Volume 18, Issue 1, Pages 37-48 (April 2005) Ataxin-3 Suppresses Polyglutamine Neurodegeneration in Drosophila by a Ubiquitin- Associated Mechanism  John M. Warrick, Lance M. Morabito, Julide Bilen, Beth Gordesky-Gold, Lynn Z. Faust, Henry L. Paulson, Nancy M. Bonini  Molecular Cell  Volume 18, Issue 1, Pages 37-48 (April 2005) DOI: 10.1016/j.molcel.2005.02.030 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Pathogenic Human Ataxin-3 Induces Progressive Neurodegeneration in Drosophila That Is Mitigated upon Coexpression of the Wild-type Protein (A–D) Top row, external eye; middle row, horizontal epon sections of the retina. (A) 7-day-old control flies bearing only the driver construct gmr-GAL4; (B) 7-day-old flies expressing the normal protein SCA3-Q27; (C) 1-day-old SCA3-Q84 flies; (D) 7-day-old SCA3-Q84 flies. (B) Expression of SCA3-Q27 has no effect on external or internal eye morphology (compare to control in [A]). Flies expressing SCA3-Q84 are born with slightly abnormal retinal morphology (C) that undergoes dramatic progressive retinal degeneration over 7 days (D). Flies of genotype w; gmr-GAL4/+, w; gmr-GAL4/UAS-SCA3-Q27, and w; gmr-GAL4/UAS-SCA3-Q84. (E and F) Retinal structure of flies coexpressing normal ataxin-3 with pathogenic ataxin-3 is restored toward normal, as shown in tangential epon sections. Flies expressing only SCA3-Q78 (E) or coexpressing normal ataxin-3 (SCA3-Q27) with SCA3-Q78 (F). Flies of genotype w; gmr-GAL4/UAS-SCA3-Q78 alone (E) or with UAS-SCA3-Q27 (F). (G) Impaired phototaxis caused by pathogenic ataxin-3 is mitigated upon coexpression of wild-type ataxin-3; 50% is a random distribution of flies between light and dark chambers. Flies bear gmr-GAL4 in trans to indicated ataxin-3 transgenes. Flies expressing normal ataxin-3 (AT3-Q27) have a robust visual response that does not differ from control flies bearing the transgenes alone (not shown). Flies expressing pathogenic ataxin-3 (AT3-Q84) by gmr-GAL4 are defective in phototaxis at 1 day and functionally blind by 4 days. Coexpression of normal ataxin-3 with pathogenic ataxin-3 restores normal vision. Data are presented as mean ± SD. Molecular Cell 2005 18, 37-48DOI: (10.1016/j.molcel.2005.02.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Normal Ataxin-3 Suppresses Retinal Neurodegeneration Induced by Pathogenic Ataxin-3 (A–C) External and (D–F) internal retinal structure. (A) Control eye of w; gmr-GAL4/+. (B) SCA3tr-Q78 induces severe eye degeneration. Fly of genotype w; gmr-GAL4/UAS-SCA3tr-Q78(S)/+. (C) Coexpression of normal ataxin-3 suppresses degeneration induced by SCA3tr-Q78. Fly of genotype w; gmr-GAL4UAS-SCA3tr-Q78(S)/UAS-SCA3-Q27. (D) Normal retinal structure of a fly expressing normal ataxin-3. (E) Degenerate retinal structure of fly expressing SCA3tr-Q78 weakly. (F) Retinal structure of fly coexpressing normal ataxin-3 with SCA3tr-Q78 is restored toward normal. Represented are flies of genotype w; gmr-GAL4/UAS-SCA3-Q27 (D), w; gmr-GAL4/+;UAS-SCA3tr-Q78(W) in trans to w (E) or UAS-SCA3-Q27 (F). Molecular Cell 2005 18, 37-48DOI: (10.1016/j.molcel.2005.02.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Ataxin-3 Delays NI Formation and Reduces Accumulation of the Pathogenic Protein (A and B) Confocal images of immunostained eye discs. Large arrow indicates the furrow and start of transgene expression; small arrow indicates the onset of NI. (A) Larva coexpressing the SCA3tr-Q78 (top, red, anti-HA) and SCA3-Q27 proteins (middle, green, anti-myc) and an overlay of the two (bottom). The wild-type ataxin-3 protein (green) delays the onset of NI formation and is recruited into NI formed by pathogenic SCA3tr-Q78 (red). (B) Expression of SCA3tr-Q78 alone for comparison. Larvae of genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S)/UAS-SCA3-Q27; and w; gmr-GAL4/UAS-SCA3tr-Q78(S). (C–E′) Immunostained retinal cryosections of flies expressing normal ataxin-3 alone (C), pathogenic full-length ataxin-3 alone (D), or coexpressing both proteins (E, E′). (C) SCA3-Q27 (anti-myc) shows diffuse staining. (E) SCA3-Q78 (anti-HA) accumulates in inclusions. (E and E′) Coexpression of SCA3-Q27 (E′, green, anti-myc) and SCA3-Q78 (E, red, anti-HA) reveals that ataxin-3, which is recruited to the inclusions, leads to a reduction in SCA3-Q78 protein levels in NI (compare to [D]). The reduction in immunostaining intensity is ∼2-fold averaged over three independent experiments of 3–5 flies/experiment. The remaining protein still forms NI based on immunostaining and SDS insolubility of the protein by Western blot. Flies of genotype w; gmr-GAL4/UAS-SCA3-Q27, w; gmr-GAL4 UAS-SCA3-Q78 in trans to w or UAS-SCA3-Q27. (F–I) Flies expressing SCA3tr-Q78 with SCA3tr-Q27 still have a severely degenerate eye (H), whereas those expressing SCA3tr-Q78 with the N-terminal domain of ataxin-3 in SCA3-delta (see Figure 7) show a suppressed external eye (I). Compare to disease eye in (F) and ataxin-3 suppressed eye in (G). Flies of genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S) in trans to w, UAS-SCA3-Q27, UAS-SCA3tr-Q27 or UAS-SCA3-delta. Molecular Cell 2005 18, 37-48DOI: (10.1016/j.molcel.2005.02.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Suppression of Neurodegeneration by Ataxin-3 Requires Proteasome Function and the Ubiquitin-Associated Activities of the Protein External eyes and immunostained cryosections of retinal tissue of flies expressing various combinations of pathogenic and normal ataxin-3. The white arrow in cryosections indicates the depth of the retina (as revealed by Hoechst staining and epon sectioning), which reflects the degree of retinal degeneration. (A) A normal eye and (B) eye section immunostained for SCA3-Q27-UIM*, which is normally diffuse and highlights the normal depth of the retina in a nondegenerate situation. (C and D) SCA3tr-Q78 causes severe retinal degeneration that is associated with severe collapse of the eye (white arrow) and inclusion formation by the pathogenic protein (D, red, anti-HA). Genotype w; gmr-GAL4/ UAS-SCA3tr-Q78(S). (E and F) Coexpression of SCA3-Q27 with SCA3tr-Q78 shows rescue of the (E) external and (F) internal eye, with the SCA3-Q27 protein now localized to the NI formed by SCA3tr-Q78 ([F], red, anti-HA, SCA3tr-Q78; [F′], green, anti-myc, SCA3-Q27). Normally, SCA3-Q27 is diffusely expressed within the eye (see Figure 3C). Genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S)/UAS-SCA3-Q27. (G and H) Limiting proteasome function using a UAS-DTS5 transgenic line abrogates the ability of ataxin-3 to suppress the toxicity of the pathogenic protein. DTS5 is a mutation in a subunit of the 20S proteasome (Saville and Belote, 1993). (G) The external eye is now degenerate, and (H) internal retinal structure is poor ([H], red, anti-HA, SCA3tr-Q78; [H′], green, anti-myc, SCA3-Q27). The DTS lines and mutations had no effect on polyglutamine toxicity in the absence of the SCA3-Q27 protein. Genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S) UAS-SCA3-Q27/ UAS-DTS5. (I and J) Mutation of the ataxin-3 UIM motifs (S236A, S256A), which eliminates ubiquitin binding, partially compromises the ability of ataxin-3 to suppress SCA3tr-Q78 toxicity. The external eye is now degenerate, and the internal retinal structure is poor. However, the SCA3-Q27-UIM* protein is still recruited to NI formed by the SCA3tr-Q78 protein ([J], red, anti-HA, SCA3tr-Q78 protein; [J′], green, anti-myc, SCA3-Q27-UIM* protein). The UIM* transgenic lines used express at levels comparable to that of normal ataxin-3 (not shown). Genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S)/UAS-SCA3-Q27-UIM*. (K and L) A point mutation of the ubiquitin protease domain to eliminate protease activity (C14A) eliminates the ability of ataxin-3 to suppress SCA3tr-Q78 toxicity. (K) The external eye is now degenerate, and (L) internal retinal structure is poor ([L], red, anti-HA, SCA3tr-Q78 protein; [L′], green, anti-myc, SCA3-Q27-C14A protein). The SCA3-Q27-C14A protein is still recruited to NI. The C14A transgenic lines used express at levels comparable to that of normal ataxin-3 (not shown). Genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S)/UAS-SCA3-Q27-C14A. Molecular Cell 2005 18, 37-48DOI: (10.1016/j.molcel.2005.02.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Activities of Pathogenic Ataxin-3 (A–E) External and internal retinal structure of flies expressing SCA3tr-Q78 alone, with SCA3-Q84, or with a second dose of SCA3tr-Q78. (A) SCA3tr-Q78 causes severe degeneration. Genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S)/+. (B) Coexpression of SCA3-Q84 with SCA3tr-Q78 shows partial suppression compared to SCA3tr-Q78 alone. Genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S)/UAS-SCA3-Q84. (C) Coexpression of SCA3tr-Q78 with itself causes a very severe phenotype. Genotype w; gmr-GAL4 UAS-SCA3tr-Q78(S)/UAS-SCA3tr-Q78(S). (D–F) Tangential epon retinal sections. (D) SCA3tr-Q78 severely disrupts internal retinal structure such that only cell debris is visible, with no photoreceptor specializations apparent. Genotype w; gmr-GAL4/+; UAS-SCA3tr-Q78(W)/+. (E) Coexpression of SCA3-Q84 with SCA3tr-Q78 results in an improved retinal structure with ommatidial units comprised of photoreceptor rhabdomere specializations now visible. Genotype w; gmr-GAL4/+; UAS-SCA3tr-Q78(W)/UAS-SCA3-Q84.(F) Mutation of the UIM domains partially compromises the ability of the pathogenic protein SCA3-Q80-UIM* to suppress SCA3tr-Q78 (compare to [E]). Genotype w; gmr-GAL4/+; UAS-SCA3tr-Q78(W)/UAS-SCA3-Q80-UIM*. (G and H) Cryosections immunostained for SCA3-Q78 protein with anti-HA (G) in trans to w and (H) in trans to pathogenic ataxin-3 protein (differentially tagged with myc). Upon coexpression of two copies of pathogenic ataxin-3, the level of the one pathogenic protein is diminished ∼2-fold (quantitated with ImageJ), further demonstrating that the pathogenic protein retains suppressor activity. Flies of genotype gmr-GAL4 UAS-SCA3-Q78 in trans to w or UAS-SCA3-Q84. (I–K) Eyes of flies expressing SCA3-Q84 (I), the pathogenic protein with UIMs mutated (J), and the pathogenic protein with a point mutation in the ubiquitin protease domain (K). The protease domain mutation generates a protein with dramatically enhanced toxicity, such that the toxicity of the full-length pathogenic protein now resembles that of strong expression of the N-terminally truncated protein lacking the N-terminal domain entirely (compare to [A]). All flies in trans to gmr-GAL4. Molecular Cell 2005 18, 37-48DOI: (10.1016/j.molcel.2005.02.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Ataxin-3 Suppresses Neurodegeneration Induced by Other PolyQ Proteins (A and B) Retinal structure of 10-day-old flies expressing only the Htt-Q120 protein is severely degenerate (A), whereas coexpression of ataxin-3 maintains normal eye structure (B). Flies of genotype elav-GAL4; gmr-Htt-Q120 in trans to w (A) or UAS-SCA3-Q27 (B). (C and D) Retinal structure of flies expressing pathogenic ataxin-1 protein is severely disrupted (C) but shows improved morphology upon coexpression of normal ataxin-3 (D). Flies of genotype UAS-SCA1-Q82; gmr-GAL4 in trans to w (C) or UAS-SCA3-Q27 (D). (E–H) Flies expressing a dominant-negative form of Hsp70 (Hsp70.K71E) have a severely disrupted eye due to compromised Hsp70 activity and induction of massive protein misfolding (E) (Bonini, 2002). Coexpression of ataxin-3 has little to no effect on this phenotype (F). In contrast, the Hsp70.K71E phenotype is mitigated by coexpression of Hsp70 (the Hsp70 transgene used here suppresses polyQ neurodegeneration the same degree as SCA3-Q27) (G) or poly-ubiquitin (H). Flies of genotype w; gmr-GAL4 UAS-Hsp70.K71E in trans to w, UAS-SCA3-Q27, UAS-Hsp70, or EP1384. (I and J) Flies expressing tau have a disrupted eye (Wittmann et al., 2001) that is not modified upon coexpression of SCA3-Q27 (J). Flies of genotype w; gmr-GAL4; UAS-tau in trans to w or UAS-SCA3-Q27. Molecular Cell 2005 18, 37-48DOI: (10.1016/j.molcel.2005.02.030) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Schematic of SCA3 Constructs Trangene constructs used included full-length human ataxin-3 with a normal length polyQ repeat of Q27 and pathogenic forms with long polyQ repeats. Pathogenic forms were tagged with myc or HA epitopes. The Josephin domain is the ubiquitin protease domain. The constructs with mutated UIMs and a mutated ubiquitin protease domain are full-length ataxin-3 with point mutations noted. The truncated constructs bear the polyQ repeat and C terminus but lack most of the N-terminal region (Warrick et al., 1998). SCA3-delta bears only the N-terminal region. Molecular Cell 2005 18, 37-48DOI: (10.1016/j.molcel.2005.02.030) Copyright © 2005 Elsevier Inc. Terms and Conditions