Ataxin-2 is a dose-sensitive modifier of TDP-43 toxicity in Drosophila. a) Expression of TDP-43 caused a dose-dependent disruption of retinal structure. Genotypes: gmr-GAL4(YH3) in trans to UAS-YFP (control), UAS-TDP-43(M) or UAS-TDP-43-YFP(S). (M) and (S) are moderate and strong TDP-43 expression, respectively (see Methods and Fig. S2). b) TDP-43 caused motility deficits when expressed in motor neurons. TDP-43.Q331K causes a more severe loss of motility than the WT protein at the same level of expression (see Fig. S2). Genotypes: D42-GAL4 in trans to +, UAS-TDP-43 or UAS-TDP-43.Q331K. c) dAtx2 modulates TDP-43 toxicity. Flies expressing TDP-43 or dAtx2 alone (dAtx2EP) have a mild effect on retinal structure. TDP-43 toxicity is more severe upon upregulation of dAtx2 (dAtx2EP3145). TDP-43 toxicity is markedly mitigated upon reduction of dAtx2 (flies in trans to null allele dAtx2X1). Genotypes: control: gmr-GAL4/UAS-YFP. TDP-43: UAS-TDP-43(M)/+, gmr-GAL4(YH3)/+. dAtx2EP: gmr-GAL4(YH3)/dAtx2EP3145. TDP43+dAtx2EP: UAS-TDP-43(M)/+, gmr-GAL4(YH3)/dAtx2EP3145. TDP-43;dAtx2X1/+: UAS-TDP-43(M)/+, gmr-GAL4(YH3)/dAtx2X1. dAtx2X1/+ has wild type retinal structure. d) dAtx2 modulates reduced lifespan conferred by TDP-43. Expression of TDP-43 in the nervous system reduces lifespan (black, compared to normal in blue). Upregulation of dAtx2 causes more rapid death (red, compared to TDP-43 in black). Upregulation of dAtx2 on its own has no effect (purple). Reduction of dAtx2 significantly extends lifespan (green, compared to TDP-43 in black). Heterozygous loss of dAtx2 on its own has no effect (not shown). quantitative-RT-PCR showed that TDP-43 expression has no effect on levels of dAtx2 transcript in dAtx2EP flies. Genotypes. elav/+: elavC155/Y. elav/dAtx2EP: elavC155/Y; +/dAtx2EP3145. elav, TDP-43/+: elavC155/Y; UAS-TDP-43(M)/+. elav, TDP-43/dAtx2EP: elavC155/Y; UAS-TDP-43(M)/+; +/dAtx2EP3145. elav, TDP-43; dAtx2X1/+: elavC155/Y; UAS-TDP-43(M)/+; +/dAtx2X1. e) TDP-43 immunoblot upon dAtx2 modulation. Reduction of dAtx2 has no effect on TDP-43 levels, whereas upregulation of dAtx2 enhances TDP-43 protein levels. dAtx2 has no effect on the transgene expression system and 2x TDP-43 does not produce the same phenotype as TDP-43+dAtx2EP (see Figs. S2 and S3). Genotypes: —, control lane. TDP-43 lanes, —: UAS-TDP-43(M)/+, gmr-GAL4(YH3)/+. dAtx2X1:UAS-TDP-43(M)/+, gmr-GAL4(YH3)/dAtx2X1. dAtx2EP: UAS-TDP-43(M)/+, gmr-GAL4(YH3)/dAtx2EP3145. Bottom, quantitation of immunoblots from independent experiments; normalized to tubulin.