Deubiquitinase USP7 contributes to the pathogenicity of spinal and bulbar muscular atrophy
Anna Pluciennik, Yuhong Liu, Elana Molotsky, Gregory B. Marsh, Bedri Ranxhi, Frederick J. Arnold, Sophie St.-Cyr, Beverly Davidson, Naemeh Pourshafie, Andrew P. Lieberman, Wei Gu, Sokol V. Todi, Diane E. Merry
Anna Pluciennik, Yuhong Liu, Elana Molotsky, Gregory B. Marsh, Bedri Ranxhi, Frederick J. Arnold, Sophie St.-Cyr, Beverly Davidson, Naemeh Pourshafie, Andrew P. Lieberman, Wei Gu, Sokol V. Todi, Diane E. Merry
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Research Article Neuroscience

Deubiquitinase USP7 contributes to the pathogenicity of spinal and bulbar muscular atrophy

Abstract

Polyglutamine (polyQ) diseases are devastating, slowly progressing neurodegenerative conditions caused by expansion of polyQ-encoding CAG repeats within the coding regions of distinct, unrelated genes. In spinal and bulbar muscular atrophy (SBMA), polyQ expansion within the androgen receptor (AR) causes progressive neuromuscular toxicity, the molecular basis of which is unclear. Using quantitative proteomics, we identified changes in the AR interactome caused by polyQ expansion. We found that the deubiquitinase USP7 preferentially interacts with polyQ-expanded AR and that lowering USP7 levels reduced mutant AR aggregation and cytotoxicity in cell models of SBMA. Moreover, USP7 knockdown suppressed disease phenotypes in SBMA and spinocerebellar ataxia type 3 (SCA3) fly models, and monoallelic knockout of Usp7 ameliorated several motor deficiencies in transgenic SBMA mice. USP7 overexpression resulted in reduced AR ubiquitination, indicating the direct action of USP7 on AR. Using quantitative proteomics, we identified the ubiquitinated lysine residues on mutant AR that are regulated by USP7. Finally, we found that USP7 also differentially interacts with mutant Huntingtin (HTT) protein in striatum and frontal cortex of a knockin mouse model of Huntington’s disease. Taken together, our findings reveal a critical role for USP7 in the pathophysiology of SBMA and suggest a similar role in SCA3 and Huntington’s disease.

Authors

Anna Pluciennik, Yuhong Liu, Elana Molotsky, Gregory B. Marsh, Bedri Ranxhi, Frederick J. Arnold, Sophie St.-Cyr, Beverly Davidson, Naemeh Pourshafie, Andrew P. Lieberman, Wei Gu, Sokol V. Todi, Diane E. Merry

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Figure 11

AR is a substrate for the deubiquitinase function of USP7.

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AR is a substrate for the deubiquitinase function of USP7. (A) Immunopre...
(A) Immunoprecipitated AR from HEK293T cells transiently expressing AR111Q, HA-tagged Ub, and FLAG-USP7 (or GFP control) was analyzed by immunoblotting with anti-AR antibody (top), HA-specific antibody (middle) and K48-linked polyubiquitin-specific antibody (bottom). Input levels are shown on the left. (B) Quantitation (from 8 independent experiments performed in duplicate or triplicate) of the relative change in HA to AR monomer signals upon FLAG-USP7 overexpression. (C) Quantitation of the relative change in K48 to AR monomer signals upon FLAG-USP7 overexpression from 4 independent experiments (each experiment performed in duplicate or triplicate). *P < 0.05; ****P < 0.0001, 2-tailed Student’s t test. Error bars represent SD. (D) Percentage of cells with nuclear inclusions in cells expressing AR112Q, AR107Q K17R, or AR108Q K17R. *P < 0.05; ***P < 0.001; ****P < 0.0001, 1-way ANOVA with post hoc Tukey’s test. Error bars represent SD. (E) To evaluate AR turnover, AR112Q, AR107Q K17R, or AR108Q K17R expression was induced with DOX for 48 hours. Following DOX washout, cells were treated with cycloheximide and either DHT or vehicle (EtOH) for an additional 24 hours. AR levels were analyzed by Western blotting (example images shown in Supplemental Figure 10E). Data were normalized to the vehicle (EtOH) treatment condition. Three independent experiments (each one in triplicate) were performed. **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA with post hoc Tukey’s test. Error bars represent SD.