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

Abstract

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...

Regional and age-dependent changes in ubiquitination in cellular and mouse models of spinocerebellar ataxia type 3

“…Mouse embryonic fibroblasts (MEFs) from both Atxn3 KO mice and WT littermates (Reina et al, 2010) were maintained in DMEM with 10% FBS, 1% Non-essential Amino Acids solution and 1% penicillin/streptomycin at 37°C and 5% CO2. Control and SCA3 neuronal progenitor cells (NPCs) were generated from the respective human embryonic stem cell (hESC) lines (CTRL, NIH registry #0147; SCA3, NIH registry # 0286), and were maintained in STEMdiff Neural Progenitor Medium (NPM), as previously described (Ashraf et al, 2020). Where indicated, cells were treated with lactacystin (15 μM; Enzo Life Sciences), Chloroquine (100 μM; Sigma-Aldrich), or DMSO (Sigma-Aldrich) for 12 h.…”

“…The SCA3 mouse lines used above overexpress wild-type or polyQ-expanded human ATXN3. To probe whether physiological levels of expanded ATXN3 affect the abundance of Ub species in human neuronal cells, we assessed the levels of Ub species in neuronal progenitor cells (NPCs) derived from a hESC line harboring one disease and one healthy allele (SCA3, NIH registry # 0286) (Moore et al, 2019;Ashraf et al, 2020). For comparison, Under basal conditions, SCA3 NPCs displayed a 75% increase in HMW pan-Ub levels compared to CTRL NPCs (Figures 6A,D).…”