Over-expression of the catalytic core of mitochondrial DNA (mtDNA) polymerase in the nervous system of Drosophila melanogaster reduces median life span by inducing mtDNA depletion

Abstract

DNA polymerase gamma (pol gamma) is the sole DNA polymerase devoted to mitochondrial DNA (mtDNA) replication. We have characterized the molecular and physiological effects of over-expression of the catalytic subunit of pol gamma, pol gamma-alpha, in the nervous system of Drosophila melanogaster using the upstream activation sequence (UAS)/yeast transcriptional activator by binding to UAS (GAL4) system. Tissue-specific over-expression of pol gamma-alpha was confirmed by immunoblot analysis, whereas the very low levels of endogenous protein are undetectable in UAS or GAL4 control lines. The transgenic flies over-expressing pol gamma-alpha in the nervous system showed a moderate increase in pupal lethality, and a significant decrease in the median life span of adult flies. Moreover, these flies displayed a decrease in the rate of synthesis of mtDNA, which is accompanied by a significant mtDNA depletion, and a corresponding decrease in the levels of mitochondrial transcription factor A (mtTFA). Biochemical analysis showed an oxidative phosphorylation (OXPHOS) defect in transgenic flies, which were more susceptible to oxidative stress. Although we did not detect apoptosis in the nervous system of adult transgenic flies, brains of larvae over-expressing pol gamma-alpha showed evidence of increased cell death that correlates with the observed phenotypes. Our data establish an animal model that mimics some of the features of human mtDNA depletion syndromes.

Fig. 1

Expression pattern of the 1407-GAL4 line revealed by crossing with UAS-GFP. (a) Brain with the ventral nerve chord to the right and the brain hemispheres to the left from third instar larvae. (b) Adult fly.

Fig. 2

Longevity curves of adult males over-expressing pol γ-α in the nervous system. Survival curves of UAS-PolG_I (open triangles), UAS-PolG_III-1 (open circles) and UAS-PolG_III-2 lines (open squares), and males expressing pol γ-α (closed symbols as above) in the nervous system. Results are expressed as a percentage of flies alive at each day of age. Median life spans of the pol γ-α expressing lines range from 39% to 52% of the control lines, as shown by the double arrow. The survival curves were fitted to Gompertz equation.

Fig. 3

Over-expression of pol γ-α protein in the nervous system. Upper panel, mitochondrial proteins isolated from adult flies were detected by immunoblot analysis using rabbit antiserum directed against Drosophila melanogaster pol γ-α subunit or nuclear-encoded mitochondrial ATPase subunit α (α-ATPase). Lower panel, steady-state levels of pol γ-α protein normalized to the α-ATPase subunit. Data are means ± SD.

Fig. 4

Time course of mitochondria DNA (mtDNA) synthesis in organello. (a) Incorporation of [α-³²P]dCMP into mtDNA at various times of incubation of isolated mitochondria at 37°C as described under Materials and methods. Mitochondria were isolated from UAS-PolG_III-2 (circles) and UAS-PolG_III-2 × 1407-GAL4 flies (squares). a.u.: arbitrary units. (b) Rate of synthesis of mtDNA derived from the data shown in (a). Data are means ± SD. *Statistical comparison was made using the Student’s t test, p < 0.003.

Fig. 5

pol γ-α over-expression induces mitochondrial DNA (mtDNA) depletion in the nervous system. (a) Immunoblot analysis of nuclear-encoded mitochondrial proteins. Upper panel, mitochondrial proteins isolated from adult flies were detected by immunoblotting with antibodies against Drosophila melanogaster pol γ-α, mitochondrial transcription factor A (mtTFA) and nuclear-encoded mitochondrial ATPase subunit α (α-ATPase). Lower panel, steady-state levels of mtTFA protein were normalized to the α-ATPase subunit. Data are means ± SD. (b) Southern blot analysis. Upper panel, total DNA was extracted from adult flies, digested with NsiI, electrophoresed, transferred to a nylon membrane and hybridized with a mtDNA fragment (mtDNA) and a fragment of the 18S rRNA gene (nDNA). Lower panel, quantitation of mtDNA/nDNA levels. Data are means ± SD. Statistical comparison was made using the Student’s t test, *p < 0.007, **p < 0.02.

Fig. 6

Transgenic flies over-expressing pol γ-α display oxidative phosphorylation deficiency and a higher susceptibility to reactive oxygen species. (a) Absolute activities of citrate synthase, complex I+III and complex IV. The activities were assayed per milligram of protein (nmol/min per mg of protein) on mitochondria from control flies UAS-PolG_III-2 (black) and flies over-expressing pol γ-α [1407-GAL4 × UAS-PolG_III-2] (white), and are represented as percentage of the control value. Data are means ± SD. Statistical comparison was made using the Student’s t test, *p ~ 0.785, **p < 0.026, ***p ~ 0.250. (b) Normalized activities of complexes. Complex I+III and complex IV activities were normalized by citrate synthase activity. Student’s t test, *p < 0.035, **p ~ 0.602. (c) Resistance to the reactive oxygen species-generating agent paraquat. Survival of male and female flies (4–5 days of age) over-expressing pol γ-α (1407-GAL4 × UAS-PolG_III-2) (white) and control flies (UAS-PolG_III-2) (black) was measured 48 h post-treatment with 20 mmol/L paraquat at 25°C. Student’s t test, *p < 0.019, **p < 0.015.

Fig. 7

Mitochondrial DNA depletion in the nervous system increases the level of apoptosis in larval brain. (a) Brains were dissected from third instar larvae over-expressing pol γ-α (1407-GAL4/UAS-GFP/ UAS-POLG_III-2) and control larvae (1407-GAL4/UAS-GFP) as described under Materials and methods. The domain of over-expression directed by the 1407-GAL4 driver was monitored by GFP fluorescence (green; upper panel). Apoptosis was detected using an antibody that detects specifically activated caspase 3 Drice (red; lower panel). The bar represents 100 μm. (b) Quantitation of the numbers of apoptotic cells in (a) lower panel. Data are means ± SD. Statistical comparison was made using the Student’s t test: UAS-PolG_III-2 (n = 18) versus UAS-PolG_III-2 × 1407-GAL4 (n = 30), *p < 0.0001.