Expression of zinc-deficient human superoxide dismutase in Drosophila neurons produces a locomotor defect linked to mitochondrial dysfunction

Abstract

More than 130 different mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been associated with amyotrophic lateral sclerosis but the mechanism of this toxicity remains controversial. To gain insight into the importance of the zinc site in the pathogenesis of SOD1 in vivo, we generated a Drosophila model with transgenic expression of a zinc-deficient human SOD1. Expression of zinc-deficient SOD1 in Drosophila resulted in a progressive movement defect with associated mitochondrial cristae vacuolization and reductions in adenosine triphosphate (ATP) levels. Furthermore, these flies are sensitized to mitochondrial toxins, paraquat, and zinc. Importantly, we show that the zinc-deficient SOD1-induced motor defect can be ameliorated by supplementing the endogenous fly respiratory chain machinery with the single-subunit NADH-ubiquinone oxidoreductase from yeast (NADH is nicotinamide adenine dinucleotide, reduced form.). These results demonstrate that zinc-deficient SOD1 is neurotoxic in vivo and suggest that mitochondrial dysfunction plays a critical role in this toxicity. The robust behavioral, pathological, and biochemical phenotypes conferred by zinc-deficient SOD1 in Drosophila have general implications for the role of the zinc ion in familial and sporadic amyotrophic lateral sclerosis.

Keywords: Amyotrophic lateral sclerosis (ALS); Fly; Mitochondria; Ndi1; Paraquat; Respiration; Superoxide dismutase (SOD1).

Fig. 1

Ubiquitous expression of zinc-deficient superoxide dismutase (SOD1, CuSOD, or D83S SOD1) in Drosophila produces locomotor defects without neurodegeneration or shortened life span. (A) Western blot analysis of SOD protein level in adult fly extracts confirmed daGAL4-mediated expression of the CuSOD transgene (third lane). The first lane indicates the knockdown of SOD protein on daGAL4-mediated activation of a UAS-SOD1-RNAi transgene. (B) Expression of CuSOD leads to formation of heterodimers between wild type SOD and CuSOD. SOD activity was measured using the in-gel nitroblue tetrazolium assay. (C) Expression of wild type human SOD1 (hSOD1) or CuSOD has a minor negative effect on the adult life span. (D) and (E) Expression of CuSOD does not promote neurodegeneration in Drosophila eye or brain (60-day-old flies; scale bar, 50 μm). (F) Expression of CuSOD significantly compromises locomotor activity (p=0.002) in 30-day-old flies. Ubiquitous expression of wild type hSOD1, however, had no adverse effect on physical activity in young (p=0.067) or old (p = 0.093) flies. (G) Representative activity pattern for 30-day-old flies during a 24-hour period. (H) Expression of CuSOD lowers ATP levels in heads (p=0.029) but not the rest of the body (p=0.21) of 10-day-old flies. Genotypes were as follows: control (da-Gal4/+), SOD1 RNAi (da-Gal4/UAS-SOD1-RNAi); and D83S SOD1 or CuSOD (da-Gal4/UAS-D83S), hSOD1 (da-Gal4/UAS-hSOD1). For (F) and (H), the significance of the difference (Note that RNAi is an abbrevation for double stranded interference RNA.) between means was analyzed using 1-way analysis of variance. Abbreviations: apoSOD, endogenous Drosophila SOD1 monomer; MnSOD, manganese superoxide dismutase (SOD2).

Fig. 2

Ubiquitous expression of zinc-deficient SOD (or CuSOD) in Drosophila deteriorates mitochondrial structure. Electron micrographs of flight muscle of 10-day-old flies (A–F) and 30-day-old flies (G–L). (A), (D), (G), and (J) control flies show normal mitochondrial ultrastructure whereas expression of CuSOD (C), (F), (I), (L) but not wild-type human superoxide dismutase (hSOD1) (B), (E), (H), (K) produces a localized rearrangement of the cristae within individual mitochondria (scale bar, 1 μm). CuSOD phenotypes were more severe on day 30 compared to day 10. Genotypes were as follows: Control (da-Gal4/+), hSOD1 (da-Gal4/UAS-hSOD1), and CuSOD (da-Gal4/UAS-D83S).

Fig. 3

Expression of zinc-deficient superoxide dismutase (SOD1, CuSOD, or D83S) in Drosophila neurons and glial cells produces locomotor defects without major changes in life span. (A–D) Expression of zinc-deficient SOD (CuSOD) or human SOD1 (hSOD1) in Drosophila neurons, glial cells, or muscles has a minor effect on adult life span. Expression of CuSOD in (E) neurons (p=0.0003), (F) motor neurons (p=0.014) and (G) glial cells (p=0.0001) but not (H) muscles (p=0.27) produces locomotor defects in 30-day-old flies as compared to wild-type hSOD1 flies. Genotypes were as follows: (A) and (E) Control: Elav-GAL4/+; hSOD1: Elav-GAL4/UAS-hSOD1; CuSOD: Elav-GAL4/UAS-D83S. (B) and (F) Control: D42-GAL4/+; hSOD1: D42-GAL4/UAS-hSOD1; CuSOD: D42-GAL4/UAS-D83S. (C) and (G) Control: Repo-GAL4/+; hSOD1: Repo-GAL4/UAS-hSOD1; CuSOD: Repo-GAL4/UAS-D83S. (D) and (H) Control: MHC-GAL4/+; hSOD1: MHC-GAL4/UAS-hSOD1; CuSOD: MHC-GAL4/UAS-D83S. For (E), (F), (G), and (H), the significance of the difference between means was analyzed using one-way analysis of variance.

Fig. 4

Neuronal expression of zinc-deficient SOD (CuSOD) sensitizes flies to zinc and paraquat toxicity. (A) Neuronal expression of zinc-deficient superoxide dismutase (SOD1 or CuSOD) leads to a decrease in ATP levels of fly heads (p=0.005). (B) and (C) Neuronal expression of CuSOD sensitizes flies to paraquat toxicity, whereas expression of wild-type human SOD1 (hSOD1) enhances resistance to paraquat (p<0.0001). (D) Neuronal expression of CuSOD sensitizes larvae to zinc (p=0.003) but not iron or copper toxicity (p>0.05). Relative survival represents eclosion ratio of females (Elav-GAL4/+ for control, Elav-GAL4/UAS-D83S for CuSOD, Elav-GAL4/UAS-hSOD1 for hSOD1) to male files (+/+ for control, +/UAS-D83S for CuSOD, +/UAS-hSOD1 for hSOD1) on different media. (E) Expression of hSOD1 does not sensitize flies to zinc toxicity. Genotypes were as follows: control (Elav-GAL4/+), hSOD1 (Elav-GAL4/UAS-hSOD1), CuSOD¹ (Elav-GAL4/UAS-D83S₁), CuSOD or CuSOD² (Elav-GAL4/UAS-D83S₂). For panels (A), (D), and (E), the significance of the difference between means was analyzed using 1-way analysis of variance. For (B) and (C), the significance of the difference between survival curves was analyzed using log-rank test.

Fig. 5

Neuronal expression of NADH–ubiquinone oxidoreductase increases ATP production and rescues locomotor defects of zinc-deficient superoxide dismutase (SOD1, CuSOD, or D83S) flies. (A) Schematic drawing of the mechanism of NADH-ubiquinone oxidoreductase (Ndi1) enhanced energy production in the electron transport chain. Coexpression of the single-subunit yeast Ndi1 with CuSOD (B) increases ATP levels in heads and (C) boosts physical activity in 30-day-old flies. The significance of the difference between means was analyzed using 1-way analysis of variance (p < 0.05). Genotypes were as follows: CuSOD (Elav-GAL4/Y; +/UAS-GFP; +/UAS-D83S), CuSOD/Ndi1¹ (Elav-GAL4/Y; +/UAS-Ndi1; +/UAS-D83S), CuSOD/Ndi1² (Elav-GAL4/Y; +; UAS-Ndi1/UAS-D83S). Abbreviations: ADP, adenosine diphosphate; C, cytochrome c; P_i, inorganic phosphate; Q, coenzyme Q; +, a wild-type chromosome containing no mutations or transgenes.