Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery - PubMed (original) (raw)

Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery

Yufeng Yang et al. Proc Natl Acad Sci U S A. 2008.

Erratum in

Proc Natl Acad Sci U S A. 2008 Nov 11;105(45):17585

Abstract

Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinson's disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. Here, we show that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. Our genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Promotion of mitochondrial fission suppresses mutant phenotypes caused by dPink1 loss of function. (A–D) Wing posture phenotypes in 5-day-old adult flies of the following genotypes: dPink1B9 (or B9) (A), dPink1B9; Drp1 extra copy (B), dPink1B9; Opa1-like/+ (C), and wild-type control (D). The arrows point to the abnormal wing posture in A and normal posture in B–D. (E–H) (Upper) Longitudinal sections of thoraces in 5-day-old adult flies of the following genotypes: dPink1B9 (E), dPink1B9; Drp1 extra copy (F), dPink1B9; Opa1-like/+ (G), and control (H). (Lower) Magnified views of corresponding IFMs. Sections from resin-embedded thoraces of 5-day-old adult flies were stained with toluidine blue to visualize tissue morphology, particularly the musculatures; anterior is to the left. The arrows point to IFMs. (I–L) Transmission electronic microscopy analysis of IFM ultrastructure in 5-day-old adult flies of the corresponding genotypes shown above. The arrows point to mitochondria. (M) Added expression of Drp1 or loss of one copy of Opa1-like suppressed the reduction of head dopamine (DA) content caused by loss of dPink1. Two-week-old flies were subject to DA measurement. *, P < 0.01, one-way ANOVA test.

Fig. 2.

Up-regulation of mitochondrial fission prevents mitochondrial clustering in DA neurons caused by loss of dPink1. (A–C) Whole-mount immunostaining of dorsolateral protocerebral posterior (PPL1 or DL1) cluster DA neurons in 5- to 7-day-old adult flies of the following genotypes: _TH-Gal4_>UAS-mitoGFP (A), _TH-Gal4_>UAS-mitoGFP; dPink1B9 (B), and _TH-Gal4_>UAS-mitoGFP; UAS-dPink1 RNAi (C). (A–C) (Left) Immunostaining of GFP to monitor mitochondria. (Center) Immunostaining of TH to mark DA neurons. (Right) Merged images. (D–I) Live imaging of mitochondria by visualizing mitoGFP within the PPL1 cluster neurons in 5- to ≈7-day-old adult flies of the following genotypes: _TH-Gal4_>UAS-mitoGFP (D), _TH-Gal4_>UAS-mitoGFP; dPink1B9 (E), _TH-Gal4_>UAS-mitoGFP; dPink1B9; Drp1 extra copy (F), _TH-Gal4_>UAS-mitoGFP; Drp1 extra copy (G), _TH-Gal4_>UAS-mitoGFP; dPink1B9, Opa1-like/+ (H), and _TH-Gal4_>UAS-mitoGFP; Opa1-like/+ (I). A′, B′, and C′ show higher magnification views of the mitochondrial networks in A–C, respectively. Arrows, aggregated mitochondria; arrowheads, tubular mitochondria. (J) Quantification of the percentage of DA neurons containing mitochondrial aggregates with diameters of 1–2 or >2 μm. Note that mitochondrial aggregates >2 μm in diameter were observed almost exclusively in dPink1 mutant. *, P < 0.01, one-way ANOVA test.

Fig. 3.

dPink1 overexpression causes mitochondrial morphological changes in DA neurons. (A) Whole-mount immunostaining of PPL1 cluster DA neurons in 5- to ≈7-day-old adult flies of the genotype _TH-Gal4_>UAS-mitoGFP, UAS-dPink1. (Left) Immunostaining of mitoGFP. (Center) Immunostaining of TH. (Right) Merged image. (B–H) Live imaging of mitochondria within the PPL1 cluster neurons in 5- to ≈7-day-old adult flies of the following genotypes: _TH-Gal4_>UAS-mitoGFP; dPink1B9; UAS-dParkin B, _TH-Gal4_>UAS-mitoGFP; UAS-dPink1; UAS-dParkin C, _TH-Gal4_>UAS-mitoGFP; UAS-dParkin D, _TH-Gal4_>UAS-mitoGFP; UAS-dPink1 (E), _TH-Gal4_>UAS-mitoGFP; UAS-dPink1; Drp1 extra copy (F), _TH-Gal4_>UAS-mitoGFP; UAS-dPink1; Drp1/+ (G), _TH-Gal4_>UAS-mitoGFP; UAS-dPink1, Opa1 like/+ (H). A′ shows higher magnification view of the mitochondrial network in A. (I and J) representative images of 1-week-old cultured wild-type fly DA neurons exhibiting punctuate-like (I) and thread-like (J) mitochondria in neuronal processes. Left images show bright-field microscopy views of the general neuronal culture (Upper) and TH+ neurons with mitochondria labeled with mito-GFP (Lower); the right images display magnified views of neuritic mitochondria of TH+ neurons. Arrows point to typical thread-like tubular mitochondria in the neurites. (K) Statistical analysis of the percentage of TH+ neurons with thread-like tubular mitochondria in their processes in the indicated genotypes. On average, ≈200 TH+ neurons for each genotype were counted. *, P < 0.01, one-way ANOVA test.

Fig. 4.

Genetic interactions between Pink1 and fission genes in modulating mitochondrial dynamics in Drosophila and mammalian cells. (A–D) Live imaging of mitochondria in S2R cells subjected to the following genetic manipulations: gfp RNAi (A), dPink1 RNAi (B), Drp1 RNAi (C), dPink1 and Drp1 double RNAi (D). Note the similar phenotype of mitochondrial aggregation (arrows) caused by dPink1 RNAi and Drp1 RNAi despite the lower percentage in the case of dPink1 RNAi. The arrowheads point to thin continuous mitochondrial tubules extending beyond mitochondrial clusters, which appeared exclusively in D. S2R cells are not homogenous in size. Two types of cells, smaller-sized (Left) and bigger-sized (Right), are shown for each genetic manipulation. The effects are similar in both sized cells. (E and F) Live imaging of mitochondria in S2R cells subject to the following genetic manipulations: dPink1 RNAi plus EGFP transfection (E); dPink1 RNAi plus EGFP-dFis1 transfection (F). EGFP-dFis1 (E) but not EGFP (F) suppressed mitochondrial clustering induced by dPink1 RNAi. The arrow marks aggregated mitochondria. (G) Seven categories of mitochondrial morphology in mammalian COS-7 cells ranging from extreme fusion (type I) to extreme fission (type VII), as revealed by MitoTracker Red staining. The categories are arbitrarily defined based on the following mitochondrial characteristics: I, perinuclear aggregates together with long continuous tubules; II, extensive, long tubular network; III, intermediate-length tubular network; IV, a mixture of short tubular mitochondria and punctate units; V, small round tubules with holes in the center; VI, small punctate dots; VII, spherical aggregates similar to that see in DA neurons overexpressing Pink1 or Parkin. To highlight the difference between categories I and VII, the long tubular mitochondria threads present in I but not VII are marked by arrows. (H and I) Statistical analysis showing that hPink1 overexpression promotes mitochondrial fission, which is prevented by the overexpression of Drp1K38A (H), whereas hPink1 RNAi promotes mitochondrial fusion in COS-7 cells, which is rescued by hFis1 or hDrp1 overexpression (I). The ratio of cells exhibiting type II and III morphology typical of fusion versus type V and VI morphology typical of fission were quantified. *, P < 0.01, one-way ANOVA test.

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Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery - PubMed (original) (raw)