A quantitative high-throughput screen identifies compounds that lower expression of the SCA2-and ALS-associated gene ATXN2

Abstract

CAG repeat expansions in the ATXN2 (ataxin-2) gene can cause the autosomal dominant disorder spinocerebellar ataxia type 2 (SCA2) as well as increase the risk of ALS. Abnormal molecular, motor, and neurophysiological phenotypes in SCA2 mouse models are normalized by lowering ATXN2 transcription, and reduction of nonmutant Atxn2 expression has been shown to increase the life span of mice overexpressing the TDP-43 (transactive response DNA-binding protein 43 kDa) ALS protein, demonstrating the potential benefits of targeting ATXN2 transcription in humans. Here, we describe a quantitative high-throughput screen to identify compounds that lower ATXN2 transcription. We screened 428,759 compounds in a multiplexed assay using an ATXN2-luciferase reporter in human embryonic kidney 293 (HEK-293) cells and identified a diverse set of compounds capable of lowering ATXN2 transcription. We observed dose-dependent reductions of endogenous ATXN2 in HEK-293 cells treated with procillaridin A, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), and heat shock protein 990 (HSP990), known inhibitors of HSP90 and Na+/K+-ATPases. Furthermore, HEK-293 cells expressing polyglutamine-expanded ATXN2-Q58 treated with 17-DMAG had minimally detectable ATXN2, as well as normalized markers of autophagy and endoplasmic reticulum stress, including STAU1 (Staufen 1), molecular target of rapamycin, p62, LC3-II (microtubule-associated protein 1A/1B-light chain 3II), CHOP (C/EBP homologous protein), and phospho-eIF2α (eukaryotic initiation factor 2α). Finally, bacterial artificial chromosome ATXN2-Q22 mice treated with 17-DMAG or HSP990 exhibited highly reduced ATXN2 protein abundance in the cerebellum. Taken together, our study demonstrates inhibition of HSP90 or Na+/K+-ATPases as potentially effective therapeutic strategies for treating SCA2 and ALS. CAG repeat expansions in the ATXN2 (ataxin-2) gene can cause the autosomal dominant disorder spinocerebellar ataxia type 2 (SCA2) as well as increase the risk of ALS. Abnormal molecular, motor, and neurophysiological phenotypes in SCA2 mouse models are normalized by lowering ATXN2 transcription, and reduction of nonmutant Atxn2 expression has been shown to increase the life span of mice overexpressing the TDP-43 (transactive response DNA-binding protein 43 kDa) ALS protein, demonstrating the potential benefits of targeting ATXN2 transcription in humans. Here, we describe a quantitative high-throughput screen to identify compounds that lower ATXN2 transcription. We screened 428,759 compounds in a multiplexed assay using an ATXN2-luciferase reporter in human embryonic kidney 293 (HEK-293) cells and identified a diverse set of compounds capable of lowering ATXN2 transcription. We observed dose-dependent reductions of endogenous ATXN2 in HEK-293 cells treated with procillaridin A, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), and heat shock protein 990 (HSP990), known inhibitors of HSP90 and Na+/K+-ATPases. Furthermore, HEK-293 cells expressing polyglutamine-expanded ATXN2-Q58 treated with 17-DMAG had minimally detectable ATXN2, as well as normalized markers of autophagy and endoplasmic reticulum stress, including STAU1 (Staufen 1), molecular target of rapamycin, p62, LC3-II (microtubule-associated protein 1A/1B-light chain 3II), CHOP (C/EBP homologous protein), and phospho-eIF2α (eukaryotic initiation factor 2α). Finally, bacterial artificial chromosome ATXN2-Q22 mice treated with 17-DMAG or HSP990 exhibited highly reduced ATXN2 protein abundance in the cerebellum. Taken together, our study demonstrates inhibition of HSP90 or Na+/K+-ATPases as potentially effective therapeutic strategies for treating SCA2 and ALS. Spinocerebellar ataxia type 2 (SCA2) is a debilitating neurodegenerative disorder characterized primarily by gait ataxia, for which there are no disease-modifying treatments (1Figueroa K.P. Pulst S.M. Identification and expression of the gene for human ataxin-2-related protein on chromosome 16.Exp. Neurol. 2003; 184: 669-678Crossref PubMed Scopus (21) Google Scholar). SCA2 is caused by a CAG repeat expansion mutation in the ATXN2 (ataxin-2) gene in an encoded region that results in polyglutamine (polyQ) expansion of the ATXN2 protein. In normal individuals, ATXN2 usually contains 22 CAGs interrupted by one or two CAA codons, whereas SCA2 occurs when ATXN2 contains pure CAG repeats numbering 33 or more (2Fernandez M. McClain M.E. Martinez R.A. Snow K. Lipe H. Ravits J. et al.Late-onset SCA2: 33 CAG repeats are sufficient to cause disease.Neurology. 2000; 55: 569-572Crossref PubMed Google Scholar). SCA2 is also characterized by anticipation where in families, CAG repeat lengths generally increase with each generation, and longer repeat lengths are associated with earlier age of onset and greater disease severity (3Pulst S.M. Nechiporuk A. Nechiporuk T. Gispert S. Chen X.N. Lopes-Cendes I. et al.Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2.Nat. Genet. 1996; 14: 269-276Crossref PubMed Google Scholar). SCA2 is included among nine total polyQ diseases, including dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Huntington’s disease, and SCAs 1 to 3, 6, 7, and 17, each of which are characterized phenotypically by progressive neurodegeneration. Ataxia in SCA2 results from progressive loss of cerebellar Purkinje cells, and pathological defects in the brain stem are also observed, mainly involving the pontine and olivary nuclei. Targeting ATXN2 may also treat ALS: intermediate CAG repeat expansions increase ALS risk, and reduction of Atxn2 expression in transactive response DNA-binding protein 43 kDa (TDP-43) transgenic mice increased life span and normalized TDP-43 aggregation (4Elden A.C. Kim H.J. Hart M.P. Chen-Plotkin A.S. Johnson B.S. Fang X. et al.Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.Nature. 2010; 466: 1069-1075Crossref PubMed Scopus (865) Google Scholar, 5Becker L.A. Huang B. Bieri G. Ma R. Knowles D.A. Jafar-Nejad P. et al.Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.Nature. 2017; 544: 367-371Crossref PubMed Scopus (286) Google Scholar). Since the discovery of ATXN2 as the SCA2 gene in 1996 (3Pulst S.M. Nechiporuk A. Nechiporuk T. Gispert S. Chen X.N. Lopes-Cendes I. et al.Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2.Nat. Genet. 1996; 14: 269-276Crossref PubMed Google Scholar, 6Imbert G. Saudou F. Yvert G. Devys D. Trottier Y. Garnier J.M. et al.Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats.Nat. Genet. 1996; 14: 285-291Crossref PubMed Google Scholar, 7Sanpei K. Takano H. Igarashi S. Sato T. Oyake M. Sasaki H. et al.Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT.Nat. Genet. 1996; 14: 277-284Crossref PubMed Google Scholar), we and others have made efforts to characterize ATXN2 function with the intention to identify therapeutic targets for SCA2. SCA2 is characterized by toxic gain-of-function mutations in the ATXN2 gene. A principal ATXN2 function is regulation of RNA processing, as the majority of known ATXN2-interacting proteins are RNA-binding proteins, including A2BP1/RBFox1, DDX6 (DEAD-box helicase 6), PABP1 (polyadenylate-binding protein 1), TDP-43, FUS (fused in sarcoma), and STAU1 (Staufen 1) (4Elden A.C. Kim H.J. Hart M.P. Chen-Plotkin A.S. Johnson B.S. Fang X. et al.Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.Nature. 2010; 466: 1069-1075Crossref PubMed Scopus (865) Google Scholar, 8Nonhoff U. Ralser M. Welzel F. Piccini I. Balzereit D. Yaspo et al.Ataxin-2 with the RNA helicase DDX6 and with and PubMed Scopus Google Scholar, H. Pulst S.M. A protein with RNA-binding with Genet. 2000; PubMed Scopus Google Scholar, G. K. A. I. K. and function of the of human U. S. A. PubMed Scopus Google Scholar, with FUS and intermediate-length polyglutamine expansions pathology in Genet. PubMed Scopus Google Scholar, S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar). ATXN2 also to and a as a of RNA or U. Ralser M. Welzel F. Piccini I. Balzereit D. Yaspo et al.Ataxin-2 with the RNA helicase DDX6 and with and PubMed Scopus Google Scholar). We that STAU1 and molecular target of are in SCA2 and ALS and mouse models associated with which can by targeting ATXN2, or S. M. T. K.P. et autophagy in Scopus Google Scholar). of cerebellar from bacterial artificial chromosome mice also the of reduced of protein as well as to an ATXN2 and the S. K.P. S. et al.Ataxin-2 in a transgenic mouse Genet. PubMed Scopus Google Scholar). ATXN2 mutation is also associated with and SCA2 Purkinje cells are characterized by is caused in by by ATXN2 with in increased from the endoplasmic reticulum J. H. et and in spinocerebellar ataxia type PubMed Scopus Google Scholar). may also from expression proteins may 1) by PubMed Google Scholar), which is a of from to inhibition of in 1996; PubMed Scopus Google Scholar). ATXN2 expression with and to in models of ALS and SCA2 S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar, P. S. K.P. et for spinocerebellar ataxia type 2017; 544: PubMed Scopus Google Scholar), and 1 are in ALS for the ATXN2 therapeutic In the we to identify that lower ATXN2 expression with a quantitative high-throughput screen a assay that ATXN2 we identified of compounds that ATXN2 transcription, including heat shock protein inhibitors and which we in and ATXN2-Q22 compounds by of SCA2 and ALS We human embryonic kidney 293 (HEK-293) to including and of which the and in the of we an and we that the and in the We a screen of compounds the cells treated with compounds for a and for an of the of the in high-throughput We observed expression by for an of We compounds two and using cells and cells and 43 including and one HSP90 of compounds also as inhibitors in the the we that by by 1 or to for for of the for compounds that function as inhibitors or inhibitors of compounds had and a that for the of the We of and using and the and with for demonstrating dose-dependent reduction of for We or from the and are in We the for for the We the a identified in the to for in a dose-dependent in HEK-293 cells with abundance by cells, we that in a dose-dependent are with as a an of or transcription or We in a using cells treated with in the and for cells treated with 1 or and the study by et A for in and of high PubMed Scopus Google Scholar), we the and also the and We a screen assay to the using A screen from to using the a set of using a multiplexed assay a assay using increase with and for of the identified compounds among with and of the and that observed with 1 the of compounds the We compounds in the screen using the assay to on and to in a multiplexed for the and the multiplexed for the of of compounds and compounds in or in reduced by more 1 or in and or in reduction of in HEK-293 cells expressing or in of total of compounds screened compounds in or in reduced by more 1 or in and or in reduction of in HEK-293 cells expressing or in in a of total of compounds screened assay of compounds from the of compounds in cells, and of had for compounds in and compounds as in cells, and for of compounds R. 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H. K. et inhibitors a and identify a of PubMed Scopus Google Scholar, J. Y. T. R. of heat shock transcription factor by HSP90 that a with PubMed Google Scholar). lowering ATXN2-Q58 in HEK-293 cells that we have are in SCA2 S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar, S. K.P. M. Pulst S.M. in human Neurol. PubMed Scopus Google Scholar), we proteins by and by 17-DMAG we observed with 17-DMAG with HSP90 target HEK-293 ATXN2-Q58 cells treated with we observed reduced ATXN2 protein with abundance of STAU1 and autophagy proteins p62, and protein 1A/1B-light chain and proteins in the protein response including homologous protein and initiation factor and protein also known as We 17-DMAG and HSP90 for lowering ATXN2 expression in our human ATXN2-Q22 transgenic mouse S. K.P. S. et al.Ataxin-2 in a transgenic mouse Genet. PubMed Scopus Google Scholar). ATXN2-Q22 mice have the and of the human ATXN2 which a polyQ repeat that is the normal in humans. ALS treated by targeting nonmutant L.A. Huang B. Bieri G. Ma R. Knowles D.A. Jafar-Nejad P. et al.Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.Nature. 2017; 544: 367-371Crossref PubMed Scopus (286) Google Scholar), we HSP90 inhibitors in mice CAG We treated ATXN2-Q22 mice with 17-DMAG two and two 3, 7, and and on expression of ATXN2 and in by which 17-DMAG reduced ATXN2 abundance associated with We a using a HSP90 of a that can also the ATXN2-Q22 mice treated two and two 3, 7, and and on the two in the HSP990 expression of ATXN2 and in by and for 17-DMAG mice treated with HSP990 had reduced ATXN2 abundance associated with efforts to from mice treated with 17-DMAG by of high to mice treated with HSP990 had reduced ATXN2 abundance in by by two and the from the ATXN2 has as a potential therapeutic target for SCA2 also ALS and neurodegenerative CAG repeat expansion mutation in ATXN2 results in a toxic of function for the encoded ATXN2 protein. ATXN2 expression phenotypes in SCA2 and ALS mice L.A. Huang B. Bieri G. Ma R. Knowles D.A. Jafar-Nejad P. et al.Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.Nature. 2017; 544: 367-371Crossref PubMed Scopus (286) Google Scholar). In we to identify compounds that can as in the of for SCA2 and potentially for ALS. by ATXN2 as a therapeutic target for SCA2 and ALS. In SCA2 lowering ATXN2 expression the expression of cerebellar proteins and Purkinje and the SCA2 P. S. K.P. et for spinocerebellar ataxia type 2017; 544: PubMed Scopus Google Scholar). Targeting ATXN2 may also an effective therapeutic for treating ALS. of ATXN2 in ALS that intermediate CAG repeat expansions in ATXN2 increased ALS risk (4Elden A.C. Kim H.J. Hart M.P. Chen-Plotkin A.S. Johnson B.S. Fang X. et al.Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.Nature. 2010; 466: 1069-1075Crossref PubMed Scopus (865) Google Scholar, K.P. Pulst S.M. risk for spinocerebellar ataxia type 2 ATXN2 CAG repeat a Neurol. PubMed Google Scholar). lowering ATXN2 abundance an effective to treating ALS. by a TDP-43 mice with Atxn2 mice P. Pulst S.M. in Purkinje and gene expression pathology in a mouse of Genet. PubMed Scopus Google Scholar), and by lowering Atxn2 expression in TDP-43 mice using an demonstrating and reduced of RNA for TDP-43 or ATXN2 proteins L.A. Huang B. Bieri G. Ma R. Knowles D.A. Jafar-Nejad P. et al.Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.Nature. 2017; 544: 367-371Crossref PubMed Scopus (286) Google Scholar). We also that ATXN2 mutation results in abundance of autophagy and In SCA2 and in cerebellar and of SCA2 mouse we observed in autophagy inhibition by increased LC3-II S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar). to increased by direct by the protein which also highly in SCA2 cells and models S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar, S. M. T. K.P. et autophagy in Scopus Google Scholar). in HEK-293 cells expressing we observed abundance of proteins in the M. K.P. S. Pulst S.M. 1 of the protein PubMed Scopus Google Scholar). We that autophagy proteins, and proteins CHOP and phospho-eIF2α are normalized in ATXN2-Q58 cells by 17-DMAG we a increase in in response to is a of the that as the of HSP90 A. A. K. and HSP90 in neurodegenerative PubMed Scopus Google Scholar, J. T. X. S. shock proteins and 2017; PubMed Scopus Google Scholar). to HSP90 that response to HSP90 inhibitors is highly inhibitors the protein response in and mice PubMed Scopus Google Scholar, G. P. A. M. S. et in in and in by of the PubMed Scopus Google Scholar, F. A. A. F. et of and in the and on the PubMed Scopus Google Scholar). In ATXN2-Q58 cells, which and autophagy the in CHOP associated with a increase a to a and of the STAU1 abundance and autophagy by targeting STAU1 or ATXN2, and by targeting STAU1 S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar, M. K.P. S. Pulst S.M. 1 of the protein PubMed Scopus Google Scholar). we observed STAU1 and with LC3-II in TDP-43 ALS and of TDP-43 which when mice for STAU1 S. M. T. K.P. et autophagy in Scopus Google Scholar). that lowering ATXN2 expression can and associated with disease in SCA2 and ALS. study that autophagy and can or normalized using compounds that lower ATXN2 in our the compounds the there including A, and of which A had the are inhibitors of and are to treat and are toxic and to can K. K. A. of and A on the regulation of in human 2010; PubMed Scopus Google Scholar). of the Purkinje in HEK-293 cells increased ATXN2 protein abundance and whereas lowering expression by reduced ATXN2 transcription that A and ATXN2 abundance in to that lower ATXN2 abundance in as our efforts to in a as for HSP90 inhibitors in ATXN2-Q22 mice ATXN2 expression using to an effective for RNA A of SCA2 is in Purkinje cells, and and observed in SCA2 S. S. M. H. T. F. of polyglutamine and DNA-binding protein 43 and in of with spinocerebellar ataxia type Neurol. PubMed Scopus Google Scholar). We have shown that the protein STAU1 highly to in SCA2 and ALS models S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar). reduction of expression of the nonmutant ATXN2 gene in TDP-43 models reduced the abundance of ATXN2 and L.A. Huang B. Bieri G. Ma R. Knowles D.A. Jafar-Nejad P. et al.Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.Nature. 2017; 544: 367-371Crossref PubMed Scopus (286) Google Scholar). In ATXN2 mutation associated with increased of RNA-binding in HEK-293 cells and in SCA2 S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar). We have also that in SCA2 mice for that the SCA2 cerebellar molecular the and Purkinje reduced in S. K.P. Pulst S.M. RNA and autophagy in a of PubMed Scopus Google Scholar). A study by Fang et S. A. P. et of TDP-43 to TDP-43 in PubMed Scopus Google to the regulation of by study identified as of phenotypes the molecular or the target of that is in reduction of ATXN2 and the In including A can also autophagy J. J. S. as autophagy PubMed Scopus Google Scholar), which on which are known to reduced by autophagy R. are by and PubMed Scopus Google Scholar). study identified compounds known to including one that identified in our identified in our we our to 17-DMAG for in of in human H. S. Y. A. A. A. et 17-DMAG as an and of the PubMed Scopus Google and 17-DMAG is and to the J. Y. A of in mice and to and J. PubMed Scopus Google Scholar, M. M. S. et of 17-DMAG a PubMed Scopus Google Scholar). 17-DMAG transcription of ATXN2 in HEK-293 cells in a dose-dependent and reduced the abundance of endogenous and ATXN2 proteins in HEK-293 cells and ATXN2-Q22 mice treated with 17-DMAG had reduction of ATXN2 protein in the We also HSP90 that also in a associated with A. Y. J. et study of HSP990 in with J. PubMed Scopus Google Scholar). in our in as mice well of the mice we treated with HSP990 for ATXN2-Q22 mice treated with HSP990 had reduced ATXN2 protein abundance in the of the molecular of 17-DMAG on ATXN2 expression from spinocerebellar ataxia on transgenic mice treated with 17-DMAG had reduced protein abundance in and reduced A. S. A. M. P. et with 17-DMAG and in a mouse of PubMed Scopus Google Scholar). inhibition by 17-DMAG associated with reduced in the and for A. S. A. M. P. et with 17-DMAG and in a mouse of PubMed Scopus Google Scholar). 17-DMAG also effective for of protein in cells M. of response A therapeutic PubMed Scopus Google and for polyQ and phenotypes in a spinal-bulbar muscular mouse K. H. M. M. M. H. et function in an Genet. PubMed Scopus Google Scholar). A of HSP90 inhibitors including 17-DMAG proteins by autophagy and can also as H. J. Y. et al.Identification of HSP90 inhibitors as a of 2017; PubMed Scopus Google Scholar). for in which HSP90 inhibitors may by ATXN2 transcription, whereas also to ATXN2 protein abundance and with benefits to In a screen of 428,759 we identified compounds including and HSP90 inhibitors that lower ATXN2 expression as well as compounds in identification of compounds on in ATXN2 expression and protein and from the may as for SCA2 or for SCA2 by which may as well treat ALS. Pulst S.M. the expression of Genet. PubMed Scopus Google contains of ATXN2 the ATXN2 and an of the ATXN2 1 the CAG of the trinucleotide is for ATXN2 transcription Pulst S.M. the expression of Genet. PubMed Scopus Google Scholar). the as well as the of the CAG repeat Pulst S.M. the expression of Genet. 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R. for the and of high-throughput PubMed Scopus Google Scholar), and normalized by to with of and of 1 where and are and lowering expression by and for compounds two and using cells, cells, and cells, with assay the of in of assay 2 compounds a with a majority of included of and for in and for and with a high-throughput to and to each well and for an on the with using a 2 on a one and two with to to the and the of each in to and in assay using the and as where 1 2 and J. A. Johnson A. A. et high-throughput a that in U. S. A. PubMed Scopus Google Scholar). in the screen in the and using cells expressing compounds that lower expression by or are as in the assay with or and a of J. A. Johnson A. A. et high-throughput a that in U. S. A. 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