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. 2013 May 21;110(21):8638-43.
doi: 10.1073/pnas.1216197110. Epub 2013 May 6.

Parkin overexpression during aging reduces proteotoxicity, alters mitochondrial dynamics, and extends lifespan

Anil Rana  1 , Michael Rera, David W Walker
Affiliations

Parkin overexpression during aging reduces proteotoxicity, alters mitochondrial dynamics, and extends lifespan

Anil Rana et al. Proc Natl Acad Sci U S A. .

Abstract

Aberrant protein aggregation and mitochondrial dysfunction have each been linked to aging and a number of age-onset neurodegenerative disorders, including Parkinson disease. Loss-of-function mutations in parkin, an E3 ubiquitin ligase that functions to promote the ubiquitin-proteasome system of protein degradation and also in mitochondrial quality control, have been implicated in heritable forms of Parkinson disease. The question of whether parkin can modulate aging or positively impact longevity, however, has not been addressed. Here, we show that ubiquitous or neuron-specific up-regulation of Parkin, in adult Drosophila melanogaster, increases both mean and maximum lifespan without reducing reproductive output, physical activity, or food intake. Long-lived Parkin-overexpressing flies display an increase in K48-linked polyubiquitin and reduced levels of protein aggregation during aging. Recent evidence suggests that Parkin interacts with the mitochondrial fission/fusion machinery to mediate the turnover of dysfunctional mitochondria. However, the relationships between parkin gene activity, mitochondrial dynamics, and aging have not been explored. We show that the mitochondrial fusion-promoting factor Drosophila Mitofusin, a Parkin substrate, increases in abundance during aging. Parkin overexpression results in reduced Drosophila Mitofusin levels in aging flies, with concomitant changes in mitochondrial morphology and an increase in mitochondrial activity. Together, these findings reveal roles for Parkin in modulating organismal aging and provide insight into the molecular mechanisms linking aging to neurodegeneration.

Keywords: energy metabolism; healthspan; mitophagy; neuronal aging; proteostasis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Ubiquitous Parkin overexpression extends lifespan. (A) Western blot analysis of Parkin expression during aging. Parkin levels are increased in female flies by transgenic expression of a parkin cDNA under control of the ubiquitous daGS promoter. Transgenic expression is induced by exposure of flies to the drug RU486. See quantification in Fig. S1A. (B and C) Survival curves of daGS>UAS-parkin flies with or without RU486-mediated transgene induction. (B) Constitutive overexpression of Parkin increases fly lifespan (P < 0.0001; log-rank test; n > 200 female flies). (C) Adult-onset overexpression of Parkin increases fly lifespan (P < 0.0001; log-rank test; n > 200 female flies). (D) Capillary feeding assay (52). Constitutive overexpression of Parkin has no significant effect on feeding behavior (n = 10; 10 female flies per replicate). Data are represented as means ± SEM. (E) Survival curves under starvation. Constitutive Parkin overexpression confers increased survival under starvation (P < 0.0001; log-rank test; n > 60 female flies). RU486 was provided in the media at 0.2 µg/mL during development and 1 µg/mL during adulthood (A, B, D, and E) and 5 µg/mL during adulthood (C).
Fig. 2.
Fig. 2.
Ubiquitous Parkin overexpression counteracts age-onset proteotoxicity. (A) Western blot detection of K48-polyubiquitinated proteins in whole-body detergent-insoluble extracts from young and aged daGS>UAS-parkin female flies with or without RU486-mediated transgene induction. Parkin overexpression leads to increased K48-polyubiquitinated proteins in aged flies (see quantification in Fig. S4A). (B–I) Immunostaining of indirect flight muscles from young and aged flies. Polyubiquitin (green) immunoreactivity reveals an age-related increase in the deposition of aggregates containing polyubiquitin proteins in uninduced control flies (B and D) and, to a lesser extent, in Parkin-overexpressing flies (C and E). F–I represent insets of B–E, respectively. Parkin overexpression counteracts the age-related increase in the cumulative area and size of protein aggregates (see quantification in J and K). **P < 0.01; *P < 0.05 (Student t test; n > 6; one fly per replicate). Phalloidin staining (red) shows F-actin, which is a component of muscle myofibrils. Data are represented as means ± SEM. (Scale bar: B–E, 20 µm; F–I, 5 µm.) (L) Western blot detection of total ubiquitin-conjugated proteins in whole-body detergent-insoluble extracts from young and aged female flies. Parkin overexpression leads to a reduced amount of detergent-insoluble ubiquitin-conjugated proteins in aged flies (see quantification in Fig. S4H). RU486 was provided in the media at 0.2 µg/mL during development and 1 µg/mL during adulthood. Young flies were 10 d of age, and old flies were 30 d of age.
Fig. 3.
Fig. 3.
Ubiquitous Parkin overexpression alters mitochondrial dynamics and activity during aging. (A) Western blot detection of dMfn in young and aged daGS>UAS-parkin female flies, with or without RU486-mediated transgene induction. dMfn levels are increased in aged flies compared with young flies. Parkin overexpression reduces dMfn levels in young and aged flies (see quantification in Fig. S5A). (B–E) Electron micrographs of indirect flight muscles from young and aged flies. Parkin overexpression leads to a more fragmented mitochondrial network. b–e are insets of B–E. (Scale bars: 5 µm.) (F) Quantification of mitochondrial area in indirect flight muscles from young and aged flies. Parkin overexpression leads to smaller mitochondria in young and aged flies (n > 81 mitochondria per sample). (G–I) Quantification of markers of mitochondrial activity in young and aged flies. Parkin overexpression leads to an increase in citrate synthase activity (G), complex I activity (H), and complex II activity (I) in young flies and an increase in citrate synthase activity in aged flies (G) (n ≥ 3; 10 flies per replicate). (J) Quantification of dPGC-1 mRNA levels in young and aged flies. Parkin overexpression leads to an increase in dPGC-1 gene expression in young flies (n = 3; five flies per replicate). Data are represented as means ± SEM. RU486 was provided in the media at 0.2 µg/mL during development and 1 µg/mL during adulthood. Young flies were 10 d of age, and old flies were 42 d of age. *P < 0.05; **P < 0.01; ***P < 0.001 (Student t test).
Fig. 4.
Fig. 4.
Neuron-specific Parkin overexpression extends lifespan. (A and B) Survival curves of ElavGS>UAS-parkin flies with or without RU486-mediated transgene induction. (A) Constitutive neuron-specific overexpression of Parkin increases fly lifespan (P < 0.0001; log-rank test; n > 180 female flies). (B) Adult-onset neuron-specific overexpression of Parkin increases fly lifespan (P <0.0001; log-rank test; n > 180 female flies). (C) Capillary feeding assay (52). Neuronal overexpression of Parkin has no significant effect on feeding behavior (n= 10; 10 female flies per replicate). (D) Survival curves under starvation. Constitutive neuron-specific overexpression of Parkin confers increased survival under starvation (P < 0.0001; log-rank test; n > 60 female flies). (E) Immunostaining of adult brains from aged (30 d) flies. Polyubiquitin (green) immunoreactivity revealed the deposition of aggregates containing polyubiquitin proteins (Ea and inset Ea′ showing only aggregates). Neuronal Parkin overexpression significantly reduces the number of protein aggregates in the aged brain (Eb and inset Eb′ showing only aggregates). See quantification in Fig. S8C. Phalloidin staining (red) outlines F-actin, and TO-PRO-3 stains nuclei (blue). (Scale bar: 10 µm.) (F and G) Quantification of markers of mitochondrial activity in heads of young and aged flies. Constitutive neuron-specific overexpression of Parkin overexpression leads to an increase in citrate synthase activity in heads of young flies (F) and complex I activity in heads of aged flies (G) (*P < 0.05; Student t test; n = 3; 10 heads from female flies per replicate). Young flies were 10 d of age, and old flies were 42 d of age. RU486 was provided in the media at 0.2 µg/mL during development and 1 µg/mL during adulthood (A, C, and D–G) and 0.5 µg/mL during adulthood (B). Data are represented as means ± SEM.
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