Phenotypic effects of familial amyotrophic lateral sclerosis mutant Sod alleles in transgenic Drosophila - PubMed (original) (raw)

Phenotypic effects of familial amyotrophic lateral sclerosis mutant Sod alleles in transgenic Drosophila

Robin J Mockett et al. Proc Natl Acad Sci U S A. 2003.

Abstract

A subset of patients suffering from familial amyotrophic lateral sclerosis (FALS) exhibit point mutations in the gene encoding Cu-Zn superoxide dismutase [superoxide:superoxide oxidoreductase, EC (SOD)]. The human wild-type and five FALS Sod mutant transgenes were introduced into the fruit fly, Drosophila melanogaster, in a Cu-Zn Sod null background. Sod null flies had dramatically decreased life span, glutathione and methionine content, fertility, locomotor activity, and resistance to hyperoxic stress, compared with wild-type controls. All of these phenotypic manifestations were rescued fully by a single human wild-type allele, expressing 5-10% of wild-type SOD activity. Full recovery of wild-type life span was also observed when human mutant and wild-type alleles were placed together in the fly Sod null background. The FALS Sod mutations alone caused a recessive phenotype, usually involving low or undetectable levels of SOD activity, in which: (i) full restoration of the wild-type phenotype was observed among young adults, and (ii) older adults exhibited a sudden increase in oxidative stress, accompanied by physiological impairment of abrupt onset, and followed by premature death. Thus, the minimal SOD activity associated with the FALS Sod mutations appears to determine longevity, not by chronically increasing oxidative stress, but by limiting the time in which a viable redox environment can be maintained. However, the dominant gain of function by mutant SOD, which occurs in human patients and in the transgenic mouse model of FALS, is not observed in Drosophila.

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Figures

Figure 1

Figure 1

SOD activity in whole-body homogenates of Drosophila. Lanes: 1, bovine erythrocyte SOD (0.06 μg); 2, Sod +/+; 3, Sod x39/+; 4, _dSod_-1; Sod x16/x39; 5, _dSod_-5; Sod x16/x39; 6, _hSod_-1; Sod x16/x39; 7, _hSod_-3; Sod x16/x39; 8, Sod _G93C_-2; Sod x16/x39; 9, Sod _G37R_-1; Sod x16/x39; 10, Sod _A4V_-1; Sod x16/x39; 11, Sod _G41D_-1; Sod x16/x39; 12, Sod _I113T_-1; Sod x16/x39; 13, (Sod-)-1; Sod x16/x39; 14, Sod x16/x39. The existence of two Drosophila Cu-Zn Sod alleles, resulting in differences in electrophoretic mobility (lanes 2–5), has been discussed previously (22).

Figure 2

Figure 2

Survivorship curves of various Drosophila lines with FALS-associated Sod mutations. Pooled data are shown for each line type. The mean life spans of the replicate lines of each line type are shown in Table 1 (Experiment 2).

Figure 3

Figure 3

Markers of oxidative stress in FALS Sod transgenic lines: GSH/GSSG (A) and methionine (B). Results are mean ± SD of 3–14 independent homogenates for each group and age. Open bars: 11–18 days. Filled bars: 45–50 days, except for groups Sod _A4V_-1; Sod x16/x39 and Sod _G41D_-1; Sod x16/x39 (20–24 days).

Figure 4

Figure 4

Negative geotaxis. The vertical walking speed was measured in 12-sec intervals, three times for each fly, and the maximum height attained was expressed as speed per second. Each point represents the average of 15–45 flies.

Figure 5

Figure 5

Female fertility. Virgin females (five per vial) were outcrossed with five outbred wild-type males 2–3 days after collection. The numbers of adult progeny were determined after full eclosion and reported as mean ± SD of three replicate crosses. In a second experiment, similar results were obtained, except that fertility diminished less rapidly with increasing age. (A) First 24 h after mating. (B) Average fertility, days 2–4 (black bars) and days 5–14 (gray bars) after mating.

Figure 6

Figure 6

Resistance to experimental oxidative stress. Flies were housed in three groups of 10 and exposed continuously to 100% oxygen beginning at 9–10 days of age. Mortality was scored once or twice daily. Results shown are mean ± SD for two independent experiments, represented by black and gray bars.

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