Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3

Abstract

Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when the enzyme is itself ubiquitinated at lysine (K) at position 117: in vitro, K117-ubiqutinated Atxn3 cleaves poly-ubiquitin markedly more rapidly compared to its unmodified counterpart. How polyQ expansion causes SCA3 remains unclear. To gather insights into the biology of disease of SCA3, here we posited the question: is K117 important for toxicity caused by pathogenic Atxn3? To answer this question, we generated transgenic Drosophila lines that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. We found that mutating K117 mildly enhances the toxicity and aggregation of pathogenic Atxn3. An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest that Atxn3 ubiquitination is a regulatory step of SCA3, in part by modulating its aggregation.

Keywords: Aggregation; Ataxia; Deubiquitinase; Polyglutamine; Ubiquitin.

Figure 1:. K117 mutation enhances Atxn3 toxicity in all tissues

A) Atxn3 protein domains and the sequences encoded by the transgenes in this study. UbS: ubiquitin-binding site. UIM: ubiquitin-binding motif. VBM: VCP-binding motif. HA tag is appended inline to each protein (sequence in italics). Josephin domain is the catalytic portion of Atxn3. B) Results of sqh-Gal4 (ubiquitous driver) expressing the indicated Atxn3 forms. All genotypes were heterozygous sqh-Gal4 in trans with the indicated UAS-Atxn3 transgene, also heterozygous. Means −/+ SD. Statistics: t-tests comparing Atxn3(Q80)-K117R to Atxn3(Q80). C) Longevity of adult flies that eclosed successfully from (B). Ctrl: sqh-Gal4 in trans with the parental line used to generate the Atxn3 transgenics. Statistics: log-rank test.

Figure 2:. K117 mutation enhances Atxn3 toxicity in neurons

A, B) Longevity of adult flies expressing the noted versions of Atxn3 in all neuronal cells. Heterozygous driver (elav-Gal4, panel A; elav-GS-Gal4, panel B) was in trans with the noted Atxn3 transgenes, also heterozygous. Statistics: log-rank tests. For adult-only expression, we utilized the GeneSwitch Gal4-UAS system, which needs the presence of the inducer, RU486 to express a specific UAS transgene. We reared flies in media without RU486 until the day that they eclosed from their pupal cases, and then moved them onto media containing RU486, enabling the expression of human Atxn3 transgenes. These flies were maintained in media with RU486 until they all died.

Figure 3:. Effects of lysine mutations on the turnover and subcellular fractionation of pathogenic Atxn3

A) Western blots from adult female flies expressing the noted versions of Atxn3 constitutively in all neurons. Flies were heterozygous for driver (elav-Gal4) and Atxn3. Day 1 adults. Each lane represents an independent repeat. Black arrow: main, unmodified Atxn3. Blue arrow: ubiquitinated Atxn3, based on our previously published work (Blount et al., 2020; Johnson et al., 2019; Sutton et al., 2017; Todi et al., 2007; Todi et al., 2010; Todi et al., 2009; Tsou et al., 2013). Gray arrow: likely proteolytic fragment we observe sometimes. Red bar: SDS-resistant Atxn3. Quantifications: images from the left were used for quantification, normalized to their respective Direct blue loading control. The entire Atxn3 signal was utilized, main band to top, including SDS-soluble and SDS-resistant species. Means −/+ SD. Statistics: one-way ANOVA with Dunnett’s post hoc. **: p<0.001. B) qRT-PCR results from 1-day-old adult females constitutively expressing the noted Atxn3 versions in all neurons, as in panel A. Means −/+ SD. Statistics: one-way ANOVA with Dunnett’s post hoc. *: p<0.05. C) Top: Western blots from adult female flies expressing the noted versions of Atxn3 in neurons for 7 days (+RU486) then allowed to degrade Atxn3 over the indicated timeline (−RU486). A higher amount of lysates from KNull was loaded to equilibrate as much as possible starting amounts at day 0. Bottom: quantification of signal from the top and additional independent repeats, normalized to respective day 0 levels. No statistical significances found were with oneway ANOVA with Dunnett’s post hoc among day 3, 5 and 7 comparisons. This experiment was repeated independently 3 times, with 5 whole flies used per group per Western blot in each repeat. Flies were heterozygous for driver (elav-GS-Gal4) and Atxn3. D) Left: Western blots of nuclear/cytoplasmic fractionations of Atxn3 versions noted expressed constitutively in all neurons. Flies were 1-day-old females. Right: quantification of images from the left and other independent repeats. Means −/+ SD. Statistics: one-way ANOVA with Dunnett’s post hoc. Flies were heterozygous for driver (elav-Gal4) and Atxn3 transgenes. ns: not significant.

Figure 4:. DnaJ-1 suppresses toxicity of pathogenic Atxn3 independently of lysine mutations

A, B) Longevity outcomes from adult flies expressing the noted versions of Atxn3 in the presence or absence of exogenous DnaJ-1, only in adult neurons. B) Statistics: log-rank tests. C) Left: Western blots from co-immunopurifications of Atxn3 expressed in female adult neurons for 7 days in the presence of exogenous FLAG-tagged DnaJ-1. Right: quantifications from the left and other independent repeats. Statistics: Wilcoxon tests. ns: not significant. FLAG-tagged DnaJ-1 was used for these studies since antibodies to detect endogenous DnaJ-1 are not available. Flies were heterozygous for the Gal4 driver and each UAS transgenes. D) qRT-PCR of endogenous levels of DnaJ-1 when Atxn3 of the noted versions was expressed. Statistics: one-way ANOVA with Dunnett’s post-hoc. ns: not significant.

Figure 5:. Lysine mutations increase levels of SDS-resistant species of pathogenic Atxn3 in adult neurons

A, B) Western blots from flies expressing the noted versions of Atxn3 in adult neurons for the indicated amounts of time. Black arrow: main, unmodified Atxn3. Blue bar: ubiquitinated Atxn3. Red bar: SDS-resistant Atxn3. Gray bar: proteolytic fragments of Atxn3. Asterisk on the right of blot in 5B is non-specific signal. Associated quantifications are from these blots. Means −/+ SD. Atxn3 signal for each lane was calculated by quantifying separately the SDS-soluble species (main band + ubiquitinated species) and the SDS-resistant species, then expressing soluble Atxn3 as percent fraction of the total Atxn3 signal in that lane, using the formula: 100% X SDS-soluble / (SDS-soluble + SDS-resistant). Statistics: one-way ANOVA with Dunnett’s post hoc. ns: not significant. *: p<0.05. ***: p<0.001. ****: p<0.0001. Flies were heterozygous for driver and Atxn3.

Figure 6:. Lysine mutations impact the toxicity of pathogenic Atxn3 in fly eyes

A) Eye scoring system. B) Means −/+ SD of eye phenotypes when the noted versions of Atxn3 are expressed in fly eyes (GMR-Gal4). ns: not significant. **: p<0.01. ***: p<0.001. ****: p<0.0001. C) Western blots and related quantifications from 1-day-old adult female fly heads. GMR-Gal4 was the driver in each case. Black arow: main ATxn3. Blue bar: ubiquitinated Atxn3. Red bar: SDS-resistant Atxn3. Gray bar: proteolytic fragments of Atxn3. Proteolytic species differ depending on the tissues in which Atxn3 is expressed in flies and also vary among blots and antibodies (Blount et al., 2020; Blount et al., 2014; Johnson et al., 2019; Johnson et al., 2021; Johnson et al., 2022a; Sutton et al., 2017; Todi et al., 2010). Quantifications are from the top and additional independent repeats. Atxn 3 signal for each lane was calculated by quantifying SDS-soluble species, SDS-resistant species, and expressed as percent soluble fraction of the total Atxn3 signal in that lane. Means −/+ SDS. Statistics: Brown-Forsythe and Welch ANOVA tests. ns: not significant; *: p<0.05. ***: p<0.001. All flies were heterozygous for driver and Atxn3 transgenes.

Figure 7:. Summary

Brief summary of the findings supported from our current data and prior publications in the discussion section.