Figure 1.
S6K and Atg1 act as genetic modifiers of PINK1 RNAi.
The flies of each indicated genotype were crossed to Mhc-Gal4>PINK1 RNAi flies (A) or Mhc-Gal4 flies (B), and the percentage of male offspring with abnormal wing posture phenotype was scored at 1-day and 14-day after eclosion. The flies were aged at 29°C. Data are presented as mean ± s.e.m. The genetic interactions between PINK1 and genes of the TOR pathway, autophagy pathway, mitochondrial fusion and fission machinery or antioxidant genes are demonstrated. The differences in abnormal wing posture phenotype between the genetic interaction flies and control flies shown in (A) are all statistically significant (P<0.005 in Student's t-test).
Figure 2.
Overexpression or knockdown of S6K strongly modifies PINK1 RNAi phenotypes in the muscle.
Overexpression of constitutively active S6Ks (S6K-TE, S6K-STDE and S6K-STDETE) in the muscle of PINK1 RNAi flies completely abolished their jump/flight ability (A), significantly decreased their muscle ATP level (B) and dramatically increased their thoracic indentation (C). In contrast, the overexpression of S6K RNAi transgene partially rescued these phenotypes in PINK1 RNAi flies. Open and closed bars represented data scored on Day 1 and Day 14 after eclosion, respectively. Data are presented as mean ± s.e.m. Significance was determined by Student's t test (*P<0.005). (D) Representative image of thoracic indentation (indicated by the white arrowhead) of Mhc-Gal4; PINK1 RNAi; S6K-TE fly at 1-day old (bottom) compared to the normal thoracic phenotype of Mhc-Gal4, PINK1 RNAi fly of the same age (top). (E) Overexpression of constitutively active S6K in PINK1 RNAi flies dramatically increased muscle degeneration. Sections from resin-embedded thoraces of 1-day-old adult flies were either stained with toluidine blue to visualize overall muscle structure (top panel) or directly visualized using TEM for mitochondrial morphology (bottom panel). WT flies or flies expressing constitutively active S6K show normal muscle structure with healthy, electron-dense mitochondria. Flies expressing PINK1 RNAi transgene had small lesions in the muscle with dysfunctional mitochondria showing broken cristae. Co-expression of S6K-TE in PINK1 RNAi flies caused more severe degeneration of mitochondria and muscle fibers, generating large lesions in the muscle that were filled with resin during embedding and are readily recognizable (indicated by black arrows).
Figure 3.
Constitutively active S6K increases mitochondrial aggregation and DA neuron loss in PINK1 mutants.
(A) Overexpression of constitutively active S6K increased the size of swollen or aggregated mitochondria in the DA neurons of PINK1 mutants. Mitochondrially targeted GFP (mitoGFP) was expressed in the DA neurons using TH-Gal4 driver [44] to help visualize mitochondrial morphology. Brains of 7-day-old adult flies of the indicated genotypes were immunostained with anti-TH antibody (red) to label DA neuron and anti-GFP antibody (green) to label mitochondria. Images of DA neurons in the PPL1 cluster were shown. Overexpression of S6K-TE in PINK1 mutant significantly increased the size of mitochondrial aggregates in DA neurons. The scale bar represents 5 µm. (B) Comparison of mitochondrial size distribution in PINK1 mutants with or without S6K-TE overexpression. Statistical significance was determined by Student's t test (**P<0.001, *P<0.05). (C) Overexpression of constitutively active S6K increased DA neuron loss in the PPL1 cluster of PINK1 mutant. DA neuron number was scored in flies aged for 14 days at 25°C. At least 7 flies were used for each genotype. Statistical significance was determined by Student's t test (**P<0.001). Data are presented as mean ± s.e.m.
Figure 4.
RpS6 or RpS9 RNAi blocks the enhancing effects of S6K-TE in PINK1 RNAi background.
Overexpression of RpS6 or RpS9 RNAi transgenes in PINK1 RNAi or PINK1 RNAi/UAS-S6K-TE flies efficiently rescued the abnormal wing posture (A), thoracic indentation (B), and energy depletion (D) phenotypes in 1-day-old flies and partially suppressed these phenotypes in 14-day-old flies. Data are presented as mean ± s.e.m. Statistical significance was determined by Student's t test (*P<0.001). (C) RpS6 or RpS9 RNAi blocked increased mitochondrial aggregation in PINK1 RNAi, UAS-S6K-TE flies. The scale bar represents 5 µm. (E) Western blot analysis comparing the levels of dS6K and phosphorylated S6K (T398) in wild type, Mhc-Gal4>PINK1 RNAi and PINK1B9 mutant flies. The phosphorylation of S6K was significantly decreased in PINK1 RNAi or mutant flies.
Figure 5.
Overexpression of Atg1 rescues PINK1 mutant phenotypes.
Overexpression of Atg1 in the muscle of PINK1B9 mutants rescued their abnormal wing posture (A), thoracic indentation (B), jump/flight activity (C) and muscle ATP level (D). Data are presented as mean ± s.e.m. Statistical significance was determined by Student's t test (*P<0.001). (E) Atg1 overexpression did not completely rescue the mitochondrial aggregation phenotype in the muscle of PINK1 RNAi flies. mitoGFP was expressed in the muscle using Mhc-Gal4 driver to visualize mitochondrial morphology by live imaging. Wild type flies showed mitochondria of relatively uniform sizes (bottom right), while PINK1 RNAi flies had bright mitochondrial aggregates. Only the co-expression of Marf RNAi or Parkin OE was able to efficiently rescue the mitochondrial aggregation phenotype in the PINK1 RNAi background.
Figure 6.
Atg1 OE rescues PINK1 RNAi phenotype by inducing autophagy.
(A, B) The rescuing effect of Atg1 OE in PINK1 RNAi flies was not dependent on S6K inhibition. Atg1 OE, as well as Parkin OE or Marf RNAi, efficiently rescued the abnormal wing posture (A) and muscle energy depletion (B) in PINK1 RNAi/S6K-TE flies. In contrast, overexpression of 4E-BP and the antioxidant genes were not as effective. Data are presented as mean ± s.e.m. Statistical significance was determined by Student's t test (*P<0.001). (C) Overexpression of Atg1 was sufficient to induce autophagy in fly muscle. UAS-LC3-GFP was expressed in the muscle of flies with the indicated genetic background, and the level of autophagy was determined by Western Blot using anti-GFP antibody. Overexpression of Atg1 significantly increased the level of LC3-II in the muscle, indicating increased autophagy. Increased autophagy was also observed in PINK1 RNAi and PINK1 mutant flies. (* indicates a cross-reaction band).
Figure 7.
Atg18 RNAi blocks the rescuing effect of Atg1 OE but not that of Parkin OE or Marf RNAi.
The rescuing effect of Atg1 OE on the abnormal wing posture (A) and muscle energy depletion (B) phenotypes could be blocked by the co-expression of Atg18 RNAi, suggesting that Atg1 functions through inducing autophagy to rescue PINK1 RNAi phenotype. In contrast, the rescue of PINK1 RNAi phenotypes by Parkin OE or Marf RNAi were largely unaffected by the disruption of Atg1 or Atg18 through RNAi. The tests were carried out in both PINK1B9 mutant and Mhc-Gal4>PINK1 RNAi/S6K-TE backgrounds. Data are presented as mean ± s.e.m. Statistical significance was determined by Student's t test (**P<0.001). (C) Atg1 or Atg18 RNAi did not abolish the rescuing effect of Parkin OE in DA neurons. mitoGFP was expressed in DA neurons using the TH-Gal4 driver to visualize mitochondrial morphology. Overexpression of Parkin efficiently rescued the mitochondrial aggregation phenotype in PINK1 mutant (top panel). Similar rescuing effect was observed when Atg1 RNAi or Atg18 RNAi was co-expressed with Parkin (middle and bottom panels). The scale bar represents 5 µm.