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.
View: Text | PDF
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

×

Figure 4

TGs regulate mutant ATXN1 levels and stability via their catalytic activity.

Options: View larger image (or click on image) Download as PowerPoint
TGs regulate mutant ATXN1 levels and stability via their catalytic activ...
(A) Cell screen data of the genes that encode catalytically active TG. Positive and negative regulators are colored with green and pink, respectively. Color scale is based on the net number of suppressor shRNAs and represented next to the table. (B) Western blot analysis of ATXN1 levels after knockdown of TG2 for 9 days in iPSC-derived neurons from patients with SCA1. Data shown as mean ±SD, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way ANOVA, post hoc Dunnett’s test. (C) Western blot analysis of ATXN1 levels after a treatment with TG2 inhibitor (LDN-27219) for 3 days on iPSC-derived neurons from patients with SCA1. Data shown as mean ± SD, **P < 0.01, 1-way ANOVA, post hoc Dunnett’s test. (D) Western blot analysis of ATXN1 levels after overexpression of either WT or catalytically inactive mutant TG2 in patient iPSC-derived neurons for 9 days. Data shown as mean ± SD, *P < 0.05, 1-way ANOVA, post hoc Tukey’s test. (E) Western blot analysis of ATXN1 levels after overexpression of either WT or catalytically inactive mutant TG5 in ATXN1 reporter cells for 3 days. Data shown as mean ± SD, ***P < 0.001, ****P < 0.0001, 1-way ANOVA, post hoc Tukey’s test. (F) Co-IP results of ATXN1[82Q] and ATXN1[30Q] with TG5 (top) or TG2 (bottom) in the HEK293T cells co-overexpressing ATXN1 and TG. V5-tagged TG5 or TG2 were pulled down and flag-tagged ATXN1 was immunoblotted. (G) Representative IF images of ATXN1[82Q] and TG5 (top) or TG2 (bottom) in Daoy cells overexpressing ATXN1[82Q] and TG5/2. Note that the 2 proteins colocalize. Scale bar: 20 μm. (H) Stability assay of inducibly expressed ATXN1 with different polyQ-length in Daoy cells expressing shTGM2 or shControl. After a 48-hour doxycycline treatment for inducing ATXN1 expression, media was exchanged into growth media without doxycycline, and cells were collected every 6 hours until 30 hours after the media change. Data shown as mean ± SD, *P < 0.05, ****P < 0.0001, 2-way ANOVA, post hoc Dunnett’s test.