PINK1-dependent recruitment of Parkin to mitochondria in mitophagy - PubMed (original) (raw)

. 2010 Jan 5;107(1):378-83.

doi: 10.1073/pnas.0911187107. Epub 2009 Dec 4.

Chun Zhou, Yong Huang, Mei Cui, Rosa L A de Vries, Jiho Kim, Jessica May, Maja Aleksandra Tocilescu, Wencheng Liu, Han Seok Ko, Jordi Magrané, Darren J Moore, Valina L Dawson, Regis Grailhe, Ted M Dawson, Chenjian Li, Kim Tieu, Serge Przedborski

Affiliations

PINK1-dependent recruitment of Parkin to mitochondria in mitophagy

Cristofol Vives-Bauza et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (DeltaPsi(m)) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of DeltaPsi(m) relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal DeltaPsi(m). We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Mitochondrial depolarization recruits WT Parkin to mitochondria. (A) Once recruited to mitochondria, Parkin associates with the mitochondrial outer membrane. HeLa cells were incubated with vehicle (DMSO) or with 10 μM CCCP for 1 h before mitochondrial isolation and protection assay by treatment with different concentrations of proteinase K (PK) (0, 0.2, 2, and 20 μg/mL). TOM20, cytochrome c, and COX-1 are markers of the outer membrane, intermembrane space, and inner membrane, respectively. (B) Parkin PD-linked mutant forms are not recruited to depolarized mitochondria. HeLa cells were transfected either with WT Parkin-myc or with mutant forms containing the pathogenic point mutations T415N and G430D. Twenty-four hours after transfection, cells were incubated with vehicle (DMSO) or with 10 μM CCCP for 1 h before fixation. Bars represent percent of cells showing Parkin-myc colocalization with TOM20 (Parkin+/TOM20+) ± SD of 3 independent experiments, determined by confocal microcopy. (B′) Western blot analysis shows that WT Parkin and its mutants T415D and G430P achieved comparable levels of protein expression. Parkin is immunodetected using anti-myc antibody. Loading is normalized by TIM23 (C) Effects on HeLa cells of 1-h incubation with different concentrations of CCCP, 24 h after transfection with Parkin-GFP. Bars represent percent of cells showing Parkin colocalization with TOM20 (Parkin+/TOM20+) ± SD of 3 independent experiments, determined by confocal microcopy. (D) Same as (B), but after 1-h incubation with 10 μM FCCP, 10 μM CCCP, 1 μM Antimycin A (AA), 1 μM Oligomycin (Oligo), or 1 μM AA and 1 μM Oligo (AA+Oligo) or after 30-min preincubation with 2 μM cyclosporine A (CsA), followed by 1-h incubation with 10 μM CCCP. Like FCCP and CCCP, AA+Oligo produce a significant percentage of cells with Parkin+/TOM20+. (E) Comparison of TMRM fluorescence acquired by live imaging and quantified as arbitrary units (A.U.) by Image J, among nontransfected and Parkin-GFP–transfected WT HeLa cells incubated with vehicle (CTL), 100 nM CCCP, or 1 μM AA + 1 μM Oligo and nontransfected and Parkin-GFP–transfected Rho0 HeLa cells. Values represent mean ± SD (n = 35–50 cells) and are representative of 3 independent experiments. **, Different from CTL. *, Different from nontransfected and transfected cells with diffuse Parkin-GFP fluorescence (Newman–Keuls post hoc test; P < 0.001).

Fig. 2.

Fig. 2.

PINK1 knockdown prevents Parkin recruitment to depolarized mitochondria. (A) Immunofluorescence of HeLa cells cotransfected with Parkin-YFP and scrambled (scr) PINK1 or DJ-1 siRNA, and incubated for 1 h with 10 μM CCCP. Mitochondria are labeled with an anti-TOM20 antibody (red). (Scale bars, 10 μM.) Zoom shows 6× magnification of the region outlined by the box. (Scale bars, 1 μM.) (B) Effects of siRNA on PINK1 and DJ1 mRNA levels. Total RNA extracted from each sample is quantified by real-time PCR (n = 3). (C) Percentages of cells from the same set of cotransfected HeLa cells as in (A) that exhibit Parkin puncta colocalizing with the mitochondrial marker TOM20 (Parkin+/TOM20+). (D) Percentages of WT and knockout (KO) PINK1 cortical neurons that exhibit Parkin+/TOM20+ puncta following 1-h incubation with or without 100 nM CCCP. Values represent means ± SD (n = 30–50 cells) and are representative of 2–3 independent experiments. **, Different from controls (Newman-Keuls post hoc test; P < 0.001).

Fig. 3.

Fig. 3.

Overexpression of PINK1 suffices to recruit Parkin to mitochondria with normal ΔΨm, as evidenced by TMRM fluorescence in living cells (see

Fig. S2_A_

). (A) Representative images illustrating the time-dependent changes in Parkin-YFP fluorescence from diffuse to punctate in immortalized mesencephalic neuronal N27 cells, after PINK1 induction. Parkin distribution is followed by live imaging of N27 cells expressing WT PINK1 driven by an inducible promoter. (Scale bar, 10 μM.) (B) Percentages of N27 cells showing Parkin translocation to mitochondria at selected time points after PINK1 induction and Parkin-YFP transfection. Mitochondria are labeled by MitoTracker Red. **, Different from non-induced N27 cells (Newman-Keuls post hoc test; P < 0.001). (C) Western blot from total cell extracts showing PINK1 induction over time. Loading is normalized with β-actin. (D) Representative images illustrating the recruitment of Parkin to mitochondria at 24 h after induction of WT PINK1 but not after induction of pathogenic PINK1 L347P and W437× mutants. (E) Percentages of N27 cells showing Parkin translocation to mitochondria at 24 h after induction of WT or mutant PINK1. Values represent means ± SD (n = 50 cells) and are representative of 3 independent experiments. **, Different from empty vector induction controls (Newman–Keuls post hoc test; P < 0.001). (F) Western blot from total cell extracts showing that PINK1 expression levels were comparable in WT PINK1 and PINK1 mutants L347P and W437×. Loading is normalized with TIM23.

Fig. 4.

Fig. 4.

Mitochondrial depolarization stabilizes both the 63-kDa full-length and the 52-kDa cleaved PINK1. Western blot analysis of the cytosolic and mitochondrial fractions of HeLa cells incubated for 1 hr with vehicle (DMSO), 10 μM CCCP, 1 μM Antimycin A (AA), 1 μM Oligomycin (Oligo), or the combination of 1 μM Antimycin A plus 1 μM Oligomycin (AA+Oligo). Treatments that dissipate ΔΨm, such as CCCP and AA+O, increase the PINK1 content in the mitochondrial fraction.

Fig. 5.

Fig. 5.

PINK1 PD mutations mitigate the formation of perinuclear mitochondrial clusters. (A) Three types of mitochondrial network morphology are defined in Parkin- and PINK1-cotransfected cells: no cluster (i.e., normal mitochondrial tubular network and distribution); incomplete cluster (i.e., mixture of perinuclear clustered mitochondria and dispersed linear mitochondria); and complete cluster (i.e., all mitochondria are clustered at the perinuclear area). Cells cotransfected with WT Parkin and PINK1 show mainly complete clusters while cells cotransfected with WT Parkin and PINK1 disease mutations or artificial dead kinase mutation (K219M) show mainly incomplete or no clusters. Bars represent percentage of cells for each type of mitochondrial morphology ± SEM; n = 200 cells counted during 3 independent experiments. **, Different from PINK1/Mock (Newman-Keuls post hoc test; P < 0.01). (Scale bars, 5 μm.) (B) Western blot from total cell extracts showing that Parkin and PINK1 expression levels were comparable in WT PINK1 and PINK1 mutants L347P and W437×. Parkin is immunodetected using anti-myc antibody. Loading is normalized with GAPDH.

Fig. 6.

Fig. 6.

Perinuclear mitochondrial clusters undergo mitochondrial autophagy. (A_–_F) SH-SY5Y cells cotransfected with Parkin and PINK1 were fixed and processed for EM with anti-Parkin or anti-PINK1 immunostaining (A, B) or without immunostaining (C, D). (A, B) With HRP-labeled secondary antibodies, immuno-EM showed perinuclear mitochondrial clusters (arrows), and both Parkin and PINK1 localized to the periphery of both individual mitochondrion (Insets) and fused mitochondria, consistent with immunofluorescent data that showed their colocalization (this figure and

Fig. S5

). (C) In some clustered mitochondria, mitochondrial outer membranes of 2 opposite mitochondria disappeared or fused but their inner membranes remained intact. (D) The autophagic vacuoles (AV) that contain mitochondria (white arrowhead) were found in the perinuclear area. (E, F) A mixture of clustered mitochondria (white arrowhead), autophagic vacuoles (black arrowhead in E), lysosomes (white arrow in F), and other nontypical vacuoles in the autophage–lysosome pathway (black arrow in E) are identified at the perinuclear area. (Scale bars, 500 nm.) (G, H) Immunofluorescence of HeLa cells transiently cotransfected with Parkin-GFP and LC3-rFP incubated with vehicle DMSO or CCCP for 1 h before cell fixation. In the perinuclear area where mitochondrial clusters accumulate, Parkin-GFP colocalized with LC3-rFP (G) and with Lamp-2 (H). (Scale bars, 10 μm.) Zoom denotes a 6× magnification of the region outlined by the box in the CCCP images. (Scale bars, 1 μm.). ER, endoplasmic reticulum; Nuc, nucleus.

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