a-f, Flies expressing similar levels of SCA3trQ78_CAG and SCA3trQ78_CAA/G protein show strikingly different degeneration. (a-c) Flies expressing SCA3 with gmr-GAL4. (d-f) Adult-onset PR retinal degeneration, with expression by rh1-GAL4 (25d). (a, d) Controls expressing non-pathogenic SCA3trQ27 protein have normal eye structure. Genotype UAS-SCA3trQ27 in trans to (a) gmr-GAL4 or (d) rh1-GAL4. (b, e) Flies expressing SCA3trQ78(s)_CAG show (b) severe retinal degeneration and (e, arrows) striking loss of rhabdomeres (4.6±0.6 PR/ommatidium). UAS-SCA3trQ78(s)_CAG in trans to (b) gmr-GAL4 and (e) rh1-GAL4. (c, f) Flies expressing SCA3trQ78_CAA/G at the same level show mild degeneration, with (c) normal external eye morphology and (f) mild PR loss (6.5±0.2 PR/ommatidium, statistically significant from e, P<0.001, two-tailed unpaired student t-test). Genotype UAS-SCA3trQ78_CAA/G in trans to (c) gmr-GAL4 or (f) rh1-GAL4. (g) Neuronal toxicity by lifespan analysis. Flies expressing SCA3trQ78(s)_CAG have a strikingly shorter lifespan than flies expressing SCA3trQ78_CAA/G at the same level (P< 0.001, log-rank test). Mean ± SD, n=150-200 flies for each. Genotypes: elav-GAL4 in trans to UAS-SCA3trQ78(s)_CAG or UAS-SCA3trQ78_CAA/G. (h) Climbing behavior with age. At 1d, both SCA3trQ78_CAG and SCA3trQ78_CAA/G flies show normal climbing compared to SCA3trQ27 control flies, with only ∼5% failing to climb with agitation (mean ± SD, n=120-200 flies total). SCA3trQ78_CAG flies show more progressive climbing defects, such that at 12d, 42.1±7.6% of the flies fail to climb (mean ± SD, n=180). In contrast, only 17.9±2.6 % of SCA3trQ78_CAA/G flies fail to climb at 12d (mean ± SD, n=200). Genotypes as in h. *, P<0.05; **, P<0.01; ***, P<0.001 (two-way ANOVA).

a-d, External eye of 1d flies. (a) Flies expressing gmr-GAL4 alone or (b) with mbl^B2-E1 have normal eye morphology. (c) Flies expressing SCA3trQ61 have a mild loss of pigmentation, and slightly disrupted internal retinal morphology. (d) Flies expressing SCA3trQ61 with mbl^B2-E1 show severe external degeneration and collapse of the retina. Genotypes w; gmr-GAL4 UAS-SCA3trQ61 in trans to (c) w (d) mbl^B2-E1. e-h, Retinal pseudopupils of 1d flies. (e) Flies expressing elav-GAL4 alone or (f) with MblA have a normal pattern of 7 photoreceptors (PR)/ommatidium. (g) Flies expressing SCA3trQ78 show mild loss of retinal integrity (arrows), with 5.8±0.4 SD PR/ommatidium (n=200 ommatidia). Genotype elav-GAL4/+; UAS-SCA3trQ78/+. (h) Co-expression of MblA with SCA3trQ78 enhances PR loss to 3.0±0.5 SD (n=200 ommatidia; significant difference from g, P< 0.0001 (two-tailed unpaired student t-test)). Genotype elav-GAL4/+;UAS-mblA/+; UAS-SCA3trQ78/+. (i) Neuronal toxicity by lifespan analysis. Upregulation of MblA further shortened the lifespan of SCA3trQ78 flies, whereas downregulation of mbl (flies heterozygous for allele mbl^E27) extended lifespan (P<0.001, SCA3trQ78 and SCA3trQ78/mbl^E27, SCA3trQ78 and SCA3trQ78/mblA, log-rank analysis). Mean ± SD; n=170-220 flies for each genotype. Genotypes for SCA3trQ78 and SCA3trQ78/mblA same as g and h, SCA3trQ78/mbl^E27 is elav-GAL4/+;mbl^E27/+;UAS-SCA3trQ78/+.

a, Constructs with untranslated CAG repeats within the 3' UTR of a transgene encoding a control protein DsRed. b-e, Flies expressing untranslated CAG repeats show neuronal degeneration. (b, c) Flies expressing CAG₁₀₀ in the eye with gmr-GAL4 showed loss of retinal integrity (arrows). Paraffin sections of 1d flies, genotypes: (b) gmr-GAL4/ UAS-CAG₁₀₀ (4x), (c) gmr-GAL4/ UAS-CAG₀. (d, e) Flies expressing CAG₁₀₀ showed progressive brain degeneration with vacuoles in the brain (arrows). Paraffin sections of 35d flies, genotypes: (c) elav-GAL4/UAS-CAG₁₀₀ (5x) and (e) elav-GAL4/UAS-CAG₀. Bar in b, 5 μm for b, c. Bar in d, 10 μm for d, e. f-g, Expression of untranslated CAG repeats induces length-dependent, progressive neural dysfunction. (f) Neurotoxicity by lifespan analysis. Flies expressing untranslated CAG repeats show length-dependent reduced lifespan. Differences in lifespan of flies expressing CAG₀, CAG₁₀₀, and CAG₂₅₀, and CAG₂₅₀/mblA are significantly different at P<0.001 (log-rank analysis). Mean ± SD, n= 250, 260, 300, 100 flies, respectively. elav-GAL4 in trans to (CAG₀) UAS-CAG₀, (CAG₁₀₀) UAS-CAG₁₀₀ (five UAS-trangenes were combined to match the RNA expression level to that of SCA3trQ78(s)_CAG), (CAG₂₅₀) UAS-CAG₂₅₀. (g) Climbing ability with age. Flies expressing CAG₀ showed normal climbing defects with age (* P<0.05). Flies expressing CAG₁₀₀ had mild climbing defects at 20d, which strikingly degenerated by 35d (*** P<0.001 compared to 1d; ** P<0.01 compared to 35d CAG₀). CAG₂₅₀ flies had moderate climbing defects at 1d, which were strikingly worse by 20d (* P<0.05). Mean ± SD, 100-200 flies per time point for each genotype in each experiment, two-way ANOVA. Genotypes as in (f).