improvement of motor coordination, learning, and memory in Sca1154Q/2Q mice. Importantly, motor improvement was seen when treatment was initiated both presymptomatically and after symptom onset. Neuropathologically, lithium treatment attenuated the reduction of dendritic branching in mutant hippocampal pyramidal neurons. We also report that lithium treatment restored the levels of isoprenylcysteine carboxyl methyltransferase (Icmt; alternatively, Pccmt), down-regulation of which is an early marker of mutant ATXN1 toxicity.
Conclusions
The effect of lithium on a marker altered early in the course of SCA1 pathogenesis, coupled with its positive effect on multiple behavioral measures and hippocampal neuropathology in an authentic disease model, make it an excellent candidate treatment for human SCA1 patients.
Huda Zoghbi and colleagues show that lithium treatment initiated before or after disease onset improves multiple symptoms of neurodegeneration in a mouse model of spinocerebellar ataxia.
Editors' Summary
Background.
Spinocerebellar ataxia type 1 (SCA1) is an inherited, incurable neurodegenerative disease in which the neurons (cells that transmit information between the brain and body) in the cerebellum (the brain region that coordinates movement) gradually die. Symptoms of the disease, which usually begins in early adult life, include poor coordination of movement (ataxia), slurred speech, and cognitive (learning and memory) problems. As more neurons die, these symptoms get worse until breathing difficulties eventually cause death. SCA1 is a “triplet repeat disease.” Information for making proteins is stored in DNA as groups of three nucleotides (codons), each specifying a different amino acid (the building blocks of proteins). In triplet repeat diseases, patients inherit a mutant gene containing abnormally long stretches of repeated codons. In SCA1, the repeated codon is CAG, which specifies glutamine. Consequently, SCA1 is a “polyglutamine disease,” a group of neurodegenerative disorders in which an abnormal protein (a different one for each disease) containing a long stretch of glutamine forms nuclear inclusions (insoluble lumps of protein) in neurons that, possibly by trapping essential proteins, cause neuronal death. In SCA1, the abnormal protein is ataxin 1, which is made in many neurons including the cerebellar neurons (Purkinje cells) that coordinate movement.
Why Was This Study Done?
Early in SCA1, the production of several messenger RNAs (the templates for protein production) decreases, possibly because transcription factors (proteins that control gene expression) interact with the mutant protein. Could the progression of SCA1 be slowed, therefore, by using an agent that affects gene expression? In this study, the researchers have investigated whether lithium can slow disease progression in an animal model of SCA1. They chose lithium for their study because this drug (best known for stabilizing mood in people with bipolar [manic] depression) affects gene expression, is neuroprotective, and has beneficial effects in animal models of Huntington disease, another polyglutamine disease.
What Did the Researchers Do and Find?
The researchers bred mice carrying one mutant gene for ataxin 1 containing a very long CAG repeat and one normal gene (Sca1154Q/2Q mice; genes come in pairs). These mice develop symptoms similar to those seen in people with SCA1. After weaning, the mice and their normal littermates were fed normal food or food supplemented with lithium for several weeks before assessing their ability to coordinate their movements and testing their cognitive skills. Dietary lithium (given before or after symptoms appeared) improved both coordination and learning and memory in the Sca1154Q/2Q mice but had little effect in the normal mice. Lithium did not change the overall appearance of the cerebellum in the Sca1154Q/2Q mice nor reduce the occurrence of nuclear inclusions, but it did partly reverse hippocampal neuron degeneration in these animals. The researchers discovered this effect by examining the shape of the hippocampal neurons in detail. These neurons normally have extensive dendrites—branch-like projections that make contact with other cells—but these gradually disappear in Sca1154Q/2Q mice; lithium partly reversed this loss. Finally, lithium also restored the level of Icmt/Pccmt mRNA in the cerebellum to near normal in the Sca1154Q/2Q mice—this mRNA is one of the first to be reduced by ataxin 1 toxicity.
What Do These Findings Mean?
These findings show that treatment with lithium slows neurodegeneration in a mouse model of SCA1, even when it is given only after the first symptoms of the disease have appeared. Unfortunately, lithium did not improve the life span of the Sca1154Q/2Q mice (although this could be because the mutant SCA1 protein has some deleterious effects outside the brain). Thus, lithium is unlikely to cure SCA1, but it could provide some help to people with this devastating disease, even if (as is usual), their condition is not diagnosed until the disease is quite advanced. However, because drugs that work in animal models of diseases do not necessarily work in people, patients with SCA1 (or other polyglutamine diseases, which might also benefit from lithium supplementation) should not be treated with lithium until human trials of this approach have been completed.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040182.
The US National Ataxia Foundation provides information for patients
International Network of Ataxia Friends has information for patients and carergivers on ataxias, including SCA1
GeneTests provides information for health care providers and researchers about SCA1
Online Mendelian Inheritance in Man (OMIM) has detailed scientific information on SCA1
Huntington's Outreach Project for Education offers information for lay people from Stanford University on trinucleotide repeat disorders including SCA1
doi:10.1371/journal.pmed.0040182
PMCID: PMC1880853  PMID: 17535104
4.  Differential Degradation of Full-length and Cleaved Ataxin-7 Fragments in a Novel Stable Inducible SCA7 Model 
Journal of Molecular Neuroscience  2012;47(2):219-233.
Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine repeats, and a common toxic gain-of-function mechanism has been proposed. Proteolytic cleavage of several polyglutamine proteins has been identified and suggested to modulate the polyglutamine toxicity. In this study, we show that full-length and cleaved fragments of the SCA7 disease protein ataxin-7 (ATXN7) are differentially degraded. We found that the ubiquitin–proteosome system (UPS) was essential for the degradation of full-length endogenous ATXN7 or transgenic full-length ATXN7 with a normal or expanded glutamine repeat in both HEK 293T and stable PC12 cells. However, a similar contribution by UPS and autophagy was found for the degradation of proteolytically cleaved ATXN7 fragments. Furthermore, in our novel stable inducible PC12 model, induction of mutant ATXN7 expression resulted in toxicity and this toxicity was worsened by inhibition of either UPS or autophagy. In contrast, pharmacological activation of autophagy could ameliorate the ATXN7-induced toxicity. Based on our findings, we propose that both UPS and autophagy are important for the reduction of mutant ataxin-7-induced toxicity, and enhancing ATXN7 clearance through autophagy could be used as a potential therapeutic strategy in SCA7.
Electronic supplementary material
The online version of this article (doi:10.1007/s12031-012-9722-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s12031-012-9722-8
PMCID: PMC3360856  PMID: 22367614
Aggregation; Ataxin-7; Autophagy; Polyglutamine; Proteasome; SCA7
5.  PolyQ Repeat Expansions in ATXN2 Associated with ALS Are CAA Interrupted Repeats 
PLoS ONE  2011;6(3):e17951.
Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27–33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1–3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.
doi:10.1371/journal.pone.0017951
PMCID: PMC3066214  PMID: 21479228
6.  ETS1 regulates the expression of ATXN2 
Human Molecular Genetics  2012;21(23):5048-5065.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 phenotype is characterized by cerebellar ataxia, neuropathy and slow saccades. SCA2 foreshortens life span and is currently without symptomatic or disease-modifying treatments. Identifying function-specific therapeutics for SCA2 is problematic due to the limited knowledge of ATXN2 function. As SCA2 is likely caused by a gain-of-toxic or gain-of-normal function like other polyglutamine disorders, targeting ATXN2 expression may represent a valid therapeutic approach. This study characterized aspects of ATXN2 expression control using an ATXN2 promoter-luciferase (luc) reporter construct. We verified the fidelity of construct expression by generating transgenic mice expressing the reporter construct. High reporter expression was seen in the cerebellum and olfactory bulb in vivo but there was relatively low expression in other tissues, similar to the expression of endogenous ataxin-2. We verified the second of two possible start codons as the functional start codon in ATXN2. By evaluating deletions in the ATXN2 promoter, we identified an E-twenty six (ETS)-binding site required for ATXN2 expression. We verified that endogenous ETS1 interacted with the ATXN2 promoter by an electromobility supershift assay and chromatin immunoprecipitation polymerase chain reaction. ETS1 overexpression increased ATXN2-luc (ATXN2-luciferase) as well as endogenous ATXN2 expression. Deletion of the putative ETS1-binding site abrogated the effects on the expression of ATXN2-luc. A dominant negative ETS1 and an ETS1 short-hairpin RNA both reduced ATXN2-luc expression. Our study broadens the understanding on the transcriptional control of ATXN2 and reveals specific regulatory features of the ATXN2 promoter that can be exploited therapeutically.
doi:10.1093/hmg/dds349
PMCID: PMC3490512  PMID: 22914732
7.  ATXN2 and Its Neighbouring Gene SH2B3 Are Associated with Increased ALS Risk in the Turkish Population 
PLoS ONE  2012;7(8):e42956.
Expansions of the polyglutamine (polyQ) domain (≥34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27–33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1–4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31–32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p = 0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway.
doi:10.1371/journal.pone.0042956
PMCID: PMC3423429  PMID: 22916186
8.  The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis 
PLoS Genetics  2015;11(1):e1004749.
DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.
Author Summary
We report that human polynucleotide kinase 3’-phosphatase (PNKP), a major DNA strand break repair enzyme, stably associates with Ataxin-3 (ATXN3). This protein contains repeats of the amino acid glutamine, and the expansion of these repeats from 14–41 to 55–82 glutamines leads to a neurological disorder called Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). However, how this expansion of glutamine leads to ataxia has remained unclear. Here we show that normal ATXN3 protein stimulates, but the expanded ATXN3 inhibits, PNKP’s DNA repair activity, causing an accumulation of DNA damage. Furthermore, a SCA3 mouse model showed decreased PNKP activity, mostly in a region that is highly affected in MJD patients’ brains. Analysis of human MJD patients’ neuronal DNA showed significant accumulation of DNA strand breaks. Collectively, the accumulation of DNA damage due to decreased PNKP repair activity is likely to induce neuronal cell death, a hallmark of SCA3/MJD pathogenesis.
doi:10.1371/journal.pgen.1004749
PMCID: PMC4310589  PMID: 25633985
9.  Machado-Joseph Disease: from first descriptions to new perspectives 
Machado-Joseph Disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), represents the most common form of SCA worldwide. MJD is an autosomal dominant neurodegenerative disorder of late onset, involving predominantly the cerebellar, pyramidal, extrapyramidal, motor neuron and oculomotor systems; although sharing features with other SCAs, the identification of minor, but more specific signs, facilitates its differential diagnosis. MJD presents strong phenotypic heterogeneity, which has justified the classification of patients into three main clinical types. Main pathological lesions are observed in the spinocerebellar system, as well as in the cerebellar dentate nucleus. MJD's causative mutation consists in an expansion of an unstable CAG tract in exon 10 of the ATXN3 gene, located at 14q32.1. Haplotype-based studies have suggested that two main founder mutations may explain the present global distribution of the disease; the ancestral haplotype is of Asian origin, and has an estimated age of around 5,800 years, while the second mutational event has occurred about 1,400 years ago. The ATXN3 gene encodes for ataxin-3, which is ubiquitously expressed in neuronal and non-neuronal tissues, and, among other functions, is thought to participate in cellular protein quality control pathways. Mutated ATXN3 alleles consensually present about 61 to 87 CAG repeats, resulting in an expanded polyglutamine tract in ataxin-3. This altered protein gains a neurotoxic function, through yet unclear mechanisms. Clinical variability of MJD is only partially explained by the size of the CAG tract, which leaves a residual variance that should be explained by still unknown additional factors. Several genetic tests are available for MJD, and Genetic Counseling Programs have been created to better assist the affected families, namely on what concerns the possibility of pre-symptomatic testing. The main goal of this review was to bring together updated knowledge on MJD, covering several aspects from its initial descriptions and clinical presentation, through the discovery of the causative mutation, its origin and dispersion, as well as molecular genetics aspects considered essential for a better understanding of its neuropathology. Issues related with molecular testing and Genetic Counseling, as well as recent progresses and perspectives on genetic therapy, are also addressed.
doi:10.1186/1750-1172-6-35
PMCID: PMC3123549  PMID: 21635785
Ataxin-3; ATXN3 gene; CAG repeats; Polyglutamine disorders; SCA3
10.  Spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces RNA foci 
Background
Spinocerebellar ataxia type 8 (SCA8) involves the expression of an expanded CTG/CAG combined repeats (CR) from opposite strands producing CUG expansion transcripts (ataxin 8 opposite strand, ATXN8OS) and a polyglutamine expansion protein (ataxin 8, ATXN8). The pathogenesis of SCA8 is complex and the spectrum of clinical presentations is broad.
Results
Using stably induced cell models expressing 0, 23, 88 and 157 CR, we study the role of ATXN8OS transcripts in SCA8 pathogenesis. In the absence of doxycycline, the stable ATXN8OS CR cell lines exhibit low levels of ATXN8OS expression and a repeat length-related increase in staurosporine sensitivity and in the number of annexin positive cells. A repeat length-dependent repression of ATXN8OS expression was also notable. Addition of doxycycline leads to 25~50 times more ATXN8OS RNA expression with a repeat length-dependent increase in fold of ATXN8OS RNA induction. ChIP-PCR assay using anti-dimethyl-histone H3-K9 and anti-acetyl-histone H3-K14 antibodies revealed increased H3-K9 dimethylation and reduced H3-K14 acetylation around the ATXN8OS cDNA gene in 157 CR line. The repeat length-dependent increase in induction fold is probably due to the increased RNA stability as demonstrated by monitoring ATXN8OS RNA decay in cells treated with the transcriptional inhibitor, actinomycin D. In cells stably expressing ATXN8OS, RNA FISH experiments further revealed ribonuclear foci formation in cells carrying expanded 88 and 157 CR.
Conclusion
The present study demonstrates that the expanded CUG-repeat tracts are toxic to human cells and may affect ATXN8OS RNA expression and stability through epigenetic and post-transcriptional mechanisms.
doi:10.1186/1471-2199-10-9
PMCID: PMC2647542  PMID: 19203395
11.  Comparison of an expanded ataxia interactome with patient medical records reveals a relationship between macular degeneration and ataxia 
Human Molecular Genetics  2010;20(3):510-527.
Spinocerebellar ataxias 6 and 7 (SCA6 and SCA7) are neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine (polyQ) tracts in CACNA1A, the alpha1A subunit of the P/Q-type calcium channel, and ataxin-7 (ATXN7), a component of a chromatin-remodeling complex, respectively. We hypothesized that finding new protein partners for ATXN7 and CACNA1A would provide insight into the biology of their respective diseases and their relationship to other ataxia-causing proteins. We identified 118 protein interactions for CACNA1A and ATXN7 linking them to other ataxia-causing proteins and the ataxia network. To begin to understand the biological relevance of these protein interactions within the ataxia network, we used OMIM to identify diseases associated with the expanded ataxia network. We then used Medicare patient records to determine if any of these diseases co-occur with hereditary ataxia. We found that patients with ataxia are at 3.03-fold greater risk of these diseases than Medicare patients overall. One of the diseases comorbid with ataxia is macular degeneration (MD). The ataxia network is significantly (P= 7.37 × 10−5) enriched for proteins that interact with known MD-causing proteins, forming a MD subnetwork. We found that at least two of the proteins in the MD subnetwork have altered expression in the retina of Ataxin-7266Q/+ mice suggesting an in vivo functional relationship with ATXN7. Together these data reveal novel protein interactions and suggest potential pathways that can contribute to the pathophysiology of ataxia, MD, and diseases comorbid with ataxia.
doi:10.1093/hmg/ddq496
PMCID: PMC3016911  PMID: 21078624
12.  14-3-3 Binding to Ataxin-1(ATXN1) Regulates Its Dephosphorylation at Ser-776 and Transport to the Nucleus* 
The Journal of Biological Chemistry  2011;286(40):34606-34616.
Background: Phosphorylation at Ser-776 of the polyglutamine disease-associated protein Ataxin-1 modulates its function.
Results: 14-3-3 binding stabilizes Ataxin-1 by blocking dephosphorylation of pS776 and impedes Ataxin-1 transport to the nucleus.
Conclusion: 14-3-3 must disassociate from Ataxin-1 for its transport to the nucleus.
Significance: 14-3-3 regulates Ataxin-1 function by protecting phosphorylation of Ser-776 and Ataxin-1 entry into the nucleus.
Spinocerebellar ataxia type 1 (SCA1) is a lethal neurodegenerative disorder caused by expansion of a polyglutamine tract in ATXN1. A prominent site of pathology in SCA1 is cerebellar Purkinje neurons where mutant ATXN1 must enter the nucleus to cause disease. In SCA1, phosphorylation of ATXN1 at Ser-776 modulates disease. Interestingly, Ser-776 is located within a region of ATXN1 that harbors several functional motifs including binding sites for 14-3-3, and splicing factors RBM17 and U2AF65. The interaction of ATXN1 with these proteins is thought to be regulated by the phosphorylation status of Ser-776. In addition, Ser-776 is adjacent to the NLS in ATXN1. Although pS776-ATXN1 is enriched in nuclear extracts of cerebellar cells, the vast majority of 14-3-3 is in the cytoplasmic fraction. We found that dephosphorylation of cytoplasmic pS776-ATXN1 is blocked by virtue of it being in a complex with 14-3-3. In addition, data suggest that binding of 14-3-3 to cytoplasmic ATXN1 impeded its transport to the nucleus, suggesting that 14-3-3 must disassociate from ATXN1 for transport of ATXN1 to the nucleus. Consistent with this hypothesis is the observation that once in the nucleus pS776 is able to be dephosphorylated. Evidence is presented that PP2A is the pS776-ATXN1 phosphatase in the mammalian cerebellum. In the nucleus, we propose that dephosphorylation of pS776-ATXN1 by PP2A regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65.
doi:10.1074/jbc.M111.238527
PMCID: PMC3186404  PMID: 21835928
Ataxia; Neurodegeneration; Nuclear Translocation; Phosphatase; PP2A; 14-3-3
13.  Partial loss of Tip60 slows mid-stage neurodegeneration in a spinocerebellar ataxia type 1 (SCA1) mouse model 
Human Molecular Genetics  2011;20(11):2204-2212.
Spinocerebellar ataxia type 1 (SCA1) is one of nine dominantly inherited neurodegenerative diseases caused by polyglutamine tract expansion. In SCA1, the expanded polyglutamine tract is in the ataxin-1 (ATXN1) protein. ATXN1 is part of an in vivo complex with retinoid acid receptor-related orphan receptor alpha (Rora) and the acetyltransferase tat-interactive protein 60 kDa (Tip60). ATXN1 and Tip60 interact directly via the ATXN1 and HMG-box protein 1 (AXH) domain of ATXN1. Moreover, the phospho-mimicking Asp amino acid at position 776, previously shown to enhance pathogenesis, increases the ability of ATXN1 to interact with Tip60. Using a genetic approach, the biological relevance of the ATXN1/Tip60 interaction was assessed by crossing ATXN1[82Q] mice with Tip60+/−animals. Partial Tip60 loss increased Rora and Rora-mediated gene expression and delayed ATXN1[82]-mediated cerebellar degeneration during mid-stage disease progression. These results suggested a specific, temporal role for Tip60 during disease progression. We also showed that genetic background modulated ATXN1[82Q]-induced phenotypes. Of interest, these latter studies showed that some phenotypes are enhanced on a mixed background while others are suppressed.
doi:10.1093/hmg/ddr108
PMCID: PMC3090197  PMID: 21427130
14.  Genetic Variance in the Spinocerebellar Ataxia Type 2 (ATXN2) Gene in Children with Severe Early Onset Obesity 
PLoS ONE  2009;4(12):e8280.
Background
Expansion of a CAG repeat in the coding region of exon 1 in the ATXN2 gene located in human chromosome 12q24.1 causes the neurodegenerative disease spinocerebellar ataxia type 2 (SCA2). In contrast to other polyglutamine (polyQ) disorders, the SCA2 repeat is not highly polymorphic in central European (CEU) controls with Q22 representing 90% of alleles, and Q23 contributing between 5–7% of alleles. Recently, the ATXN2 CAG repeat has been identified as a target of adaptive selection in the CEU population. Mouse lines deficient for atxn2 develop marked hyperphagia and obesity raising the possibility that loss-of-function mutations in the ATXN2 gene may be related to energy balance in humans. Some linkage studies of obesity related phenotypes such as antipsychotic induced weight gain have reported significant lod scores on chromosome 12q24. We tested the hypothesis that rare loss-of-function ATXN2 variants cause obesity analogous to rare mutations in the leptin, leptin receptor and MC4R genes.
Methodology/Principal Findings
We sequenced the coding region of ATXN2 including intron-exon boundaries in 92 severely obese children with a body mass index (BMI) >3.2 standard deviations above age- and gender-adjusted means. We confirmed five previously identified single nucleotide polymorphisms (SNPs) and three new SNPs resulting in two synonymous substitutions and one intronic polymorphism. Alleles encoding >Q22 were overrepresented in our sample of obese children and contributed 15% of alleles in children identified by their parents as white. SNP rs695872 closely flanking the CAG repeat showed a greatly increased frequency of C/C homozygotes and G/C heterozygotes compared with reported frequencies in the CEU population.
Conclusions/Significance
Although we did not identify variants leading to novel amino acid substitutions, nonsense or frameshift mutations, this study warrants further examination of variation in the ATXN2 gene in obesity and related phenotypes in a larger case-control study with emphasis on rs695872 and CAG repeat structure.
doi:10.1371/journal.pone.0008280
PMCID: PMC2791421  PMID: 20016785
15.  Inactivation of PNKP by Mutant ATXN3 Triggers Apoptosis by Activating the DNA Damage-Response Pathway in SCA3 
PLoS Genetics  2015;11(1):e1004834.
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3’-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.
Author Summary
Spinocerebellar ataxia type 3 (SCA3) is an untreatable neurodegenerative disease, and the most common dominantly inherited ataxia worldwide. SCA3 is caused by expansion of a CAG tri-nucleotide repeat sequence in the ATXN3 gene’s coding region. The expanded CAG sequences encode a run of the amino acid glutamine; the mutant ATXN3 interacts with multiple proteins in vivo to create insoluble aggregates in SCA3 brains. It is thought that the loss of function of the aggregated proteins contributes to cellular toxicity and neurodegeneration in SCA3. Despite significant progress in understanding SCA3’s etiology, the molecular mechanism by which the mutant protein triggers the death of neurons in SCA3 brains remains unknown. We now report that the mutant ATXN3 protein interacts with and inactivates PNKP (polynucleotide kinase 3’-phosphatase), an essential DNA strand break repair enzyme. This inactivation results in persistent accumulation of DNA damage, and chronic activation of the DNA damage-response ATM signaling pathway in SCA3. Our work suggests that persistent DNA damage/strand breaks and chronic activation of ATM trigger neuronal death in SCA3. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death pathways provides a molecular basis for neurodegeneration in SCA3, and perhaps ultimately for its treatment.
doi:10.1371/journal.pgen.1004834
PMCID: PMC4295939  PMID: 25590633
16.  Requirement for Zebrafish Ataxin-7 in Differentiation of Photoreceptors and Cerebellar Neurons 
PLoS ONE  2012;7(11):e50705.
The expansion of a polyglutamine (polyQ) tract in the N-terminal region of ataxin-7 (atxn7) is the causative event in spinocerebellar ataxia type 7 (SCA7), an autosomal dominant neurodegenerative disorder mainly characterized by progressive, selective loss of rod-cone photoreceptors and cerebellar Purkinje and granule cells. The molecular and cellular processes underlying this restricted neuronal vulnerability, which contrasts with the broad expression pattern of atxn7, remains one of the most enigmatic features of SCA7, and more generally of all polyQ disorders. To gain insight into this specific neuronal vulnerability and achieve a better understanding of atxn7 function, we carried out a functional analysis of this protein in the teleost fish Danio rerio. We characterized the zebrafish atxn7 gene and its transcription pattern, and by making use of morpholino-oligonucleotide-mediated gene inactivation, we analysed the phenotypes induced following mild or severe zebrafish atxn7 depletion. Severe or nearly complete zebrafish atxn7 loss-of-function markedly impaired embryonic development, leading to both early embryonic lethality and severely deformed embryos. More importantly, in relation to SCA7, moderate depletion of the protein specifically, albeit partially, prevented the differentiation of both retina photoreceptors and cerebellar Purkinje and granule cells. In addition, [1–232] human atxn7 fragment rescued these phenotypes showing strong function conservation of this protein through evolution. The specific requirement for zebrafish atxn7 in the proper differentiation of cerebellar neurons provides, to our knowledge, the first in vivo evidence of a direct functional relationship between atxn7 and the differentiation of Purkinje and granule cells, the most crucial neurons affected in SCA7 and most other polyQ-mediated SCAs. These findings further suggest that altered protein function may play a role in the pathophysiology of the disease, an important step toward the development of future therapeutic strategies.
doi:10.1371/journal.pone.0050705
PMCID: PMC3511343  PMID: 23226359
17.  dAtaxin-2 Mediates Expanded Ataxin-1-Induced Neurodegeneration in a Drosophila Model of SCA1 
PLoS Genetics  2007;3(12):e234.
Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders sharing atrophy of the cerebellum as a common feature. SCA1 and SCA2 are two ataxias caused by expansion of polyglutamine tracts in Ataxin-1 (ATXN1) and Ataxin-2 (ATXN2), respectively, two proteins that are otherwise unrelated. Here, we use a Drosophila model of SCA1 to unveil molecular mechanisms linking Ataxin-1 with Ataxin-2 during SCA1 pathogenesis. We show that wild-type Drosophila Ataxin-2 (dAtx2) is a major genetic modifier of human expanded Ataxin-1 (Ataxin-1[82Q]) toxicity. Increased dAtx2 levels enhance, and more importantly, decreased dAtx2 levels suppress Ataxin-1[82Q]-induced neurodegeneration, thereby ruling out a pathogenic mechanism by depletion of dAtx2. Although Ataxin-2 is normally cytoplasmic and Ataxin-1 nuclear, we show that both dAtx2 and hAtaxin-2 physically interact with Ataxin-1. Furthermore, we show that expanded Ataxin-1 induces intranuclear accumulation of dAtx2/hAtaxin-2 in both Drosophila and SCA1 postmortem neurons. These observations suggest that nuclear accumulation of Ataxin-2 contributes to expanded Ataxin-1-induced toxicity. We tested this hypothesis engineering dAtx2 transgenes with nuclear localization signal (NLS) and nuclear export signal (NES). We find that NLS-dAtx2, but not NES-dAtx2, mimics the neurodegenerative phenotypes caused by Ataxin-1[82Q], including repression of the proneural factor Senseless. Altogether, these findings reveal a previously unknown functional link between neurodegenerative disorders with common clinical features but different etiology.
Author Summary
The spinocerebellar ataxias (SCAs) are a group of ∼30 neurodegenerative disorders caused by different types of mutations in a variety of unrelated genes. For example, SCA1 and SCA2 are caused by mutations in Ataxin-1 and Ataxin-2, two proteins related in name only. Despite these differences, most SCAs share a number of striking clinical and neuropathological similarities, such as ataxia, tremor, speech difficulties, and atrophy of the cerebellum and brainstem. In addition, many ataxia-causing proteins share interacting protein partners. Together, these observations suggest that many SCAs also share common mechanisms of pathogenesis. Here, we report previously unknown functional interactions between the genes and proteins responsible for SCA1 and SCA2. We find that Ataxin-1 and Ataxin-2 physically interact, and that mutant Ataxin-1 forces Ataxin-2 to accumulate in the nucleus instead of the cytoplasm. Most importantly, using an animal model, we discovered that the Drosophila Ataxin-2 gene is a strong suppressor of Ataxin-1-induced neurotoxicity. Thus, neuronal degeneration may take place through common mechanisms in different SCAs. These findings open the possibility of future common therapies for these neurodegenerative disorders for which there is no effective treatment.
doi:10.1371/journal.pgen.0030234
PMCID: PMC2323314  PMID: 18166084
18.  De Novo Mutations in Ataxin-2 Gene and ALS Risk 
PLoS ONE  2013;8(8):e70560.
Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2) is the genetic cause of spinocerebellar ataxia type 2 (SCA2). Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS). Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.
doi:10.1371/journal.pone.0070560
PMCID: PMC3735591  PMID: 23936447
19.  Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1 
Polyglutamine diseases are dominantly inherited neurodegenerative diseases caused by an expansion of a CAG trinucleotide repeat encoding a glutamine tract in the respective disease-causing proteins. Extensive studies have been performed to unravel disease pathogenesis and to develop therapeutics. Here, we report on several lines of evidence demonstrating that Nemo-like kinase (NLK) is a key molecule modulating disease toxicity in spinocerebellar ataxia type 1 (SCA1), a disease caused by a polyglutamine expansion in the protein ATAXIN1 (ATXN1). Specifically, we show that NLK, a serine/threonine kinase that interacts with ATXN1, modulates disease phenotypes of polyglutamine-expanded ATXN1 in a Drosophila model of SCA1. Importantly, the effect of NLK on SCA1 pathology is dependent upon NLK’s enzymatic activity. Consistent with this, reduced Nlk expression suppresses the behavioral and neuropathological phenotypes in SCA1 knock-in mice. These data clearly indicate that either reducing NLK enzymatic activity or decreasing NLK expression levels can have beneficial effects against the toxicity induced by polyglutamine-expanded ATXN1.
doi:10.1523/JNEUROSCI.3465-12.2013
PMCID: PMC3710458  PMID: 23719801
20.  Atxn1 protein family and Cic regulate extracellular matrix remodeling and lung alveolarization 
Developmental Cell  2011;21(4):746-757.
Summary
Although expansion of CAG repeats in ATAXIN1 (ATXN1) causes Spinocerebellar ataxia type 1, the functions of ATXN1 and ATAXIN1-Like (ATXN1L) remain poorly understood. To investigate the function of these proteins, we generated and characterized Atxn1L−/− and Atxn1−/−; Atxn1L−/− mice. Atxn1L−/− mice have hydrocephalus, omphalocoele and lung alveolarization defects. These phenotypes are more penetrant and severe in Atxn1−/−; Atxn1L−/− mice, suggesting that Atxn1 and Atxn1L are functionally redundant. Upon pursuing the molecular mechanism, we discovered that several Matrix metalloproteinase (Mmp) genes are overexpressed and that the transcriptional repressor Capicua (Cic) is destabilized in Atxn1L−/− lungs. Consistent with this, Cic deficiency causes lung alveolarization defect. Loss of either Atxn1L or Cic derepresses Etv4, an activator for Mmp genes, thereby mediating Mmp9 overexpression. These findings demonstrate a critical role of ATXN1/ATXN1L-CIC complexes in extracellular matrix (ECM) remodeling during development and their potential roles in pathogenesis of disorders affecting ECM remodeling.
doi:10.1016/j.devcel.2011.08.017
PMCID: PMC3253850  PMID: 22014525
21.  Phosphorylation of ATXN1 at Ser776 in the cerebellum 
Journal of neurochemistry  2009;110(2):675-686.
Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative disorders caused by a mutant protein with an expanded polyglutamine tract. Phosphorylation of ataxin-1 (ATXN1) at serine 776 is implicated in SCA1 pathogenesis. Previous studies, utilizing transfected cell lines and a Drosophila photoreceptor model of SCA1, suggest that phosphorylating ATXN1 at S776 renders it less susceptible to degradation. This work also indicated that oncogene from AKR mouse thymoma (Akt) promotes the phosphorylation of ATXN1 at S776 and severity of neurodegeneration. Here, we examined the phosphorylation of ATXN1 at S776 in cerebellar Purkinje cells, a prominent site of pathology in SCA1. We found that while phosphorylation of S776 is associated with a stabilization of ATXN1 in Purkinje cells, inhibition of Akt either in vivo or in a cerebellar extract-based phosphorylation assay did not decrease the phosphorylation of ATXN1-S776. In contrast, immunodepletion and inhibition of cyclic AMP-dependent protein kinase decreased phosphorylation of ATXN1-S776. These results argue against Akt as the in vivo kinase that phosphorylates S776 of ATXN1 and suggest that cyclic AMP-dependent protein kinase is the active ATXN1-S776 kinase in the cerebellum.
doi:10.1111/j.1471-4159.2009.06164.x
PMCID: PMC2754139  PMID: 19500214
ataxin-1; cyclic AMP-dependent protein kinase; oncogene from AKR mouse thymoma; phosphorylation; spinocerebellar ataxia type 1
22.  Characterization of the Zebrafish atxn1/axh Gene Family 
Journal of neurogenetics  2008;23(3):313-323.
In mammals, ataxin-1 (ATXN1) is a member of a family of proteins in which each member contains an AXH domain. Expansion of the polyglutamine tract in ATXN1 causes the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) with prominent cerebellar pathology. Toward a further characterization of the genetic diversification of the ATXN1/AXH gene family, we identified and characterized members of this gene family in zebrafish, a lower vertebrate with a cerebellum. The zebrafish genome encodes two ATXN1 homologs, atxn1a and atxn1b, and one ATXN1L homolog, atxn1l. Key biochemical features of the human ATXN1 protein not seen in the invertebrate homologs (a nuclear localization sequence and a site of phosphorylation at serine 776) are conserved in the zebrafish homologs, and all three zebrafish Atxn1/Axh proteins behave similarly to their human counterparts in tissue culture cells. Importantly, each of the three homologs is expressed in the zebrafish cerebellum, which in humans is a prominent site of SCA1 pathogenesis. In addition, atxn1a and atxn1b are expressed in the developing zebrafish cerebellum. These data show that in zebrafish, a lower vertebrate, the complexity of the atxn1/axh gene family is more similar to higher vertebrates than invertebrates with a simple central nervous system and suggests a relationship between the diversification of the ATXN1/AXH gene family and the development of a complex central nervous system including a cerebellum.
doi:10.1080/01677060802399976
PMCID: PMC2722686  PMID: 19085187
ataxin-1; ataxin-1 like; AXH; SCA1; Danio rerio
23.  The Role of Interruptions in polyQ in the Pathology of SCA1 
PLoS Genetics  2013;9(7):e1003648.
At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.
Author Summary
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder resulting in loss of coordination and balance. It is caused by an expanded repeated DNA sequence (CAG) in the gene ATXN1. The CAG repeat region is normally interrupted by the DNA sequence CAT. Loss of this interruption is believed to cause instability whereby the CAG repeat expands beyond a key threshold resulting, ultimately, in polyglutamine protein aggregation and cell death. Here we examine how interruptions influence pathology in patients and establish a cellular model to support our findings. We distinguish our patients into two sub-groups based on whether or not their expanded CAG repeat stretches contained an interruption. This is not possible with conventional diagnostic techniques. Differentiating the sub-group with no interruptions led to improved accuracy in predicting their age at onset. The other sub-group, with interruptions, reveals a delay in age at onset that shows greater alignment with the longest stretch of CAG repeats. These findings are significant for genetic counselling and prognosis. Our cellular model and in vitro studies confirmed the relationship between disease severity and uninterrupted repeat length and showed that interruptions do not significantly affect the polyglutamine protein aggregation, but do slow down the aggregation rate.
doi:10.1371/journal.pgen.1003648
PMCID: PMC3723530  PMID: 23935513
24.  Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS 
Nature  2010;466(7310):1069-1075.
Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disease. The causes of ALS are poorly understood, although the protein TDP-43 has been suggested to play a critical role in disease pathogenesis. Here we show that Ataxin-2, a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2 (SCA2), is a potent modifier of TDP-43 toxicity in animal and cellular models. The proteins associate in a complex that depends on RNA. Ataxin-2 is abnormally localized in spinal cord neurons of ALS patients. Likewise, TDP-43 shows mislocalization in SCA2. To assess a role in ALS, we analyzed the Ataxin-2 gene (ATXN2) in 915 ALS patients. We found intermediate-length polyQ expansions (27–33 Qs) in ATXN2 significantly associated with ALS. These data establish ATXN2 as a relatively common ALS disease susceptibility gene. Further, these findings indicate that the TDP-43/Ataxin-2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.
doi:10.1038/nature09320
PMCID: PMC2965417  PMID: 20740007
25.  Ataxin-2 Modulates the Levels of Grb2 and Src but Not Ras Signaling 
Ataxin-2 (ATXN2) is implicated mainly in mRNA processing. Some ATXN2 associates with receptor tyrosine kinases (RTK), inhibiting their endocytic internalization through interaction of proline-rich domains (PRD) in ATXN2 with SH3 motifs in Src. Gain of function of ATXN2 leads to neuronal atrophy in the diseases spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS). Conversely, ATXN2 knockout (KO) mice show hypertrophy and insulin resistance. To elucidate the influence of ATXN2 on trophic regulation, we surveyed interactions of ATXN2 with SH3 motifs from numerous proteins and observed a novel interaction with Grb2. Direct binding in glutathione S-transferase (GST) pull-down assays and coimmunoprecipitation of the endogenous proteins indicated a physiologically relevant association. In SCA2 patient fibroblasts, Grb2 more than Src protein levels were diminished, with an upregulation of both transcripts suggesting enhanced protein turnover. In KO mouse embryonal fibroblasts (MEF), the protein levels of Grb2 and Src were decreased. ATXN2 absence by itself was insufficient to significantly change Grb2-dependent signaling for endogenous Ras levels, Ras-GTP levels, and kinetics as well as MEK1 phosphorylation, suggesting that other factors compensate for proliferation control. In KO tissue with postmitotic neurons, a significant decrease of Src protein levels is prominent rather than Grb2. ATXN2 mutations modulate the levels of several components of the RTK endocytosis complex and may thus contribute to alter cell proliferation as well as translation and growth.
doi:10.1007/s12031-012-9949-4
PMCID: PMC3739869  PMID: 23335000
SCA2; Ataxin-2; Grb2; Src; Ras; Receptor tyrosine kinases; Endocytosis; Proliferation

Results 1-25 (1097609)