Interaction of the polyglutamine protein ataxin-3 with Rad23 regulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3

Abstract

Polyglutamine (polyQ) repeat expansion in the deubiquitinase ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.

Degron capability of the hydrophobic C‐terminus of the polyglutamine disease protein, ataxin‐3

“…Examples of other proteins that espouse degrons but are degraded by the proteasome in a ubiquitination‐independent manner include ornithine decarboxylase, Rpn4, and thymidylate synthase, as well as p21, p53, and α‐synuclein (Ella et al., 2019; Erales & Coffino, 2014; Sanchez‐Lanzas & Castano, 2014). Our prior and current data indicate that ataxin‐3 belongs to the group of proteins that can be degraded without the necessity of its own ubiquitination (Blount et al., 2014; Sutton et al., 2017; Todi et al., 2010; Tsou et al., 2013). This is not to say that circumstances might not exist where ubiquitination of ataxin‐3 could enhance its targeting to the proteasome and subsequent degradation.…”

Druggable genome screen identifies new regulators of the abundance and toxicity of ATXN3, the Spinocerebellar Ataxia Type 3 disease protein

“…To assess whether the above findings in two cell models are physiologically relevant in vivo , we tested the efficacy of 10 orthologs of identified genes to alter mutant ATXN3-mediated toxicity in a Drosophila model of SCA3 [13, 28, 31]: CACNG7 , CHD4 , CDK8 , FASTK , FBXL3 , FES , HR , MC3R , PKD2 , and TACR1 . For simplicity, we focused on the fly eye expression model which is commonly used to examine the role and pathogenicity of various misfolded proteins [32].…”

“…For simplicity, we focused on the fly eye expression model which is commonly used to examine the role and pathogenicity of various misfolded proteins [32]. Expression of pathogenic ATXN3 (Q77) in fly eyes is insufficiently toxic to cause marked degeneration of external structures [28, 31], necessitating the examination of internal eye structures for degenerative phenotypes or the use of a membrane-targeted GFP molecule (CD8-GFP) as a simple readout of the loss of the functional unit of the fly eye, the ommatidium [23]. Through this second assay, a toxic protein such as pathogenic ATXN3 is expressed in fly eyes independently of CD8-GFP; whereas the outside part of the fly eye seems unperturbed by the presence of the toxic protein, internal structures degenerate and photoreceptor cells disappear, resulting in the loss of GFP fluorescence [23].…”

“…The GMR-Gal4 driver (#8605) and UAS-mCD8-GFP (#5137) were from the Bloomington Drosophila Stock center. The UAS-ataxin-3Q77 line has been described before [13, 28]. For histological sections, adult fly wings and proboscises were removed, and flies were fixed in 2% glutaraldehyde/ 2% paraformaldehyde in Tris-buffered saline with 0.1% Triton X-100.…”

Diagnosis of polyglutamine spinocerebellar ataxias by polymerase chain reaction amplification and Sanger sequencing

“…The SCAs caused by the CAG repeat expansion mutation are classified as polyglutamine SCAs. These include SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17, and dentatorubral-pallidoluysian atrophy (DRPLA), in which the expansion within the glutamine-encoding CAG trinucleotide repeats accounts for SCA (3). Healthy individuals usually carry the heterozygous CAG repeats at a number below the predicted pathological threshold.…”