Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases.

Aerosol and Particle Technology Laboratory Chemical Process and Energy Resources Institute Centre for Research and Technology Hellas 57001 Thessaloniki Greece

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University 62500 Brno Czech Republic

Division of Medical Biotechnology Paul Ehrlich Institute 63225 Langen Germany

Faculty of Biology Johannes Gutenberg University Mainz 55122 Mainz Germany

Faculty of Biology Johannes Gutenberg University Mainz 55122 Mainz Germany; Human Embryo and Stem Cell Laboratory The Francis Crick Institute NW1 1AT London UK

Institute of Applied Biosciences Centre for Research and Technology Hellas 57001 Thessaloniki Greece

Institute of Applied Biosciences Centre for Research and Technology Hellas 57001 Thessaloniki Greece; Department of Molecular Medicine and Surgery Karolinska Institutet 17177 Stockholm Sweden

Institute of Biology Medicinal Chemistry and Biotechnology National Hellenic Research Foundation 11365 Athens Greece

Max Delbrueck Center for Molecular Medicine in the Helmholtz Association Berlin 13125 Germany

Max Delbrueck Center for Molecular Medicine in the Helmholtz Association Berlin 13125 Germany; Department of General Pediatrics Neonatology and Pediatric Cardiology University Children's Hospital Heinrich Heine University 40225 Düsseldorf Germany

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Orr H.T., Zoghbi H.Y. SCA1 molecular genetics: a history of a 13 year collaboration against glutamines. Hum. Mol. Genet. 2001;10(20):2307–2311. PubMed

Skinner P.J., Koshy B.T., Cummings C.J., Klement I.A., Helin K., Servadio A., et al. Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures. Nature. 1997;389(6654):971–974. PubMed

Takahashi T., Kikuchi S., Katada S., Nagai Y., Nishizawa M., Onodera O. Soluble polyglutamine oligomers formed prior to inclusion body formation are cytotoxic. Hum. Mol. Genet. 2008;17(3):345–356. PubMed

Ramdzan Y.M., Trubetskov M.M., Ormsby A.R., Newcombe E.A., Sui X., Tobin M.J., et al. Huntingtin inclusions trigger cellular quiescence, deactivate apoptosis, and lead to delayed necrosis. Cell Rep. 2017;19(5):919–927. PubMed

Bertoni A., Giuliano P., Galgani M., Rotoli D., Ulianich L., Adornetto A., et al. Early and late events induced by polyQ-expanded proteins: identification of a common pathogenic property of polYQ-expanded proteins. J. Biol. Chem. 2011;286(6):4727–4741. PubMed PMC

Burke K.A., Legleiter J. Atomic force microscopy assays for evaluating polyglutamine aggregation in solution and on surfaces. Methods Mol. Biol. 2013;1017:21–40. PubMed

Dahlgren P.R., Karymov M.A., Bankston J., Holden T., Thumfort P., Ingram V.M., et al. Atomic force microscopy analysis of the Huntington protein nanofibril formation. Nanomed. Nanotechnol. Biol. Med. 2005;1(1):52–57. PubMed

Duim W.C., Chen B., Frydman J., Moerner W.E. Sub-diffraction imaging of huntingtin protein aggregates by fluorescence blink-microscopy and atomic force microscopy. ChemPhysChem : a European journal of chemical physics and physical chemistry. 2011;12(13):2387–2390. PubMed PMC

Bauerlein F.J.B., Saha I., Mishra A., Kalemanov M., Martinez-Sanchez A., Klein R., et al. In situ architecture and cellular interactions of PolyQ inclusions. Cell. 2017;171(1):179–187 e10. PubMed

Gruber A., Hornburg D., Antonin M., Krahmer N., Collado J., Schaffer M., et al. Molecular and structural architecture of polyQ aggregates in yeast. Proc. Natl. Acad. Sci. U. S. A. 2018;115(15):E3446–E3453. PubMed PMC

Koch P., Breuer P., Peitz M., Jungverdorben J., Kesavan J., Poppe D., et al. Excitation-induced ataxin-3 aggregation in neurons from patients with Machado-Joseph disease. Nature. 2011;480(7378):543–546. PubMed

Hansen S.K., Stummann T.C., Borland H., Hasholt L.F., Tumer Z., Nielsen J.E., et al. Induced pluripotent stem cell - derived neurons for the study of spinocerebellar ataxia type 3. Stem Cell Res. 2016;17(2):306–317. PubMed

Siska E.K., Koliakos G., Petrakis S. Stem cell models of polyglutamine diseases and their use in cell-based therapies. Front. Neurosci. 2015;9:247. PubMed PMC

Huda F., Fan Y., Suzuki M., Konno A., Matsuzaki Y., Takahashi N., et al. Fusion of human fetal mesenchymal stem cells with "degenerating" cerebellar neurons in spinocerebellar ataxia type 1 model mice. PloS One. 2016;11(11) PubMed PMC

Mates L., Chuah M.K., Belay E., Jerchow B., Manoj N., Acosta-Sanchez A., et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 2009;41(6):753–761. PubMed

Hudecek M., Izsvak Z., Johnen S., Renner M., Thumann G., Ivics Z. Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side. Crit. Rev. Biochem. Mol. Biol. 2017;52(4):355–380. PubMed

Kapetanou M., Chondrogianni N., Petrakis S., Koliakos G., Gonos E.S. Proteasome activation enhances stemness and lifespan of human mesenchymal stem cells. Free Radic. Biol. Med. 2016;103:226–235. PubMed

Siska E.K., Weisman I., Romano J., Ivics Z., Izsvak Z., Barkai U., et al. Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring. PloS One. 2017;12(9) PubMed PMC

Trachana V., Petrakis S., Fotiadis Z., Siska E.K., Balis V., Gonos E.S., et al. Human mesenchymal stem cells with enhanced telomerase activity acquire resistance against oxidative stress-induced genomic damage. Cytotherapy. 2017;19(7):808–820. PubMed

Saarikangas J., Barral Y. Protein aggregates are associated with replicative aging without compromising protein quality control. eLife. 2015;4 PubMed PMC

Petrakis S., Rasko T., Mates L., Ivics Z., Izsvak Z., Kouzi-Koliakou K., et al. Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells. Biotechnol. J. 2012;7(7):891–897. PubMed

Petrakis S., Rasko T., Russ J., Friedrich R.P., Stroedicke M., Riechers S.P., et al. Identification of human proteins that modify misfolding and proteotoxicity of pathogenic ataxin-1. PLoS Genet. 2012;8(8) PubMed PMC

Lorenz C., Lesimple P., Bukowiecki R., Zink A., Inak G., Mlody B., et al. Human iPSC-derived neural progenitors are an effective drug discovery model for neurological mtDNA disorders. Cell stem cell. 2017;20(5):659–674 e9. PubMed

Reinhardt P., Glatza M., Hemmer K., Tsytsyura Y., Thiel C.S., Hoing S., et al. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PloS One. 2013;8(3) PubMed PMC

Wanker E.E., Scherzinger E., Heiser V., Sittler A., Eickhoff H., Lehrach H. Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates. Methods Enzymol. 1999;309:375–386. PubMed

Ami D., Natalello A., Doglia S.M. Fourier transform infrared microspectroscopy of complex biological systems: from intact cells to whole organisms. Methods Mol. Biol. 2012;895:85–100. PubMed

Chondrogianni N., Petropoulos I., Franceschi C., Friguet B., Gonos E.S. Fibroblast cultures from healthy centenarians have an active proteasome. Exp. Gerontol. 2000;35(6–7):721–728. PubMed

Gilmore J.L., Yoshida A., Takahashi H., Deguchi K., Kobori T., Louvet E., et al. Analyses of nuclear proteins and nucleic acid structures using atomic force microscopy. Methods Mol. Biol. 2015;1262:119–153. PubMed

Golan M., Jelinkova S., Kratochvilova I., Skladal P., Pesl M., Rotrekl V., et al. AFM monitoring the influence of selected cryoprotectants on regeneration of cryopreserved cells mechanical properties. Front. Physiol. 2018;9:804. PubMed PMC

Nardone G., Oliver-De La Cruz J., Vrbsky J., Martini C., Pribyl J., Skladal P., et al. YAP regulates cell mechanics by controlling focal adhesion assembly. Nat. Commun. 2017;8:15321. PubMed PMC

Necas D., Klapetek P. Gwyddion: an open-source software for SPM data analysis. Cent. Eur. J. Phys. 2012;10(1):181–188.

Wisniewski J.R., Zougman A., Nagaraj N., Mann M. Universal sample preparation method for proteome analysis. Nat. Methods. 2009;6(5):359–362. PubMed

Cox J., Hein M.Y., Luber C.A., Paron I., Nagaraj N., Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Molecular & cellular proteomics. MCP. 2014;13(9):2513–2526. PubMed PMC

Pertea M., Kim D., Pertea G.M., Leek J.T., Salzberg S.L. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 2016;11(9):1650–1667. PubMed PMC

Yu G., Wang L.G., Han Y., He Q.Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS A J. Integr. Biol. 2012;16(5):284–287. PubMed PMC

Kuleshov M.V., Jones M.R., Rouillard A.D., Fernandez N.F., Duan Q., Wang Z., et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016;44(W1):W90–W97. PubMed PMC

Alanis-Lobato G., Andrade-Navarro M.A., Schaefer M.H. HIPPIE v2.0: enhancing meaningfulness and reliability of protein-protein interaction networks. Nucleic Acids Res. 2017;45(D1):D408–D414. PubMed PMC

Arrondo J.L., Goni F.M. Structure and dynamics of membrane proteins as studied by infrared spectroscopy. Prog. Biophys. Mol. Biol. 1999;72(4):367–405. PubMed

Heimburg T., Schuenemann J., Weber K., Geisler N. Specific recognition of coiled coils by infrared spectroscopy: analysis of the three structural domains of type III intermediate filament proteins. Biochemistry. 1996;35(5):1375–1382. PubMed

Natalello A., Frana A.M., Relini A., Apicella A., Invernizzi G., Casari C., et al. A major role for side-chain polyglutamine hydrogen bonding in irreversible ataxin-3 aggregation. PloS One. 2011;6(4) PubMed PMC

Tanaka M., Morishima I., Akagi T., Hashikawa T., Nukina N. Intra- and intermolecular beta-pleated sheet formation in glutamine-repeat inserted myoglobin as a model for polyglutamine diseases. J. Biol. Chem. 2001;276(48):45470–45475. PubMed

Rekas A., Alattia J.R., Nagai T., Miyawaki A., Ikura M. Crystal structure of venus, a yellow fluorescent protein with improved maturation and reduced environmental sensitivity. J. Biol. Chem. 2002;277(52):50573–50578. PubMed

Zandomeneghi G., Krebs M.R., McCammon M.G., Fandrich M. FTIR reveals structural differences between native beta-sheet proteins and amyloid fibrils. Protein Sci. 2004;13(12):3314–3321. PubMed PMC

Trache A., Meininger G.A. Atomic force microscopy (AFM) Curr Protoc Microbiol. 2008 2C.2.1-2C.2.17. PubMed

Vahabi S., Nazemi Salman B., Javanmard A. Atomic force microscopy application in biological research: a review study. Iran. J. Med. Sci. 2013;38(2):76–83. PubMed PMC

Thomas G., Burnham N.A., Camesano T.A., Wen Q. Measuring the mechanical properties of living cells using atomic force microscopy. JoVE. 2013;76 PubMed PMC

Kim Y.E., Hosp F., Frottin F., Ge H., Mann M., Hayer-Hartl M., et al. Soluble oligomers of PolyQ-expanded huntingtin target a multiplicity of key cellular factors. Mol. Cell. 2016;63(6):951–964. PubMed

Kim J., Waldvogel H.J., Faull R.L., Curtis M.A., Nicholson L.F. The RAGE receptor and its ligands are highly expressed in astrocytes in a grade-dependant manner in the striatum and subependymal layer in Huntington's disease. J. Neurochem. 2015;134(5):927–942. PubMed

Tong X., Gui H., Jin F., Heck B.W., Lin P., Ma J., et al. Ataxin-1 and Brother of ataxin-1 are components of the Notch signalling pathway. EMBO Rep. 2011;12(5):428–435. PubMed PMC

Yue S., Serra H.G., Zoghbi H.Y., Orr H.T. The spinocerebellar ataxia type 1 protein, ataxin-1, has RNA-binding activity that is inversely affected by the length of its polyglutamine tract. Hum. Mol. Genet. 2001;10(1):25–30. PubMed

Sahin U., Ferhi O., Jeanne M., Benhenda S., Berthier C., Jollivet F., et al. Oxidative stress-induced assembly of PML nuclear bodies controls sumoylation of partner proteins. J. Cell Biol. 2014;204(6):931–945. PubMed PMC

Hosp F., Vossfeldt H., Heinig M., Vasiljevic D., Arumughan A., Wyler E., et al. Quantitative interaction proteomics of neurodegenerative disease proteins. Cell Rep. 2015;11(7):1134–1146. PubMed PMC

Koeppen A.H. The pathogenesis of spinocerebellar ataxia. Cerebellum. 2005;4(1):62–73. PubMed

Buijsen R.A.M., Gardiner S.L., Bouma M.J., van der Graaf L.M., Boogaard M.W., Pepers B.A., et al. Generation of 3 spinocerebellar ataxia type 1 (SCA1) patient-derived induced pluripotent stem cell lines LUMCi002-A, B, and C and 2 unaffected sibling control induced pluripotent stem cell lines LUMCi003-A and B. Stem Cell Res. 2018;29:125–128. PubMed

Ishida Y., Kawakami H., Kitajima H., Nishiyama A., Sasai Y., Inoue H., et al. Vulnerability of Purkinje cells generated from spinocerebellar ataxia type 6 patient-derived iPSCs. Cell Rep. 2016;17(6):1482–1490. PubMed

Mertens J., Reid D., Lau S., Kim Y., Gage F.H. Aging in a dish: iPSC-derived and directly induced neurons for studying Brain aging and age-related neurodegenerative diseases. Annu. Rev. Genet. 2018;52:271–293. PubMed PMC

Victor M.B., Richner M., Olsen H.E., Lee S.W., Monteys A.M., Ma C., et al. Striatal neurons directly converted from Huntington's disease patient fibroblasts recapitulate age-associated disease phenotypes. Nat. Neurosci. 2018;21(3):341–352. PubMed PMC

de Chiara C., Menon R.P., Dal Piaz F., Calder L., Pastore A. Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein. J. Mol. Biol. 2005;354(4):883–893. PubMed

Petrakis S., Schaefer M.H., Wanker E.E., Andrade-Navarro M.A. Aggregation of polyQ-extended proteins is promoted by interaction with their natural coiled-coil partners. Bioessays. 2013;35(6):503–507. PubMed PMC

Belay E., Matrai J., Acosta-Sanchez A., Ma L., Quattrocelli M., Mates L., et al. Novel hyperactive transposons for genetic modification of induced pluripotent and adult stem cells: a nonviral paradigm for coaxed differentiation. Stem Cell. 2010;28(10):1760–1771. PubMed

Jackel C., Nogueira M.S., Ehni N., Kraus C., Ranke J., Dohmann M., et al. A vector platform for the rapid and efficient engineering of stable complex transgenes. Sci. Rep. 2016;6:34365. PubMed PMC

Kowarz E., Loscher D., Marschalek R. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Biotechnol. J. 2015;10(4):647–653. PubMed

Hartley J.L., Temple G.F., Brasch M.A. DNA cloning using in vitro site-specific recombination. Genome Res. 2000;10(11):1788–1795. PubMed PMC

Vodyanik M.A., Yu J., Zhang X., Tian S., Stewart R., Thomson J.A., et al. A mesoderm-derived precursor for mesenchymal stem and endothelial cells. Cell stem cell. 2010;7(6):718–729. PubMed PMC

Scuteri A., Miloso M., Foudah D., Orciani M., Cavaletti G., Tredici G. Mesenchymal stem cells neuronal differentiation ability: a real perspective for nervous system repair? Curr. Stem Cell Res. Ther. 2011;6(2):82–92. PubMed

Guo L., Giasson B.I., Glavis-Bloom A., Brewer M.D., Shorter J., Gitler A.D., et al. A cellular system that degrades misfolded proteins and protects against neurodegeneration. Mol. Cell. 2014;55(1):15–30. PubMed PMC

Wagner W., Bork S., Horn P., Krunic D., Walenda T., Diehlmann A., et al. Aging and replicative senescence have related effects on human stem and progenitor cells. PloS One. 2009;4(6) PubMed PMC

Ruggeri F.S., Longo G., Faggiano S., Lipiec E., Pastore A., Dietler G. Infrared nanospectroscopy characterization of oligomeric and fibrillar aggregates during amyloid formation. Nat. Commun. 2015;6:7831. PubMed PMC

Ruggeri F.S., Vieweg S., Cendrowska U., Longo G., Chiki A., Lashuel H.A., et al. Nanoscale studies link amyloid maturity with polyglutamine diseases onset. Sci. Rep. 2016;6:31155. PubMed PMC

Sathasivam K., Lane A., Legleiter J., Warley A., Woodman B., Finkbeiner S., et al. Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease. Hum. Mol. Genet. 2010;19(1):65–78. PubMed PMC

Wacker J.L., Zareie M.H., Fong H., Sarikaya M., Muchowski P.J. Hsp 70 and Hsp 40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer. Nat. Struct. Mol. Biol. 2004;11(12):1215–1222. PubMed

Hands S., Sajjad M.U., Newton M.J., Wyttenbach A. In vitro and in vivo aggregation of a fragment of huntingtin protein directly causes free radical production. J. Biol. Chem. 2011;286(52):44512–44520. PubMed PMC

Beal M.F. Aging, energy, and oxidative stress in neurodegenerative diseases. Ann. Neurol. 1995;38(3):357–366. PubMed

Hetman M., Pietrzak M. Emerging roles of the neuronal nucleolus. Trends Neurosci. 2012;35(5):305–314. PubMed PMC

Lu M., Boschetti C., Tunnacliffe A. Long term aggresome accumulation leads to DNA damage, p53-dependent cell cycle arrest, and steric interference in mitosis. J. Biol. Chem. 2015;290(46):27986–28000. PubMed PMC

Bhaskar K., Miller M., Chludzinski A., Herrup K., Zagorski M., Lamb B.T. The PI3K-Akt-mTOR pathway regulates Abeta oligomer induced neuronal cell cycle events. Mol. Neurodegener. 2009;4:14. PubMed PMC

Wu Y.T., Tan H.L., Huang Q., Ong C.N., Shen H.M. Activation of the PI3K-Akt-mTOR signaling pathway promotes necrotic cell death via suppression of autophagy. Autophagy. 2009;5(6):824–834. PubMed

Schaefer M.H., Wanker E.E., Andrade-Navarro M.A. Evolution and function of CAG/polyglutamine repeats in protein-protein interaction networks. Nucleic Acids Res. 2012;40(10):4273–4287. PubMed PMC

Pelassa I., Cora D., Cesano F., Monje F.J., Montarolo P.G., Fiumara F. Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction. Hum. Mol. Genet. 2014;23(13):3402–3420. PubMed PMC

Wagner A.S., Politi A.Z., Ast A., Bravo-Rodriguez K., Baum K., Buntru A., et al. Self-assembly of mutant huntingtin exon-1 fragments into large complex fibrillar structures involves nucleated branching. J. Mol. Biol. 2018;430(12):1725–1744. PubMed

Yang W., Dunlap J.R., Andrews R.B., Wetzel R. Aggregated polyglutamine peptides delivered to nuclei are toxic to mammalian cells. Hum. Mol. Genet. 2002;11(23):2905–2917. PubMed

Itahana K., Bhat K.P., Jin A., Itahana Y., Hawke D., Kobayashi R., et al. Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation. Mol. Cell. 2003;12(5):1151–1164. PubMed

Blum E.S., Abraham M.C., Yoshimura S., Lu Y., Shaham S. Control of nonapoptotic developmental cell death in Caenorhabditis elegans by a polyglutamine-repeat protein. Science. 2012;335(6071):970–973. PubMed PMC

Slomnicki L.P., Pietrzak M., Vashishta A., Jones J., Lynch N., Elliot S., et al. Requirement of neuronal ribosome synthesis for growth and maintenance of the dendritic tree. J. Biol. Chem. 2016;291(11):5721–5739. PubMed PMC

Experimental Treatment with Edaravone in a Mouse Model of Spinocerebellar Ataxia 1