Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3

Costa, Maria do Carmo; Ashraf, Naila S.; Fischer, Svetlana; Yang, Yemen; Schapka, Emily; Joshi, Gnanada S.; McQuade, Thomas J.; Dharia, Rahil M; Dulchavsky, Mark; Ouyang, Michelle; Cook, David G.; Sun, Duxin; Larsen, Martha J.; Gestwicki, Jason E.; Todi, Sokol V.; Ivanova, Magdalena I.; Paulson, Henry L.

doi:10.1093/brain/aww228

Cited by 39 publications

(43 citation statements)

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“…Heat shock proteins Hsp40, Hsp70, and Hsp90β, which play important roles in maintaining protein homeostasis in cells 50 , are also altered in SCA3 post-mortem brains and disease model systems [50][51][52][53] . Immunoblot analysis revealed Hsp40 and Hsp70 to be significantly decreased by 50% ± 21% (p = 0.037) and 42% ± 18% (p = 0.039), respectively, in SCA3-hESC relative to WT-hESC (n = 3 passages per line, 2-3 replicates per passage) ( Figure 3G and 3H).…”

Section: Altered Expression Of Key Protein Clearance Pathway Proteinsmentioning

confidence: 99%

Antisense oligonucleotide therapy rescues aggresome formation in a novel Spinocerebellar Ataxia type 3 human embryonic stem cell line

Moore

Keller

Bushart

et al. 2019

Preprint

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Highlights Generated first NIH-approved SCA3 human embryonic stem cell line (SCA3-hESC)  SCA3-hESC exhibit robust ATXN3 aggregation pathology and form p62+ aggresomes  Anti-ATXN3 antisense oligonucleotides rescue aggresome formation in SCA3-hESC  Derived SCA3 neurons form aggregates and exhibit impaired protein quality control Abstract Spinocerebellar ataxia type 3 (SCA3) is a fatal, late-onset neurodegenerative disorder characterized by selective neuropathology in the brainstem, cerebellum, spinal cord, and substantia nigra. Here, we characterize the first NIH-approved human embryonic stem cell (hESC) line derived from an embryo harboring the SCA3 mutation. Referred here as SCA3-hESC, this line is heterozygous for the mutant polyglutamine-encoding CAG repeat expansion in the ATXN3 gene within the pathogenic repeat range for SCA3. We observed relevant molecular hallmarks of the human disease at all differentiation stages from stem cells to cortical neurons, including robust ATXN3 aggregation and altered expression of key components of the protein quality control machinery. Finally, antisense oligonucleotide-mediated reduction of ATXN3 prevented the formation of p62-positive aggresomes in SCA3-hESCs. The SCA3-hESC line offers a unique and highly relevant human disease model that holds strong potential to advance understanding of SCA3 disease mechanisms and facilitate the evaluation of possible SCA3 therapies. Keywords  Aggresome  Antisense oligonucleotide  Ataxin-3  Machado-Joseph disease  Neurodegeneration  Polyglutamine disease 2.10 Key resources table.Key resource information is provided in a separate table.

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Section: Altered Expression Of Key Protein Clearance Pathway Proteinsmentioning

confidence: 99%

Antisense oligonucleotide therapy rescues aggresome formation in a novel Spinocerebellar Ataxia type 3 human embryonic stem cell line

Moore

Keller

Bushart

et al. 2019

Preprint

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“…From 1,250 FDA‐approved drugs, it was found that the atypical antipsychotic drug aripiprazole reduces the levels of mutant ATXN3 in both systems. This compound was further tested in vivo in the mouse and fly models of MJD/SCA3 increasing the longevity of the flies and reducing mutant ATXN3 species in the brain of the transgenic mouse model …”

Section: Therapeutic Strategies For Polyq Diseasesmentioning

confidence: 99%

Discovery of Therapeutic Approaches for Polyglutamine Diseases: A Summary of Recent Efforts

Duarte‐Silva

Maciel

2016

Medicinal Research Reviews

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Polyglutamine (PolyQ) diseases are a group of neurodegenerative disorders caused by the expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the coding region of specific genes. This leads to the production of pathogenic proteins containing critically expanded tracts of glutamines. Although polyQ diseases are individually rare, the fact that these nine diseases are irreversibly progressive over 10 to 30 years, severely impairing and ultimately fatal, usually implicating the full-time patient support by a caregiver for long time periods, makes their economic and social impact quite significant. This has led several researchers worldwide to investigate the pathogenic mechanism(s) and therapeutic strategies for polyQ diseases. Although research in the field has grown notably in the last decades, we are still far from having an effective treatment to offer patients, and the decision of which compounds should be translated to the clinics may be very challenging. In this review, we provide a comprehensive and critical overview of the most recent drug discovery efforts in the field of polyQ diseases, including the most relevant findings emerging from two different types of approaches-hypothesis-based candidate molecule testing and hypothesis-free unbiased drug screenings. We hereby summarize and reflect on the preclinical studies as well as all the clinical trials performed to date, aiming to provide a useful framework for increasingly successful future drug discovery and development efforts.

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“…To identify genes that regulate levels of pathogenic ATXN3 in mammalian cells, we used our previously developed ATXN3-Luc cellular assay (Figure 1A,B) [13] to screen the druggable genome subset of the human siGENOME siRNA library (Dharmacon). This library comprises SMARTpools of four individual siRNAs targeting 2742 genes that are considered potential therapeutic targets, including G-protein coupled receptors (GPCRs), ion channels, protein kinases, proteases, phosphatases and ubiquitin conjugation enzymes (https://dharmacon.horizondiscovery.com/rnai/).…”

Section: Resultsmentioning

confidence: 99%

“…This library comprises SMARTpools of four individual siRNAs targeting 2742 genes that are considered potential therapeutic targets, including G-protein coupled receptors (GPCRs), ion channels, protein kinases, proteases, phosphatases and ubiquitin conjugation enzymes (https://dharmacon.horizondiscovery.com/rnai/). The ATXN3-Luc assay measures chemiluminescence in HEK293 cells stably overexpressing FLAG-tagged human ATXN3 harboring an expanded polyQ repeat in the disease range (Q81) fused to firefly Luciferase, under the control of a CMV promoter (Figure 1A,B) [13]. We reasoned that by screening for steady state levels of the ATXN3/Luciferase fusion protein (reported as chemiluminescence), and because its expression is driven by the CMV promoter, we would identify genes that regulate ATXN3 abundance at post-transcriptional steps (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…The GMR-Gal4 driver (#8605) and UAS-mCD8-GFP (#5137) were from the Bloomington Drosophila Stock center. The UAS-ataxin-3Q77 line has been described before [13, 28]. For histological sections, adult fly wings and proboscises were removed, and flies were fixed in 2% glutaraldehyde/ 2% paraformaldehyde in Tris-buffered saline with 0.1% Triton X-100.…”

Section: Methodsmentioning

confidence: 99%

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Druggable genome screen identifies new regulators of the abundance and toxicity of ATXN3, the Spinocerebellar Ataxia Type 3 disease protein

Ashraf

Sutton

Yang

et al. 2019

Preprint

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27Background: Spinocerebellar Ataxia type 3 (SCA3, also known as Machado-Joseph disease) 28 is a neurodegenerative disorder caused by a CAG repeat expansion encoding an abnormally 29 long polyglutamine (polyQ) tract in the disease protein, ataxin-3 (ATXN3). No preventive 30treatment is yet available for SCA3. Because SCA3 is likely caused by a toxic gain of ATXN3 31 function, a rational therapeutic strategy is to reduce mutant ATXN3 levels by targeting pathways 32 that control its production or stability. Here, we sought to identify genes that modulate ATXN3 33 levels as potential therapeutic targets in this fatal disorder. 34 Methods:We screened a collection of siRNAs targeting 2742 druggable human genes using a 35 cell-based assay based on luminescence readout of polyQ-expanded ATXN3. From 317 36 candidate genes identified in the primary screen, 100 genes were selected for validation. 37Among the 33 genes confirmed in secondary assays, 15 were validated in an independent cell 38 model as modulators of pathogenic ATXN3 protein levels. Ten of these genes were then 39 assessed in a Drosophila model of SCA3, and one was confirmed as a key modulator of 40 physiological ATXN3 abundance in SCA3 neuronal progenitor cells. 41 Results: Among the 15 genes shown to modulate ATXN3 in mammalian cells, orthologs of 42 CHD4, FBXL3, HR and MC3R regulate mutant ATXN3-mediated toxicity in fly eyes. Further 43 mechanistic studies of one of these genes, FBXL3, encoding a F-box protein that is a 44 component of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex, showed that it reduces 45 levels of normal and pathogenic ATXN3 in SCA3 neuronal progenitor cells, primarily via a SCF 46 complex-dependent manner. Bioinformatic analysis of the 15 genes revealed a potential 47 molecular network with connections to tumor necrosis factor-a/nuclear factor-kappa B (TNF/NF-48 kB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways. 49 Conclusions:We identified 15 druggable genes with diverse functions to be suppressors or 50 enhancers of pathogenic ATXN3 abundance. Among identified pathways highlighted by this 51 screen, the FBXL3/SCF axis represents a novel molecular pathway that regulates physiological 52 levels of ATXN3 protein. 53 54 Keywords: polyglutamine, spinocerebellar ataxia, Machado-Joseph disease, 55 neurodegeneration, high-throughput screen, human embryonic stem cells, Drosophila 56 57 Introduction 58The polyglutamine (polyQ) diseases are inherited neurodegenerative diseases caused by 59 expanded CAG repeats that encode abnormally long glutamine repeats in the disease proteins 60[1, 2]. Spinocerebellar Ataxia type 3 (SCA3) is one of nine known polyQ disorders and the most 61 common dominant ataxia, primarily manifesting with degeneration of the cerebellum, brainstem, 62spinal cord, and basal ganglia [3][4][5][6][7]. The CAG repeat in the ATXN3 gene, which normally is 12 63 to 44 triplets, becomes expanded to ~60 to 87 repeats in SCA3 [8, 9]. Despite sharing a 64 propensity to misfold and aggregate, polyQ disease p...

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Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3

Cited by 39 publications

References 64 publications

Antisense oligonucleotide therapy rescues aggresome formation in a novel Spinocerebellar Ataxia type 3 human embryonic stem cell line

Antisense oligonucleotide therapy rescues aggresome formation in a novel Spinocerebellar Ataxia type 3 human embryonic stem cell line

Discovery of Therapeutic Approaches for Polyglutamine Diseases: A Summary of Recent Efforts

Druggable genome screen identifies new regulators of the abundance and toxicity of ATXN3, the Spinocerebellar Ataxia Type 3 disease protein

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