RNA toxicity is a component of ataxin-3 degeneration in Drosophila

Abstract

Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of a CAG repeat encoding glutamine within the coding region of the respective genes. The molecular and cellular pathways underlying polyQ-induced neurodegeneration are the focus of much research, and it is widely considered that toxic activities of the protein, resulting from the abnormally long polyQ tract, cause pathogenesis. Here we provide evidence for a pathogenic role of the CAG repeat RNA in polyQ toxicity using Drosophila. In a Drosophila screen for modifiers of polyQ degeneration induced by the spinocerebellar ataxia type 3 (SCA3) protein ataxin-3, we isolated an upregulation allele of muscleblind (mbl), a gene implicated in the RNA toxicity of CUG expansion diseases. Further analysis indicated that there may be a toxic role of the RNA in polyQ-induced degeneration. We tested the role of the RNA by altering the CAG repeat sequence to an interrupted CAACAG repeat within the polyQ-encoding region; this dramatically mitigated toxicity. In addition, expression of an untranslated CAG repeat of pathogenic length conferred neuronal degeneration. These studies reveal a role for the RNA in polyQ toxicity, highlighting common components in RNA-based and polyQ-protein-based trinucleotide repeat expansion diseases.

Figure 1. Upregulation of mbl enhances ataxin-3 toxicity

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/+.

Figure 2. Interruptions of the CAG repeat mitigate SCA3 protein pathogenesis

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).

Figure 3. Untranslated CAG repeats induce progressive neural dysfunction

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).