Polyglutamine genes interact to modulate the severity and progression of neurodegeneration in Drosophila

Abstract

The expansion of polyglutamine tracts in a variety of proteins causes devastating, dominantly inherited neurodegenerative diseases, including six forms of spinal cerebellar ataxia (SCA). Although a polyglutamine expansion encoded in a single allele of each of the responsible genes is sufficient for the onset of each disease, clinical observations suggest that interactions between these genes may affect disease progression. In a screen for modifiers of neurodegeneration due to SCA3 in Drosophila, we isolated atx2, the fly ortholog of the human gene that causes a related ataxia, SCA2. We show that the normal activity of Ataxin-2 (Atx2) is critical for SCA3 degeneration and that Atx2 activity hastens the onset of nuclear inclusions associated with SCA3. These activities depend on a conserved protein interaction domain of Atx2, the PAM2 motif, which mediates binding of cytoplasmic poly(A)-binding protein (PABP). We show here that PABP also influences SCA3-associated neurodegeneration. These studies indicate that the toxicity of one polyglutamine disease protein can be dramatically modulated by the normal activity of another. We propose that functional links between these genes are critical to disease severity and progression, such that therapeutics for one disease may be applicable to others.

Figure 1. Atx2 Enhances Atx3-Dependent Neurodegeneration

External eye (top) and internal retina sections (bottom) of 1-d-old males. Internal degeneration is reflected in the thickness of the retina, indicated by the yellow double-headed arrows. (A) Control fly with driver only; eye is normal with a highly regular structure and normal pigmentation. Genotype: Gmr-Gal4/+. (B) Flies with a weakly expressing insertion of SCA3trQ78 have a normal external eye, although internally the retina is disorganized (compare to panel [A]). Genotype: Gmr-Gal4/+; UAS-SCA3trQ78(w)/+. (C) Flies expressing Atx2 have a mildly rough external eye surface, but no obvious degeneration externally or internally. Genotype: Gmr-Gal4/UAS-Atx2. (D) Expression of Atx2 with SCA3trQ78 results in severe degeneration, with loss of pigmentation externally and collapse of the retina internally. Genotype: Gmr-Gal4/UAS-Atx2; UAS-SCA3trQ78(w)/+.

Figure 2. Adult-Onset Degeneration due to Pathogenic Atx3 Is Synergistically Enhanced by Atx2

(A–H) Pseudopupil preparations: each panel shows a field of seven unit eyes (ommatidia); each ommatidium has seven visible photoreceptors (PR) with light-gathering organelles (rhabdomeres) in a characteristic trapezoidal pattern (e.g., green arrowhead in [A]). Examples of degenerate ommatidia are highlighted in (E) by a yellow arrowhead (4 PR) and in (G) by a white arrowhead (1 PR). The mean number of PR ± SEM per ommatidium (n = 10 flies) is indicated at the bottom right of each panel. (A–D) shows 6-d-old adults; (E–H) 18-d-old adults. (A and E) Expression of SCA3trQ78 causes adult-onset PR loss over 18 d (from a mean of 7.0 at 1 d to 3.7). Genotype UAS-SCA3trQ78(s)/+; Rh1-Gal4/+. (B and F) Mild degeneration occurs with up-regulation of Atx2, to a mean of 5.9 PR at 18 d. Genotype UAS-Atx2/+; Rh1-Gal4/+. (C and G) Coexpression of SCA3trQ78 with Atx2 causes severe PR loss (to a mean of 4.7 as early as 6 d and to 1.3 at 18 d). Genotype UAS-Atx2/UAS-SCA3trQ78(s); Rh1-Gal4/+. (D and H) Strong expression of Atx2 alone causes severe degeneration (to a mean of 1.5 PR at 18 d). Genotype: UAS-Atx2(s)/+; Rh1-Gal4/+. (I) Distribution of ommatidia in 6-d flies. The mean numbers of PR are indicated schematically by the positions of arrowheads at top relative to the x-axis (* p < 0.001 vs. SCA3trQ78 alone, p < 0.05 vs. Atx2 alone). Genotypes as in (A–C), and mean PR counts are listed in lower right corners of (A–C). (J) The full-length pathogenic Atx3 protein also interacts synergistically with Atx2: distribution of PR at 18–23 d; see text for mean PR counts, also indicated by triangles at the top relative to the x-axis (* p < 0.001 vs. SCA3Q84 and p < 0.05 vs. Atx2). Genotypes: (green) UAS-SCA3Q84/+; Rh1-Gal4/+, (grey) UAS-Atx2/+; Rh1-Gal4/+, and (red) UAS-Atx2/UAS-SCA3Q84; Rh1-Gal4/+.

Figure 3. Atx2 Function Is Required for Atx3-Induced Neurodegeneration

(A) Heterozygous loss of Atx2 suppresses SCA3trQ78 degeneration in the adult-onset model (*** p = 0.0007). Distribution of ommatidia in 12-d flies with either normal Atx2 function or with heterozygous loss of Atx2 activity. Grey bars: UAS-SCA3trQ78(s)/+; Rh1-Gal4/+. Green bars: UAS-SCA3trQ78(s)/+; Rh1-Gal4/atx2^X1. (B) Clones of sensory neurons in the anterior wing margin labeled with GFP by the MARCM technique [32]. Top, membrane-bound GFP marks neurons expressing UAS-SCA3trQ78(s); bottom, GFP signal merged with bright-field image. (C) Loss of Atx2 function in neuronal clones homozygous for atx2⁻ protects neurons from SCA3trQ78 toxicity. The retention of GFP⁺ neurons present in newly born adults is plotted over time (mean ± SEM, n = 10 flies per genotype). Whereas SCA3trQ78 expression alone results in rapid loss of neurons (red), expression of SCA3trQ78 together with homozygous loss of atx2 results in significantly delayed neuron loss (green). Loss of atx2 alone has a minimal effect (black). (D and E) Images of two wings, at two time points each (<8 h and 3 d). Arrowheads flank GFP⁺ neurons in the anterior margin in young animals. (D) Wing from a fly expressing SCA3trQ78 in GFP⁺ neurons with normal Atx2 activity. Neurons visible when the fly was less than 8 h old (top) are no longer detectable at 3 d (bottom). (E) Wing from a fly expressing SCA3trQ78 in GFP⁺ neurons that also lack all Atx2 activity (homozygous for a null allele, atx2 ^X1/atx2 ^X1); the GFP signal is maintained over 3 d. See Materials and Methods for genotypes for (B–E). Proximal is to the left in (B, D, and E).

Figure 4. Atx2 Alters the Time Course of and Colocalizes with Pathogenic Atx3 Inclusions

Retinal cryosections at (A, E, and G) 24 h and (B–D, F, and H) 4 d. (A–D) Flies expressing SCA3trQ78 only (genotype UAS-SCA3trQ78(s)/+; Rh1-Gal4/+). (E and F) Flies coexpressing SCA3trQ78 and Atx2 (UAS-SCA3trQ78(s)/UAS-Atx2; Rh1-Gal4/+). (G and H) Flies coexpressing SCA3trQ78 and Atx2ΔP (UAS-SCA3trQ78(s)/+; Rh1-Gal4/UAS-Atx2ΔP). (A, B, and E–H) Pathogenic Atx3 protein is tagged with the HA epitope and visualized with anti-HA (magenta). Up-regulation of Atx2, but not of Atx2ΔP lacking the PAM2 motif, results in the early onset of inclusions at 24 h. (C) Same section as in (B), stained for endogenous Atx2 (green). (D) Merged images show colocalization of Atx2 in the inclusions. See Figure S3 for larger images, including DAPI staining, that show nuclear localization and orientation within the retina. (I) Coexpression of SCA3trQ78 with Atx2 does not significantly change SCA3 mRNA levels (p = 0.21). Quantitative real-time PCR comparing mean relative levels ± SEM of SCA3trQ78 mRNA from heads of 1–2-d flies, from two separate cDNA preparations for each group, normalized to coexpression with control transgene UAS-eGFP. Genotypes: UAS-SCA3trQ78(s)/UAS-eGFP; Rh1-Gal4/+, and UAS-SCA3trQ78(s)/UAS-Atx2; Rh1-Gal4/+. (J) Immunoblot of the Atx3 protein (anti-HA, top) and loading control β-actin (bottom). Lanes 1–3, head extracts from 24-h flies; lanes 4–6, heads extracts from 4-d flies. Lanes 1 and 4, coexpression of SCA3trQ78 and control transgene (UAS-eGFP); lanes 2 and 5, coexpression of SCA3trQ78 and Atx2; lanes 3 and 6, coexpression of SCA3trQ78 and Atx2ΔP (genotypes in [A], [E], [G]). At 24 h, only flies coexpressing Atx2, but not Atx2ΔP, have high-molecular weight complexes containing Atx3 visible in the stacking gel portion (s) of the blot.

Figure 5. The PAM2 Motif of Atx2 Is Required for Enhancement of Atx3 Degeneration and for Atx2 Toxicity

(A and B) External eye and internal retinal sections. Retinal thickness is indicated by double-headed yellow arrows. (A) Expression of Atx2 lacking the PAM2 motif (Atx2ΔP) causes a disrupted eye with (top) external roughness and dorsal discoloration and (below) disorganized internal structure. Genotype: Gmr-Gal4/+; UAS-Atx2ΔP/+. (B) Co-expression of Atx2ΔP with SCA3trQ78 does not enhance SCA3trQ78 degeneration; the eye looks identical to that of flies expressing Atx2ΔP only. Genotype: Gmr-Gal4/+; UAS-SCA3trQ78(w)/UAS-Atx2ΔP. (C and D) Pseudopupil preparations to highlight internal retinal degeneration. (C) Flies expressing Atx2ΔP maintain a normal retina over time, with a mean of seven PR (18 d shown, genotype: Rh1-Gal4/UAS-Atx2ΔP). (D) Coexpression of Atx2ΔP with SCA3trQ78 has minimal effect on SCA3trQ78 degeneration, with a mean of 6.7 PR (6 d, UAS-SCA3trQ78(s)/+; Rh1-Gal4/UAS-Atx2ΔP); normally SCA3trQ78 expression by itself results in a mean of 6.9 PR at 6 d (see Figure 2A).

Figure 6. The Atx2 Binding Partner PABP Modulates Neurodegeneration

Distributions of ommatidia from pseudopupil analysis; mean PR counts indicated by the positions of triangles above the graphs (see text for precise values). (A) Flies expressing pathogenic Atx3 at 18d. Top: red bars show distribution of ommatidia in flies heterozygous for a deletion removing pabp (Df(pabp)) compared to flies heterozygous for a P element used to synthesize the deletion (grey; P[SZ]). The loss of 50% of PABP activity (red) results in significant enhancement of degeneration (*** p < 10⁻⁴). Genotypes: UAS-SCA3trQ78(s)/P[5-SZ-3325]; Rh1-Gal4/+ or UAS-SCA3trQ78(s)/Df(2R)ED3610; Rh1-Gal4/+. Bottom: up-regulation of PABP (green) suppresses degeneration induced by SCA3trQ78 (*** p < 10⁻⁴). Genotypes: UAS-SCA3trQ78(s)/+; Rh1-Gal4/+ or UAS-SCA3trQ78(s)/+; Rh1-Gal4/UAS-PABP. (B) Strong expression of Atx2 in 6-d flies. Top, degeneration by Atx2 (grey) is enhanced by reduced levels of PABP (red; ** p = 0.003). Bottom, up-regulation of PABP (green) suppresses Atx2-mediated degeneration (*** p < 10⁻⁴). Genotypes as in (A), except with UAS-Atx2(s) instead of UAS-SCA3trQ78.