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• W2097978324 abstract "Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. We examined soluble protein complexes from mouse cerebellum and found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions. Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. We examined soluble protein complexes from mouse cerebellum and found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions. SCA1 (Spinocerebellar ataxia type 1) is one of nine unrelated genes in which expansion of a glutamine-encoding triplet repeat causes a dominantly inherited neurodegenerative disease. Despite broad expression of polyglutamine proteins, distinct subsets of neurons are vulnerable in each disease. Genetic studies have shown that expansion of the polyglutamine tract confers a toxic gain of function (Duyao et al., 1995Duyao M.P. Auerbach A.B. Ryan A. Persichetti F. Barnes G.T. McNeil S.M. Ge P. Vonsattel J.P. Gusella J.F. Joyner A.L. et al.Inactivation of the mouse Huntington's disease gene homolog Hdh.Science. 1995; 269: 407-410Crossref PubMed Scopus (528) Google Scholar, Ikeda et al., 2005Ikeda Y. Aihara K. Sato T. Akaike M. Yoshizumi M. Suzaki Y. Izawa Y. Fujimura M. Hashizume S. Kato M. et al.Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis.J. Biol. Chem. 2005; 280: 29661-29666Crossref PubMed Scopus (111) Google Scholar, Matilla et al., 1998Matilla A. Roberson E.D. Banfi S. Morales J. Armstrong D.L. Burright E.N. Orr H.T. Sweatt J.D. Zoghbi H.Y. Matzuk M.M. Mice lacking ataxin-1 display learning deficits and decreased hippocampal paired-pulse facilitation.J. Neurosci. 1998; 18: 5508-5516Crossref PubMed Google Scholar, Matsumoto et al., 2005Matsumoto T. Takeyama K. Sato T. Kato S. Study of androgen receptor functions by genetic models.J. Biochem. 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Poirier M.A. Protein aggregation and neurodegenerative disease.Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2201) Google Scholar), there is considerable debate as to the biophysical and biochemical state of the disease protein that renders it neurotoxic and the mechanism that leads to selective neuronal vulnerability in each of these disorders. Emerging data reveal that neurotoxicity is modulated by the context of the polyglutamine expansion. For example, the S776A mutation of polyglutamine-expanded ATAXIN-1 (ATXN1) prevents the ataxia and neurodegeneration caused by expression of the SCA1 disease protein in mouse Purkinje cells (Emamian et al., 2003Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice.Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Likewise, in Huntington disease, phosphorylation, SUMO modification, or mutation of the Caspase-6 cleavage site modulate the neurotoxicity of mutant Huntingtin (Graham et al., 2006Graham R.K. Deng Y. Slow E.J. Haigh B. Bissada N. Lu G. Pearson J. Shehadeh J. Bertram L. Murphy Z. et al.Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin.Cell. 2006; 125: 1179-1191Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, Luo et al., 2005Luo S. Vacher C. Davies J.E. Rubinsztein D.C. Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity.J. Cell Biol. 2005; 169: 647-656Crossref PubMed Scopus (134) Google Scholar, Pardo et al., 2006Pardo R. Colin E. Regulier E. Aebischer P. Deglon N. Humbert S. Saudou F. Inhibition of calcineurin by FK506 protects against polyglutamine-huntingtin toxicity through an increase of huntingtin phosphorylation at S421.J. 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Full-length, but not truncated, polyglutamine-expanded androgen receptor reproduces the sex-specific neuropathology of SBMA (Chevalier-Larsen et al., 2004Chevalier-Larsen E.S. O'Brien C.J. Wang H. Jenkins S.C. Holder L. Lieberman A.P. Merry D.E. Castration restores function and neurofilament alterations of aged symptomatic males in a transgenic mouse model of spinal and bulbar muscular atrophy.J. Neurosci. 2004; 24: 4778-4786Crossref PubMed Scopus (179) Google Scholar, Katsuno et al., 2002Katsuno M. Adachi H. Kume A. Li M. Nakagomi Y. Niwa H. Sang C. Kobayashi Y. Doyu M. Sobue G. Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy.Neuron. 2002; 35: 843-854Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, McManamny et al., 2002McManamny P. Chy H.S. Finkelstein D.I. Craythorn R.G. Crack P.J. Kola I. Cheema S.S. Horne M.K. Wreford N.G. O'Bryan M.K. et al.A mouse model of spinal and bulbar muscular atrophy.Hum. Mol. Genet. 2002; 11: 2103-2111Crossref PubMed Scopus (62) Google Scholar). The discovery that the AXH domain of ATXN1 mediates SCA1 neurotoxicity further emphasized the importance of cis-acting domains in pathogenesis and the relationship between such domains and the polyglutamine tract (Tsuda et al., 2005Tsuda H. Jafar-Nejad H. Patel A.J. Sun Y. Chen H.K. Rose M.F. Venken K.J. Botas J. Orr H.T. Bellen H.J. et al.The AXH domain of Ataxin-1 mediates neurodegeneration through its interaction with Gfi-1/Senseless proteins.Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Lastly, studies from Huntington and SCA3 models have revealed a genetic interaction between the wild-type proteins and their polyglutamine-expanded counterparts (Cattaneo et al., 2005Cattaneo E. Zuccato C. Tartari M. Normal huntingtin function: an alternative approach to Huntington's disease.Nat. Rev. Neurosci. 2005; 6: 919-930Crossref PubMed Scopus (471) Google Scholar, Van Raamsdonk et al., 2005Van Raamsdonk J.M. Pearson J. Rogers D.A. Bissada N. Vogl A.W. Hayden M.R. Leavitt B.R. Loss of wild-type huntingtin influences motor dysfunction and survival in the YAC128 mouse model of Huntington disease.Hum. Mol. Genet. 2005; 14: 1379-1392Crossref PubMed Scopus (117) Google Scholar, Warrick et al., 2005Warrick J.M. Morabito L.M. Bilen J. Gordesky-Gold B. Faust L.Z. Paulson H.L. Bonini N.M. Ataxin-3 suppresses polyglutamine neurodegeneration in Drosophila by a ubiquitin-associated mechanism.Mol. Cell. 2005; 18: 37-48Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, Zuccato et al., 2003Zuccato C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. et al.Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes.Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (686) Google Scholar). These observations suggest a relationship between the selective neurotoxic properties of the polyglutamine-expanded protein and the normal functions of the wild-type protein. Aberrant protein interactions are thought to mediate neurotoxicity in polyglutamine diseases (Gatchel and Zoghbi, 2005Gatchel J.R. Zoghbi H.Y. Diseases of unstable repeat expansion: mechanisms and common principles.Nat. Rev. Genet. 2005; 6: 743-755Crossref PubMed Scopus (608) Google Scholar, Harjes and Wanker, 2003Harjes P. Wanker E.E. The hunt for huntingtin function: interaction partners tell many different stories.Trends Biochem. Sci. 2003; 28: 425-433Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, Li and Li, 2004Li S.H. Li X.J. Huntingtin-protein interactions and the pathogenesis of Huntington's disease.Trends Genet. 2004; 20: 146-154Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, MacDonald, 2003MacDonald M.E. Huntingtin: alive and well and working in middle management.Sci. STKE. 2003; 207: pe48Google Scholar). Polyglutamine expansion alters the interactions of Huntingtin with HAP1/p150Glued/dynein complexes, as well as numerous transcription factors, leading to their functional impairment (Chen-Plotkin et al., 2006Chen-Plotkin A.S. Sadri-Vakili G. Yohrling G.J. Braveman M.W. Benn C.L. Glajch K.E. DiRocco D.P. Farrell L.A. Krainc D. Gines S. et al.Decreased association of the transcription factor Sp1 with genes downregulated in Huntington's disease.Neurobiol. Dis. 2006; 22: 233-241Crossref PubMed Scopus (82) Google Scholar, Dunah et al., 2002Dunah A.W. Jeong H. Griffin A. Kim Y.M. Standaert D.G. Hersch S.M. Mouradian M.M. Young A.B. Tanese N. Krainc D. Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease.Science. 2002; 296: 2238-2243Crossref PubMed Scopus (559) Google Scholar, Gauthier et al., 2004Gauthier L.R. Charrin B.C. Borrell-Pages M. Dompierre J.P. Rangone H. Cordelieres F.P. De Mey J. MacDonald M.E. Lessmann V. Humbert S. et al.Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules.Cell. 2004; 118: 127-138Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar, Kegel et al., 2002Kegel K.B. Meloni A.R. Yi Y. Kim Y.J. Doyle E. Cuiffo B.G. Sapp E. Wang Y. Qin Z.H. Chen J.D. et al.Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription.J. Biol. Chem. 2002; 277: 7466-7476Crossref PubMed Scopus (210) Google Scholar, Li et al., 2002Li S.H. Cheng A.L. Zhou H. Lam S. Rao M. Li H. Li X.J. Interaction of Huntington disease protein with transcriptional activator Sp1.Mol. Cell. Biol. 2002; 22: 1277-1287Crossref PubMed Scopus (271) Google Scholar, Schaffar et al., 2004Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation.Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, Zhai et al., 2005Zhai W. Jeong H. Cui L. Krainc D. Tjian R. In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets.Cell. 2005; 123: 1241-1253Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, Zuccato et al., 2003Zuccato C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. et al.Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes.Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (686) Google Scholar). Other examples of altered protein-protein interactions include Ataxin-7 with CRX and the STAGA/TFTC complexes and Ataxin-3 with components of the ubiquitin proteasome system (Chen et al., 2004Chen S. Peng G.H. Wang X. Smith A.C. Grote S.K. Sopher B.L. La Spada A.R. Interference of Crx-dependent transcription by ataxin-7 involves interaction between the glutamine regions and requires the ataxin-7 carboxy-terminal region for nuclear localization.Hum. Mol. Genet. 2004; 13: 53-67Crossref PubMed Scopus (66) Google Scholar, Doss-Pepe et al., 2003Doss-Pepe E.W. Stenroos E.S. Johnson W.G. Madura K. Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis.Mol. Cell. 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USA. 2005; 102: 8472-8477Crossref PubMed Scopus (182) Google Scholar, Strom et al., 2005Strom A.L. Forsgren L. Holmberg M. A role for both wild-type and expanded ataxin-7 in transcriptional regulation.Neurobiol. Dis. 2005; 20: 646-655Crossref PubMed Scopus (34) Google Scholar, Warrick et al., 2005Warrick J.M. Morabito L.M. Bilen J. Gordesky-Gold B. Faust L.Z. Paulson H.L. Bonini N.M. Ataxin-3 suppresses polyglutamine neurodegeneration in Drosophila by a ubiquitin-associated mechanism.Mol. Cell. 2005; 18: 37-48Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Nevertheless, it is unknown if neurotoxicity of the mutant proteins occurs through associations with these native protein complexes, loss of these associations, or aberrant interactions and novel complexes formed by misfolded or aggregating disease protein. To test the hypothesis that polyglutamine expansion mediates pathology by modulating the native protein interactions of the disease protein, we sought to characterize the endogenous ATXN1 protein complexes in mouse cerebellum and identify ATXN1-associated proteins by biochemical copurification from stable cell lines. We report that ATXN1 isolated from mouse cerebellum predominantly associates into large complexes containing the mammalian homolog of Drosophila Capicua (CIC), a transcriptional repressor containing a Sox-like high mobility group (HMG) box. Both major isoforms of CIC (CIC-L and CIC-S) are subunits of these complexes. We demonstrate that expression of polyglutamine-expanded ATXN1 alters the repressor activity of CIC, establishing a functional link between the two proteins. Finally, we provide evidence that SCA1 neuropathology depends on incorporation of polyglutamine-expanded ATXN1 into its native CIC-containing complexes. To identify the major ATXN1 protein complexes in vivo, we examined soluble ATXN1-associated complexes in wild-type mouse cerebellum using gel-filtration and ion exchange chromatography. Gel filtration revealed that the majority of endogenous wild-type ATXN1 (∼75%) elutes as large protein complexes (estimated size ∼1.8 MDa) (Figures 1A and 1B). Only about 25% of ATXN1 elutes as smaller protein complexes (observed as a shoulder on the larger complexes elution profile, estimated size ∼300 kDa) (Figure 1B, right panel). To identify proteins that interact with ATXN1 in its native complexes, we purified ATXN1-associated proteins by tandem affinity purification (TAP) (Rigaut et al., 1999Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2225) Google Scholar) and mass spectrometry (Figure S1). We found two novel interacting proteins with apparent molecular masses of ∼160 kDa and ∼250 kDa, both corresponding to the human homolog of Drosophila Capicua (Cic). Northern blot analysis of mouse brain RNA for Cic identified two major transcripts (Figure S5A) corresponding to two Cic mRNA transcripts predicted to encode proteins of 258 kDa for CIC-L and 164 kDa for CIC-S (Figure S2A). We also identified these alternatively spliced CIC mRNA transcripts for both human and Drosophila (Figures S2A and S2B). To detect the endogenous CIC protein, we generated antisera against the common C-terminal region of the mouse CIC isoforms (Figure S3A). This antibody specifically recognized CIC-L and CIC-S from mouse cerebellar and HeLa cell extracts (Figures 1 and S3B). To determine whether CIC is a stable ATXN1-interacting partner, we analyzed the elution profile of CIC by gel-filtration chromatography. We observed that CIC-S and CIC-L coeluted with the large ATXN1 protein complexes (Figures 1A and 1B). Quantification of the relative elution profiles of CIC and ATXN1 using 1 ml fractions, which allows the entire profile to be analyzed on a single gel, revealed a near perfect match between the elution profiles of CIC and the large ATXN1 complexes (Figure 1B). If CIC and ATXN1 are components of the same protein complexes, loss of ATXN1 will alter the elution profiles of CIC. Indeed, we found that elution of the CIC isoforms is shifted toward later fractions in cerebellar extracts from Sca1 null mice (Figure S4). Furthermore, we observed a strict cofractionation of ATXN1 with the CIC isoforms using anion exchange chromatography (Figure 1C). Although no significant population of ATXN1 fractionated independently of CIC, we did detect low levels of ATXN1 in the flow-through fractions in the absence of detectable CIC (data not shown), suggesting the smaller ATXN1 complexes have minimal binding to the column matrix. In sum, the nearly identical elution patterns of the large ATXN1 complexes with CIC-L and CIC-S, and the shift of the CIC profiles in the absence of ATXN1, suggest that the majority of endogenous CIC in the mouse cerebellum stably associates into ATXN1-CIC protein complexes. Also, the smaller ATXN1 protein complexes likely do not contain CIC. To determine whether ATXN1 and CIC interact in the tissue most vulnerable in SCA1, we performed coimmunoprecipitation (coIP) of cerebellar extracts from wild-type and Sca1 null mice using anti-ATXN1 antibody (11NQ). The anti-ATXN1 antibody specifically immunoprecipitated both CIC isoforms from cerebellar extracts of wild-type but not Sca1 null mice, indicating that CIC and ATXN1 interact in vivo (Figure 2A; input and loading controls for this coIP are in Figure 3A). This is in agreement with our recent work, in which we identified CIC as a binding partner of ATXN1 in an unbiased yeast two-hybrid screen (Lim et al., 2006Lim J. Hao T. Shaw C. Patel A.J. Szabo G. Rual J.F. Fisk C.J. Li N. Smolyar A. Hill D.E. et al.A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.Cell. 2006; 125: 801-814Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). Having established the ATXN1-CIC interaction, we compared their expression patterns in mouse brain. Northern blot analysis showed that the Cic isoforms are highly expressed in the cerebellum and olfactory bulb (Figure S5A, upper panel). In situ hybridization of adult mouse brain for Cic and Sca1 revealed strikingly similar expression patterns, with high expression in Purkinje cells and in the hippocampus (Figures S5B–S5I). Immunohistochemistry for CIC and ATXN1 also displayed matching protein expression patterns in most brain regions (data not shown). Importantly, both proteins are highly expressed in the nuclei of the Purkinje cells, where polyglutamine-expanded ATXN1 does the most damage (Figure 2B). To look for interdependence between CIC and ATXN1, we examined CIC protein levels in Sca1 null mice. We found that both CIC isoforms had significantly reduced levels in Sca1 null mouse cerebellum (Figures 2C and 2D) and cerebrum (data not shown). As the mRNA levels of the Cic isoforms are unaltered in Sca1 null mice, the dependency of CIC on ATXN1 is posttranscriptional (Figure 2E). Thus, CIC is less stable in the absence of ATXN1 in vivo. To assess the degree of CIC association with ATXN1 in vivo, we immunoprecipitated ATXN1 from cerebellar extracts and measured the codepletion of CIC from the post-IP supernatants. Both CIC isoforms were substantially depleted following ATXN1 immunodepletion, demonstrating that the majority of endogenous CIC associates with ATXN1 (Figure 3A). In contrast, the levels of neither Gapdh (data not shown) or AKT (Figure 3A), an ATXN1 kinase thought to interact transiently with ATXN1 (Chen et al., 2003Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. et al.Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), are decreased by ATXN1 immunodepletion. Averaging data from independent experiments, we observed that when an average of 72% of ATXN1 is immunodepleted, an average of 62% of CIC-L and 56% of CIC-S are codepleted from the cerebellar extracts. We therefore estimate that ∼80% of endogenous CIC associates with ATXN1 (Figure 3B). The depleted CIC isoforms are found in the pellet of the immunoprecipitate (see for example Figure 2A). To establish whether CIC also interacts with mutant forms of ATXN1, we performed immunoprecipitation on lysates from HeLa cells transiently transfected with variants of FLAG-tagged ATXN1 containing 2Q, 82Q, or 82Q with a S776A mutation (which abolishes the interaction of ATXN1 with 14-3-3) or 2Q with a deletion of the AXH domain. We found that both endogenous CIC isoforms coimmunoprecipitated with each of these ATXN1 variants, except the one lacking the AXH domain (Figure 3C). We conclude that the ATXN1-CIC interaction requires the AXH domain and is independent of 14-3-3 binding. To identify the domains responsible for interaction between the two proteins, we performed yeast two-hybrid interaction assays using a series of ATXN1 and CIC deletion constructs. We found that two N-terminal fragments of CIC-S (amino acids 1–300 and 1–205) interact with the C-terminal half of ATXN1 and that the AXH domain of ATXN1 is sufficient for its interaction with CIC (Figure 3D). To further map the ATXN1 binding domain of CIC, we generated serial deletions at the N terminus of mouse CIC-S for pull-down assays with GST-tagged full-length wild-type ATXN1 (Figures S6A and S6B). We narrowed the interaction region to 31 amino acids (amino acids 16–46) of CIC-S (Figure S6C). Comparison of this 31-amino acid sequence across species revealed a conserved stretch of eight amino acids present in both CIC-S and CIC-L isoforms with the consensus sequence WXX(L/I)(V/L)PX(L/M) (Figure 3E). We then confirmed in vitro that human ATXN1 binds to Drosophila Cic through the consensus eight amino acids (Figure S6D). ATXN1 and CIC thus appear to be in vivo binding partners that interact directly through evolutionarily conserved domains. Having established that polyglutamine-expanded ATXN1 interacts with CIC, we asked whether the expanded polyglutamine tract alters the incorporation of ATXN1 into its native protein complexes. We examined cerebellar extracts from Sca1154Q/+ animals that express polyglutamine-expanded ATXN1[154Q] from its endogenous locus and are an accurate model of SCA1 (Watase et al., 2002Watase K. Weeber E.J. Xu B. Antalffy B. Yuva-Paylor L. Hashimoto K. Kano M. Atkinson R. Sun Y. Armstrong D.L. et al.A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration.Neuron. 2002; 34: 905-919Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). As previously reported, the levels of soluble expanded protein are significantly lower than wild-type ATXN1 levels in these animals, although expression of the mutant allele is identical to the wild-type allele (Watase et al., 2002Watase K. Weeber E.J. Xu B. Antalffy B. Yuva-Paylor L. Hashimoto K. Kano M. Atkinson R. Sun Y. Armstrong D.L. et al.A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration.Neuron. 2002; 34: 905-919Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Gel-filtration chromatography of extracts from early symptomatic animals (10–15 weeks of age) revealed that soluble ATXN1[154Q] and wild-type ATXN1 (expressed from the untargeted allele) eluted into the same fractions as ATXN1 from wild-type extracts (Figure 4A). The elution profile of CIC from Sca1154Q/+ mice is indistinguishable from that seen in wild-type animals (Figure 4A). A subtle change in the elution profile of the smaller ATXN1 complexes (elution fractions from 13 ml to 16 ml) was observed in the mutant animals: both ATXN1[154Q] and wild-type ATXN1 showed a relative loss from elution fractions at 14 ml and beyond, with a corresponding increase in elution fractions at 13 ml and 13.5 ml (estimated size ∼500 kDa) compared to wild-type animals (compare Figure 1A to 4A). The basis for this change is unclear at present, but it could represent an alternate conformation of the smaller ATXN1 protein complexes coupled with the slight increase of molecular weight due to polyglutamine expansion. The similar elution patterns of wild-type and expanded ATXN1 by gel-filtration chromatography suggest that polyglutamine expansion does not prevent the incorporation of ATXN1 into its normal endogenous protein complexes. To confirm that polyglutamine-expanded ATXN1 incorporates into similar complexes as wild-type ATXN1, we analyzed cerebellar extracts from Sca1154Q/+ animals by anion-exchange chromatography. We found that ATXN1[154Q] coelutes with wild-type ATXN1 and with both CIC isoforms (Figure 4B). The elution profiles of ATXN1, CIC-L, and CIC-S in extracts by anion-exchange chromatography are quite broad; this argues against a single complex of uniform structure and suggests that the ATXN1-CIC protein complexes adopt multiple conformational or compositional states separable by differences in their charge (Figures 1C and 4B). We previously showed that mutating serine 776 of ATXN1 to alanine (S776A) dramatically suppresses the neurotoxicity of the polyglutamine-expanded protein in Purkinje cells (Emamian et al., 2003Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice.Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). This mutation causes loss of interaction with the chaperone protein 14-3-3 (Chen et al., 2003Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. et al.Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.Cell. 2003; 113: 457-468Abstract Full Text Full Tex" @default.
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• W2097978324 date "2006-12-01" @default.
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• W2097978324 title "ATAXIN-1 Interacts with the Repressor Capicua in Its Native Complex to Cause SCA1 Neuropathology" @default.
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• W2097978324 doi "https://doi.org/10.1016/j.cell.2006.11.038" @default.
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