Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells

Abstract

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado-Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.

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“…Indeed, animal models carrying mutant HTT and those lacking HTT normal function show some similarities at pathological and behavioral levels, suggesting a contribution of a reduction in the normal function of HTT to the HD phenotype . In accordance, a study in MJD/SCA3 showed that the lack of ATXN3 produced molecular alterations in a neuronal cell model that overlapped those seen in cells overexpressing expanded ATXN3, and also in neurons from a mouse model of MJD/SCA3, suggesting a contribution of an impaired function of ATXN3 to MJD/SCA3 pathogenesis . In SCA1, in which the toxic gain of function hypothesis is well accepted, additional data suggest that perturbation of a normal ATXN1 function contributes to pathogenesis, namely through changes in specific interactions .…”

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“…Increased levels of ubiquitinated substrates have been observed in human SCA3 brain tissue and mouse models of SCA3, possibly reflecting altered mutATXN3 deubiquitinase activity 47 and downstream impairment in proteolytic clearance pathways 48,49 . Using a panubiquitin antibody, we found that poly-ubiquitinated protein levels were slightly but significantly increased by 17% ± 4% in SCA3-hESC relative to WT-hESC (p = 0.015, n = 3 passages per line) ( Figure 3E and 3F), suggesting impairment in ubiquitin-dependent proteasomal clearance.…”

Regulation of neuronal mRNA splicing and Tau isoform ratio by ATXN3 through deubiquitylation of splicing factors

“…As shown in Figure 2A-D, knockdown of ATXN3 significantly altered the processing of the three splicing reporters, suggesting a general deregulation of splicing in the absence of this protein. Interestingly, overexpression of a catalytically inert version of ATXN3 in SH-SY5Y cells (ATXN3_C14A), previously shown to have a dominant-negative effect 25 also yielded similar alterations (Figure 2E). Consistent with a global deregulation of splicing in the absence of ATXN3, microarray analysis (Figure 2F) using specific arrays containing additional probes for exon/exon junctions, confirmed that 1993 genes (43%), from the 7450 differentially expressed probes in ATXN3 shRNA cells, presented differentially regulated alternative splicing events (Table EV1).…”

“…With the goal of identifying substrates of the DUB activity in neuronal cells, we searched for changes in the ubiquitome of neuronally differentiated SH-SY5Y cells lacking ATXN3 (ATXN3 shRNA cells – Figure EV1A-B) 25 , taking advantage of a recently developed methodology that combines capture of the whole spectrum of polyubiquitylated proteins in a cell extract using an enrichment by Tandem Binding Ubiquitin Entities (TUBEs) followed by mass spectrometry analysis (TUBEs-LC-MS/MS) 30 . Figure 1A summarizes the steps followed for the purification and identification of the polyubiquitylated proteins in ATXN3 shRNA and scrambled control shRNA (SCR shRNA ) cells.…”

“…Indeed, since many of these diseases exhibit ubiquitylated protein aggregates, loss of ubiquitin homeostasis may be an important contributor for disease. Considering the importance of DUB enzymes in maintaining the ubiquitylation balance, we focused on the characterization of the ubiquitome of neuronal cells lacking ATXN3 25 . Using TUBEs, that enable the pulldown of polyubiquitylated proteins without further genetic manipulation or inhibition of the proteasome 30 in combination with LC-MS/MS, we were able to consistently identify 615 proteins per condition, a yield comparable to those described in other studies 35,36 .…”