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 5

TGs preferentially cross-link mutant ATXN1 and regulate its solubility and oligomerization.

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TGs preferentially cross-link mutant ATXN1 and regulate its solubility a...
(A) In vitro TG assay of ATXN1[82Q] with recombinant human TG2 under various conditions: with normal or heat-inactivated enzyme; with or without Ca2+ ion, pan-TG inhibitor cystamine (40 mM), or TG2-specific inhibitor LDN-27219 (2 mM). The proportion of ATXN1 high MW (HMW) species in each lane represented in bottom graph. Data shown as mean ± SD, ****P < 0.0001, 1-way ANOVA, post hoc Dunnett’s test. (B) In vitro TG assay of ATXN1[82Q], ATXN1[30Q], and ATXN1[2Q] with recombinant human TG2. Either 1× (+) dose or 2× (++) doses of ATXN1 was used for the reaction. The proportion of ATXN1 HMW species in lane 10–17 represented in bottom graph. Data shown as mean ± SD, ****P < 0.0001, 1-way ANOVA, post hoc Tukey’s test. (C) Western blot analysis of Triton X-100–soluble or –insoluble ATXN1 HMW species and monomer extracted from HEK293T cells overexpressing ATXN1[82Q] and TG5 or LacZ. Empty, empty vector; LacZ, overexpression control. Triton X-100–insoluble ATXN1 (HMW species + monomer) to Triton X-100–soluble ATXN1 (monomer) ratio in each overexpression group represented in bottom right graph. Asterisk indicates nonspecific bands. Data shown as mean ± SD, ***P < 0.001, ****P < 0.0001, 2-tailed t test for bottom right graph; 1-way ANOVA for other graphs with post hoc Dunnett’s test. (D) Western blot analysis of Triton X-100–soluble or –insoluble ATXN1 HMW species and monomer extracted from HEK293T cells overexpressing ATXN1[82Q] and shRNA against TG5. Triton X-100–insoluble ATXN1 (HMW species + monomer) to Triton X-100–soluble ATXN1 (monomer) ratio in each experimental group represented in middle right graph. 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. (E) Western blot analysis of soluble ATXN1 oligomers in cerebellar lysate of WT mice and SCA1 mice expressing shTgm5 (same lysate in Figure 3D) using oligomer-specific F11G3 antibody. Data shown as mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001, 1-way ANOVA, post hoc Dunnett’s test.