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

Abstract

Spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, is the most common dominantly inherited ataxia. SCA3 is caused by a CAG repeat expansion in the ATXN3 gene that encodes an expanded tract of polyglutamine in the disease protein ataxin-3 (ATXN3). As a deubiquitinating enzyme, ATXN3 regulates numerous cellular processes including proteasome- and autophagy-mediated protein degradation. In SCA3 disease brain, polyQ-expanded ATXN3 accumulates with other cellular constituents, including ubiquitin (Ub)-modified proteins, in select areas like the cerebellum and the brainstem, but whether pathogenic ATXN3 affects the abundance of ubiquitinated species is unknown. Here, in mouse and cellular models of SCA3, we investigated whether elimination of murine Atxn3 or expression of wild-type or polyQ-expanded human ATXN3 alters soluble levels of overall ubiquitination, as well as K48-linked (K48-Ub) and K63-linked (K63-Ub) chains. Levels of ubiquitination were assessed in the cerebellum and brainstem of 7- and 47-week-old Atxn3 knockout and SCA3 transgenic mice, and also in relevant mouse and human cell lines. In older mice, we observed that wild-type ATXN3 impacts the cerebellar levels of K48-Ub proteins. In contrast, pathogenic ATXN3 leads to decreased brainstem abundance of K48-Ub species in younger mice and changes in both cerebellar and brainstem K63-Ub levels in an age-dependent manner: younger SCA3 mice have higher levels of K63-Ub while older mice have lower levels of K63-Ub compared to controls. Human SCA3 neuronal progenitor cells also show a relative increase in K63-Ub proteins upon autophagy inhibition. We conclude that wild-type and mutant ATXN3 differentially impact K48-Ub- and K63-Ub-modified proteins in the brain in a region- and age-dependent manner.

The deubiquitinase function of ataxin-3 and its role in the pathogenesis of Machado-Joseph disease and other diseases

“…PolyQ expansion does not significantly alter ataxin-3's polyubiquitin binding capacity in vitro [ 84 ], however, polyQ-expanded ataxin-3 appears to be less efficient than its wild-type counterpart at reducing ubiquitinated proteins in cells, which indicates that expansion may somehow impair its DUB function [ 88 ]. Further suggestion of altered DUB function of polyQ-expanded ataxin-3 stems from a recent study reporting that the amount of K48- and K63-ubiquitinated proteins varies between brain regions and with disease stage within the brain of MJD mice [ 100 ].…”

“…While a growing body of evidence suggests that polyQ expansion in ataxin-3 may impact its DUB function [ 88 , 100 ], whether this results in a gain or loss of DUB function and the impact of such changes on ataxin-3's targets is yet to be comprehensively evaluated, as detailed in Table 1 . The broader implications of changes in ataxin-3's DUB function due to polyQ expansion in the context of MJD pathophysiology also remain to be clarified.…”

“…Despite the fact that inclusions are a pathological hallmark of MJD, and high expression of full-length ataxin-3 causes cell death and formation of intranuclear inclusions, there is no clear correlation between the presence of intranuclear inclusions and cell death [ 153 ]. A recent study demonstrated region-specific changes in the abundance of ubiquitinated proteins in MJD mice, with expression of polyQ-expanded ataxin-3 resulting in significantly increased abundance of K63-ubiquitinated proteins in the cerebellum and brainstem, and decreased abundance of K48-ubiquitinated proteins in the brainstem of 7-week-old MJD mice [ 100 ]. It would therefore be interesting to further explore whether changes in the DUB function of ataxin-3 due to polyQ expansion affects some cell types more than others, and whether this correlates with the cell types that are particularly impacted in MJD.…”

Oxidative Stress in Spinocerebellar Ataxia Type 3 and Its Attenuation by Herbal Remedies in Traditional Chinese Medicine: A Systematic Review

“…The ATXN3 protein is a ubiquitously expressed deubiquitinating enzyme that is responsible for the regulation of transcription, proteasome- and autophagy-mediated protein degradation pathways, DNA damage repair, and modulating cytoskeletal dynamics [ 18 , 58 , 59 , 60 ]. In polyQ disorders, including SCA3, diminished protection against free radicals caused by increased oxidative stress gives rise to mitochondrial dysfunction and cellular damage.…”

Spinocerebellar Ataxia Type 3 Pathophysiology—Implications for Translational Research and Clinical Studies

“…Post-translational modifications like ubiquitination, were shown to increase the activity of ATXN3 but did not affect the translocation rate. However, it was observed that the enzymatically active protein was located more often in the nuclei [13,20].…”