Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant - PubMed (original) (raw)

Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant

Hyongjong Koh et al. J Biol Chem. 2012.

Abstract

PTEN-induced kinase 1 (PINK1), which is associated with early onset Parkinson disease, encodes a serine-threonine kinase that is critical for maintaining mitochondrial function. Moreover, another Parkinson disease-linked gene, parkin, functions downstream of PINK1 in protecting mitochondria and dopaminergic (DA) neuron. In our fly genetic screening, knockdown of Sir2 blocked PINK1 overexpression-induced phenotypes. Consistently, ectopic expression of Sir2 successfully rescued mitochondrial defects in PINK1 null mutants, but unexpectedly, failed in parkin mutants. In further genetic analyses, deletion of FOXO nullified the Sir2-induced mitochondrial restoration in PINK1 null mutants. Moreover, overexpression of FOXO or its downstream target gene such as SOD2 or Thor markedly ameliorated PINK1 loss-of-function defects, suggesting that FOXO mediates the mitochondrial protecting signal induced by Sir2. Consistent with its mitochondria-protecting role, Sir2 expression prevented the DA neuron loss of PINK1 null mutants in a FOXO-dependent manner. Loss of Sir2 or FOXO induced DA neuron degeneration, which is very similar to that of PINK1 null mutants. Furthermore, PINK1 deletion had no deleterious effect on the DA neuron loss in Sir2 or FOXO mutants, supporting the idea that Sir2, FOXO, and PINK1 protect DA neuron in a common pathway. Overall, these results strongly support the role of Sir2 and FOXO in preventing mitochondrial dysfunction and DA neuron loss, further suggesting that Sir2 and FOXO function downstream of PINK1 and independently of Parkin.

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Figures

FIGURE 1.

FIGURE 1.

Expression of Sir2 rescues PINK1 null mutant phenotypes in a _FOXO_-dependent manner. A, light stereo micrographs of the thoraces of PINK1 null mutants (B9, arm), Sir2-expressing PINK1 null mutants (_B9, arm_>Sir2), and Sir2-expressing PINK1 and FOXO double mutants (_B9, arm_>_Sir2, FOXO_−). _arm_-GAL4/+ (arm) flies were used as wild type controls. White arrows indicate collapsed-thorax phenotypes. B, percentage of defective thorax and wing phenotypes. C, toluidine blue-stained longitudinal sections (top panels) and merged images of TUNEL (red) and DAPI (blue) staining (bottom panels) of indirect flight muscle in the thoraces. D, quantification of the mtDNA of thoraces (n = 3). Cox I, cytochrome c oxidase subunit I; Cox III, cytochrome c oxidase subunit III; Cyt B, cytochrome b. E, comparison of the ATP content of thoraces (n = 3). F, comparison of climbing ability (n = 4). Significance was determined by one-way ANOVA with Bonferroni's correction (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars indicate mean ± S.D. Scale bars: yellow, 5 μm; white, 10 μm. Details of all the indicated genotypes in this and other figures are described in the

supplemental Experimental Procedures

.

FIGURE 2.

FIGURE 2.

FOXO expression rescues PINK1 null mutant phenotypes. A, light stereo micrographs of the thoraces of PINK1 null mutants (B9, hs), FOXO-expressing PINK1 null mutants (_B9, hs_>FOXO), and PINK1 and FOXO double mutants (_B9, FOXO_−). _hs_-GAL4/+ (hs) flies were used as wild type controls. White arrows indicate collapsed-thorax phenotypes. B, percentage of defective thorax and wing phenotypes. C, comparison of climbing ability (n = 4). D, toluidine blue-stained longitudinal sections (top panels) and merged images of TUNEL (red) and DAPI (blue) staining (bottom panels) of indirect flight muscle in the thoraces. E, quantification of the mtDNA of thoraces (n = 3). Cox I, cytochrome c oxidase subunit I; Cox III, cytochrome c oxidase subunit III; Cyt B, cytochrome b. F, comparison of the ATP content of thoraces (n = 3). Significance was determined by one-way ANOVA with Bonferroni's correction (*, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant). Error bars indicate mean ± S.D. Scale bars: yellow, 5 μm; white, 10 μm.

FIGURE 3.

FIGURE 3.

FOXO target genes SOD2 and Thor rescue PINK1 null mutant phenotypes. A, comparison of SOD2 mRNA level in the thoraces from wild type controls (hs), PINK1 null mutants (B9, hs), and FOXO-expressing PINK1 null mutants (_B9, hs_>FOXO) (n = 3). B, comparison of Thor mRNA level in the thoraces (n = 3). C, light stereo micrographs of the thoraces of wild type controls (hs), PINK1 null mutants (B9, hs), SOD2-expressing PINK1 null mutants (_B9, hs_>SOD2), and Thor-expressing PINK1 null mutants (_B9, hs_>Thor). A white arrow indicates collapsed-thorax phenotypes. D, percentage of defective thorax and wing phenotypes. E, comparison of climbing ability (n = 4). F, quantification of the mtDNA of thoraces (n = 3). Cox I, cytochrome c oxidase subunit I; Cox III, cytochrome c oxidase subunit III; Cyt B, cytochrome b. G, comparison of the ATP content of thoraces (n = 3). Significance was determined by one-way ANOVA with Bonferroni's correction (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars indicate mean ± S.D.

FIGURE 4.

FIGURE 4.

Sir2 and FOXO suppress mitochondrial enlargement in DA neurons of PINK1 null mutants. A, examination of the mitochondria in DA neurons within the DL1 cluster of adult brain from wild type control (WT), PINK1 null mutants (B9), Sir2-expressing PINK1 null mutants (B9, _TH_>Sir2), Sir2-expressing PINK1 null mutants with a heterozygous FOXO mutation (B9, TH_>Sir2, FOXO_−/+), SOD2-expressing PINK1 null mutants (B9, _TH_>SOD2), and Thor-expressing PINK1 null mutants (B9, _TH_>Thor). _TH-_GAL4-drived expression of mitochondria-targeted green fluorescent protein (_TH_>mitoGFP, green) showed mitochondrial shape and size in the DA neurons stained with anti-TH antibody (red). B, graph showing the percentage of the number of DA cells with mitochondria larger than 2 μm in diameter over the total number of DA cells in DL1 clusters (n = 3). Significance was determined by one-way ANOVA with Bonferroni's correction (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars indicate mean ± S.D. Scale bars: yellow, 5 μm.

FIGURE 5.

FIGURE 5.

Sir2 and FOXO ameliorate DA neuronal degeneration in PINK1 null mutants. A, images of the DA neurons within the DL1 cluster of adult brain from wild type control (TH), PINK1 mutants (B9, TH), Sir2-expressing PINK1 null mutants (B9, _TH_>Sir2), Sir2-expressing PINK1 null mutants with a heterozygous FOXO mutation (B9, TH_>Sir2, FOXO_−/+), SOD2-expressing PINK1 null mutants (B9, _TH_>SOD2), and Thor-expressing PINK1 null mutants (B9, _TH_>Thor). DA neurons were stained with anti-TH antibody (green) B, graph showing the average number of DA neurons in DL1 clusters (n = 40). Significance was determined by one-way ANOVA with Bonferroni's correction (***, p < 0.001). Error bars indicate mean ± S.D. Scale bars: white, 20 μm.

FIGURE 6.

FIGURE 6.

PINK1, Sir2, and FOXO act in same pathway in preventing DA neuron loss. A, images of the DA neurons within the DL1 cluster of adult brain from wild type control (WT), PINK1 null mutants (B9), Sir2 mutants (_Sir2_−), PINK1 and Sir2 double mutants (_B9, Sir2_−), FOXO mutants (_FOXO_−), and PINK1 and FOXO double mutants (_B9, FOXO_−). DA neurons were stained with anti-TH antibody (green). B, graph showing the average number of DA neurons in DL1 clusters (n = 40). Significance was determined by one-way ANOVA with Bonferroni's correction (***, p < 0.001; NS, not significant). Error bars indicate mean ± S.D. Scale bar: white, 20 μm.

FIGURE 7.

FIGURE 7.

Dual roles of PINK1 in mitochondria protection. Ub, ubiquitin.

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