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, 107 (21), 9747-52

DJ-1 Is Critical for Mitochondrial Function and Rescues PINK1 Loss of Function

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DJ-1 Is Critical for Mitochondrial Function and Rescues PINK1 Loss of Function

Ling-Yang Hao et al. Proc Natl Acad Sci U S A.

Abstract

Mutations or deletions in PARKIN/PARK2, PINK1/PARK6, and DJ-1/PARK7 lead to autosomal recessive parkinsonism. In Drosophila, deletions in parkin and pink1 result in swollen and dysfunctional mitochondria in energy-demanding tissues. The relationship between DJ-1 and mitochondria, however, remains unclear. We now report that Drosophila and mouse mutants in DJ-1 show compromised mitochondrial function with age. Flies deleted for DJ-1 manifest similar defects as pink1 and parkin mutants: male sterility, shortened lifespan, and reduced climbing ability. We further found poorly coupled mitochondria in vitro and reduced ATP levels in fly and mouse DJ-1 mutants. Surprisingly, up-regulation of DJ-1 can ameliorate pink1, but not parkin, mutants in Drosophila; cysteine C104 (analogous to C106 in human) is critical for this rescue, implicating the oxidative functions of DJ-1 in this property. These results suggest that DJ-1 is important for proper mitochondrial function and acts downstream of, or in parallel to, pink1. These findings link DJ-1, pink1, and parkin to mitochondrial integrity and provide the foundation for therapeutics that link bioenergetics and parkinsonism.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
DKO mutant flies show multiple defects with age. (A) Lifespan of control flies compared with DKO flies. More than 200 flies for each genotype were used (P < 0.001, log–rank test). (B) Climbing ability of control and DKO flies at different ages. The fraction of total flies that climbed out of the required distance in the given time is shown (mean ± SEM; n > 100 flies). P = 0.0004 (t test, two tailed) between control and DKO at 7 weeks. (C) Electron micrographic images of control and DKO spermatogenesis. The mitochondrial derivatives are regular and orderly in the control but are aberrant in DKO (white arrows). (Lower) Higher magnification of squared areas above. (D) Progression of sperm individualization, highlighted with phalloidin, which labels sperm head investment cones (white arrows), is orderly and compact in the control but disorganized in DKO testes (3-day males).
Fig. 2.
Fig. 2.
Mitochondrial dysfunction in DKO mutant flies. (A) The relative level of mtDNA in young (3 days) and old (30 days) control (white) and DKO (gray) thoraces (mean ± SEM, three DNA extractions for 3 days, four for 30 days). The ND5 locus was used to measure mtDNA levels, normalized to the nuclear let-7 locus. P = 0.0055 (t test, two tailed). (B) Western immunoblot for level of complex I subunit NDUFS3 (Upper) and actin shows similar levels in control and DKO with age. (C) Mitochondrial RCR of control (white) and DKO (gray). (Left) Whole flies (mean ± SEM, three preparations of mitochondria for 3 days, four for 30 days) (two-tailed t test, P = 0.005 between 30-day control and DKO samples). (Right) Heads (mean ± SEM, seven preparations of mitochondria). Mitochondrial RCR showed significance in aged whole fly and a modest but insignificant decrease in aged heads RCR (two-tailed t test, P = 0.096 between 30-day control and DKO). (D) ATP levels (mole/mg) of control (white) and DKO (gray) flies (mean ± SEM, three sample preparations) (two-tailed t test, P = 0.011 between 30-day control and DKO samples from whole flies).
Fig. 3.
Fig. 3.
DJ-1 KO mice show motor and mitochondrial dysfunction. (A) Rotorod test of control (solid) and DJ-1 KO (dashed) animals of different ages, showing a significant (P < 0.01) difference by 15 months (mean ± SD, n = 6–10 animals per time point). (B, Upper) ATP levels (moles/mg) in hindlimb skeletal muscle (a mixture of soleus and gastrocnemius muscle) of control (white) and DJ-1 null (gray) mice (mean ± SEM, n = 6 animals) (two-tailed t test, P = 0.017 between 14-month control and KO samples). (Lower) ATP levels from brain extracts (mean ± SEM, n = 6 animals).
Fig. 4.
Fig. 4.
Up-regulation of DJ-1 rescues Pink1 muscle defects. (A) Cryosections of the thorax stained with phalloidin to highlight muscle structure of young (<3 days) male flies. Visible vacuoles (arrows) are observed in the pink1 deleted mutant, pink1B9. Both DJ-1a and parkin up-regulation ameliorate the defects; compare with the pink1Rev control, a normal revertant of the original pink1B9 mutation. (B) Widespread apoptosis revealed by TUNEL staining (Upper) in thorax sections of pink1B9 flies is rescued by DJ-1a up-regulation. (C) Relative mtDNA levels from male thoraces was assayed by real-time PCR and adjusted to the levels in the control pink1Rev (mean ± SEM, four to six independent experiments). The mtDNA level was rescued by DJ-1a and parkin up-regulation (two-tailed t test, P = 0.004 between pink1 with driver and pink1 with parkin up-regulated samples, P = 0.006 between pink1 with driver and pink1 with DJ-1a up-regulated samples. A p value of <0.0167 was considered significant after correction for multiple tests). (D) Real-time PCR assays for levels of parkin (Upper), drp1 (white, Lower), and dMfn2 (gray, Lower) mRNA in young (<5 days) male flies (mean ± SEM, three to four independent experiments). The RNA levels were adjusted to pink1Rev levels. Flies overexpressing parkin had dramatically higher (≈650×) parkin mRNA levels, whereas up-regulation of DJ-1a did not change parkin mRNA levels. DJ-1a up-regulation did not change the mRNA levels of drp1 or dMfn2. (E) Up-regulation of DJ-1 does not rescue parkin mutation. Cryosections of thoraces stained with phalloidin (red) for muscle structure (white arrows, vacuoles highlighted deteriorated structure). TUNEL for apopotosis (green) and DAPI (blue). Vacuoles and TUNEL signal of parkin deleted mutant, parkind25, was unchanged with up-regulation of DJ-1a.

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