Cross-species genetic screens identify transglutaminase 5 as a regulator of polyglutamine-expanded ataxin-1
Won-Seok Lee, … , Juan Botas, Huda Y. Zoghbi
Won-Seok Lee, … , Juan Botas, Huda Y. Zoghbi
Published May 2, 2022
Citation Information: J Clin Invest. 2022;132(9):e156616. https://doi.org/10.1172/JCI156616.
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Research Article Genetics Neuroscience

Cross-species genetic screens identify transglutaminase 5 as a regulator of polyglutamine-expanded ataxin-1

Abstract

Many neurodegenerative disorders are caused by abnormal accumulation of misfolded proteins. In spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded (polyQ-expanded) ataxin-1 (ATXN1) causes neuronal toxicity. Lowering total ATXN1, especially the polyQ-expanded form, alleviates disease phenotypes in mice, but the molecular mechanism by which the mutant ATXN1 is specifically modulated is not understood. Here, we identified 22 mutant ATXN1 regulators by performing a cross-species screen of 7787 and 2144 genes in human cells and Drosophila eyes, respectively. Among them, transglutaminase 5 (TG5) preferentially regulated mutant ATXN1 over the WT protein. TG enzymes catalyzed cross-linking of ATXN1 in a polyQ-length–dependent manner, thereby preferentially modulating mutant ATXN1 stability and oligomerization. Perturbing Tg in Drosophila SCA1 models modulated mutant ATXN1 toxicity. Moreover, TG5 was enriched in the nuclei of SCA1-affected neurons and colocalized with nuclear ATXN1 inclusions in brain tissue from patients with SCA1. Our work provides a molecular insight into SCA1 pathogenesis and an opportunity for allele-specific targeting for neurodegenerative disorders.

Authors

Won-Seok Lee, Ismael Al-Ramahi, Hyun-Hwan Jeong, Youjin Jang, Tao Lin, Carolyn J. Adamski, Laura A. Lavery, Smruti Rath, Ronald Richman, Vitaliy V. Bondar, Elizabeth Alcala, Jean-Pierre Revelli, Harry T. Orr, Zhandong Liu, Juan Botas, Huda Y. Zoghbi

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Figure 1

Cross-species screen of druggable genes reveals potential regulators of ATXN1.

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Cross-species screen of druggable genes reveals potential regulators of ...
(A) Schematic representation of the cell-based screen. ATXN1 reporter cells produce RFP-ATXN1[82Q] and YFP from the same transcript through separate translation processes. ATXN1[82Q] levels are monitored by RFP/YFP ratio whereby YFP normalizes ATXN1 levels. After retroviral transduction of the reporter cells with pooled shRNA libraries targeting 7787 genes, cells were subjected to FACS to collect the cells with the lowest 5% and highest 5% RFP/YFP ratio. Genomic DNAs of these cells were extracted, and Illumina sequencing revealed relative enrichment of each shRNA in the sorted cells compared with the non-sorted bulk population. Identifying the genes targeted by the enriched or depleted shRNAs in each group revealed regulators of ATXN1 protein levels, which were filtered and prioritized (see Methods). (B) A diagram for modifier screen in Drosophila. Ectopic expression of human mutant ATXN1[82Q] in Drosophila eyes induces retinal degeneration. This fly was crossed with shRNA fly lines that target 1102 Drosophila genes corresponding to 2144 human homologs for identifying genes that suppress or exacerbate external eye phenotype. (C) The number of genes that overlapped between cell-based screen and Drosophila-based screen. The 156 positive regulators, including 70 genes that overlapped in the 2 screens and 86 top cell screen hits were selected for validation (See Methods for criteria applied). (D) A diagram for tiered validation of potential ATXN1 regulators. The number of genes before and after each validation step is displayed. (E) Summary of ATXN1[82Q] ELISA result presented as averaged percentage changes of ATXN1[82Q] levels after knockdown of 156 genes by 3 shRNAs in the ATXN1 reporter cells. MSK1 and ATXN1 were included as positive controls. (F) A representative ATXN1[82Q] ELISA result after knockdown of the genes that belong to the “others” and ”kinase/phosphatase” libraries. Individual bar displays ATXN1 levels of each shRNA. Blue-colored genes are selected for next validations. Data shown as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way ANOVA, post hoc Dunnett’s test.