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1.  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.
PMCID: PMC3735591  PMID: 23936447
2.  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.
PMCID: PMC3360856  PMID: 22367614
Aggregation; Ataxin-7; Autophagy; Polyglutamine; Proteasome; SCA7
3.  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.
PMCID: PMC3423429  PMID: 22916186
4.  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.
PMCID: PMC2900305  PMID: 20628574
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.
PMCID: PMC3066214  PMID: 21479228
6.  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.
PMCID: PMC2965417  PMID: 20740007
7.  Spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces RNA foci 
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.
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.
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.
PMCID: PMC2647542  PMID: 19203395
8.  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.
PMCID: PMC2754139  PMID: 19500214
ataxin-1; cyclic AMP-dependent protein kinase; oncogene from AKR mouse thymoma; phosphorylation; spinocerebellar ataxia type 1
9.  LANP mediates neuritic pathology in Spinocerebellar ataxia type 1 
Neurobiology of disease  2012;48(3):526-532.
Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease that results from a pathogenic glutamine-repeat expansion in the protein ataxin-1 (ATXN1). Although the functions of ATXN1 are still largely unknown, there is evidence to suggest that ATXN1 plays a role in regulating gene expression, the earliest process known to go awry in SCA1 mouse models. In this study, we show that ATXN1 reduces histone acetylation, a post-translational modification of histones associated with enhanced transcription, and represses histone acetyl transferase-mediated transcription. In addition, we find that depleting the Leucine-rich Acidic Nuclear Protein (LANP)—an ATXN1 binding inhibitor of histone acetylation—reverses aspects of SCA1 neuritic pathology.
PMCID: PMC3987943  PMID: 22884877
LANP; pp32; ANP32-A; Spinocerebellar ataxia type 1; SCA1; Neurite outgrowth
10.  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.
PMCID: PMC3090197  PMID: 21427130
11.  Exercise and Genetic Rescue of SCA1 via the Transcriptional Repressor Capicua* 
Science (New York, N.Y.)  2011;334(6056):690-693.
Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). To determine the long-term effects of exercise, we implemented a mild exercise regimen in a mouse model of SCA1 and found a considerable improvement in survival accompanied by upregulation of epidermal growth factor and consequential downregulation of Capicua, an ATXN1 interactor. Offspring of Capicua mutant mice bred to SCA1 mice showed significant improvement of all disease phenotypes. Although polyglutamine-expanded Atxn1 caused some loss of Capicua function, further reducing Capicua levels, either genetically or by exercise, mitigated the disease phenotypes. Thus, exercise might have long-term beneficial effects in other ataxias and neurodegenerative diseases.
PMCID: PMC3232424  PMID: 22053053
12.  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.
PMCID: PMC3710458  PMID: 23719801
13.  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.
PMCID: PMC3016911  PMID: 21078624
14.  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.
PMCID: PMC2323314  PMID: 18166084
15.  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.
PMCID: PMC3739869  PMID: 23335000
SCA2; Ataxin-2; Grb2; Src; Ras; Receptor tyrosine kinases; Endocytosis; Proliferation
16.  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.
PMCID: PMC2722686  PMID: 19085187
ataxin-1; ataxin-1 like; AXH; SCA1; Danio rerio
17.  Caring for Machado-Joseph Disease: current understanding and how to help patients 
Machado-Joseph disease or spinocerebellar ataxia 3 (MJD/SCA3) is a clinically heterogeneous, neurodegenerative disorder characterized by varying degrees of ataxia, ophthalmoplegia, peripheral neuropathy, pyramidal dysfunction and movement disorder. MJD/SCA3 is caused by a CAG repeat expansion mutation in the protein coding region of the ATXN3 gene located at chromosome 14q32.1. Current hypotheses regarding pathogenesis favor the view that mutated ataxin-3, with its polyglutamine expansion, is prone to adopt an abnormal conformation, engage in altered protein-protein interactions and aggregate. Expanded CAG repeat length correlates with the range and severity of the clinical manifestations and inversely correlates with age of disease onset. Though MJD/SCA3 is classically described as affecting the cerebellum, brain stem and basal ganglia, recent neuropathology and neuroimaging series demonstrate involvement of other areas such as the thalamus and cerebral cortex. Clinically, much emphasis has been placed in the description and recognition of the non-motor symptoms observed in these patients, such as pain, cramps, fatigue and depression. Currently, no disease modifying treatment exists for MJD/SCA3. Standard of care includes genetic counseling, exercise/physical therapy programs, and speech and swallow evaluation. Symptomatic treatment for clinical findings such as depression, sleep disorders, parkinsonism, dystonia, cramps, and pain is important to improve the quality of life for those with MJD/SCA3.
PMCID: PMC2818316  PMID: 19811945
Spinocerebellar ataxia type 3; SCA3; Machado-Joseph disease; CAG
18.  Functional Annotation of Small Noncoding RNAs Target Genes Provides Evidence for a Deregulated Ubiquitin-Proteasome Pathway in Spinocerebellar Ataxia Type 1 
Journal of Nucleic Acids  2012;2012:672536.
Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by the expansion of CAG repeats in the ataxin 1 (ATXN1) gene. In affected cerebellar neurons of patients, mutant ATXN1 accumulates in ubiquitin-positive nuclear inclusions, indicating that protein misfolding is involved in SCA1 pathogenesis. In this study, we functionally annotated the target genes of the small noncoding RNAs (ncRNAs) that were selectively activated in the affected brain compartments. The primary targets of these RNAs, which exhibited a significant enrichment in the cerebellum and cortex of SCA1 patients, were members of the ubiquitin-proteasome system. Thus, we identified and functionally annotated a plausible regulatory pathway that may serve as a potential target to modulate the outcome of neurodegenerative diseases.
PMCID: PMC3471453  PMID: 23094141
19.  Genetic Variance in the Spinocerebellar Ataxia Type 2 (ATXN2) Gene in Children with Severe Early Onset Obesity 
PLoS ONE  2009;4(12):e8280.
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.
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.
PMCID: PMC2791421  PMID: 20016785
20.  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.
PMCID: PMC3186404  PMID: 21835928
Ataxia; Neurodegeneration; Nuclear Translocation; Phosphatase; PP2A; 14-3-3
21.  Lithium Therapy Improves Neurological Function and Hippocampal Dendritic Arborization in a Spinocerebellar Ataxia Type 1 Mouse Model 
PLoS Medicine  2007;4(5):e182.
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.
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
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
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
PMCID: PMC1880853  PMID: 17535104
22.  Transgenic Models of Spinocerebellar Ataxia Type 10: Modeling a Repeat Expansion Disorder 
Genes  2012;3(3):481-491.
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease with a spectrum of phenotypes. SCA10 is caused by a pentanucleotide repeat expansion of the ATTCT motif within intron 9 of ATAXIN 10 (ATXN10). Patients present with cerebellar ataxia; however, a subset also develops epileptic seizures which significantly contribute to the morbidity and mortality of the disease. Past research from our lab has demonstrated that epileptic SCA10 patients predominantly originate from or have ancestral ties to Mexico. In addition, a large proportion of epileptic SCA10 patients carry repeat interruptions within their SCA10 expansion. This paper outlines the variability in SCA10 phenotypes and our attempts to model these phenotypes using transgenic mouse models and highlights the benefits of using a transgenic model organism to understand the pathological mechanisms of a human disease.
PMCID: PMC3899997  PMID: 24533179
RNA-mediated gain-of-function; genotype-phenotype correlations; autosomal dominant cerebellar ataxia; repeat expansion
23.  Transgenic Models of Spinocerebellar Ataxia Type 10: Modeling a Repeat Expansion Disorder 
Genes  2012;3(3):481-491.
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease with a spectrum of phenotypes. SCA10 is caused by a pentanucleotide repeat expansion of the ATTCT motif within intron 9 of ATAXIN 10 (ATXN10). Patients present with cerebellar ataxia; however, a subset also develops epileptic seizures which significantly contribute to the morbidity and mortality of the disease. Past research from our lab has demonstrated that epileptic SCA10 patients predominantly originate from or have ancestral ties to Mexico. In addition, a large proportion of epileptic SCA10 patients carry repeat interruptions within their SCA10 expansion. This paper outlines the variability in SCA10 phenotypes and our attempts to model these phenotypes using transgenic mouse models and highlights the benefits of using a transgenic model organism to understand the pathological mechanisms of a human disease.
PMCID: PMC3899997  PMID: 24533179
RNA-mediated gain-of-function; genotype-phenotype correlations; autosomal dominant cerebellar ataxia; repeat expansion
24.  Compromised Mitochondrial Complex II in Models of Machado-Joseph Disease 
Biochimica et Biophysica Acta  2011;1822(2):139-149.
Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of CAG repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend towards reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.
PMCID: PMC3338188  PMID: 22037589
Machado-Joseph disease; ataxin-3; protein aggregation; mitochondria; cell death; succinate dehydrogenase
25.  Mouse Ataxin-3 Functional Knock-Out Model 
Neuromolecular Medicine  2010;13(1):54-65.
Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.
Electronic supplementary material
The online version of this article (doi:10.1007/s12017-010-8137-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3044828  PMID: 20945165
Ataxin-3; Mouse model; Knock-in; Knock-out; CAG repeats; Splicing

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