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Cited by 371Parkinson's Disease Mechanisms and TreatmentsAutophagy in Disease and TherapyMitochondrial Function and Pathology

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Parkinson's Disease–Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria

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Liu, Song, Sawada, Tomoyo, Lee, Seongsoo, Yu, Wendou, Silverio, George, Alapatt, Philomena, Millan, Ivan, Shen, Alice, Saxton, William, Kanao, Tomoko, Takahashi, Ryosuke, Hattori, Nobutaka, Imai, Yuzuru, Lu, Bingwei·2012·PLoS Genetics
DOI 10.1371/journal.pgen.1002537PMC PMC3291531
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Abstract

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.

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Figures

Figure 1 Genetic interaction between PINK1 and the mitochondrial transport machinery in the muscle. Effects of knockdown or overexpression of Miro, Milton or Khc on the abnormal wing posture (A), jump/fly ability (B), and thoracic ATP level (C) of PINK1B9 mutant are shown. Male flies of the corresponding genotypes were scored at 1-day or 7-day of age at 25°C. Miro-RNAi106683 was used for the rest of studies. Data are from three independent experiments and the results are presented as mean ± s.e.m.
Figure 1 Genetic interaction between PINK1 and the mitochondrial transport machinery in the muscle. Effects of knockdown or overexpression of Miro, Milton or Khc on the abnormal wing posture (A), jump/fly ability (B), and thoracic ATP level (C) of PINK1B9 mutant are shown. Male flies of the corresponding genotypes were scored at 1-day or 7-day of age at 25°C. Miro-RNAi106683 was used for the rest of studies. Data are from three independent experiments and the results are presented as mean ± s.e.m.CC BY · PubMed Central
Figure 2 Genetic interaction between PINK1 and Miro in DA neurons. (A–E) Mitochondrial morphology in DA neurons of different genotyped flies visualized with mitoGFP expressed under TH-Gal4 control. Red: TH, green: GFP. DA neurons of the protocerebral posterior lateral 1 (PPL1) cluster were shown. Scale bar: 5 µm. (F) Effects of Miro-RNAi and Miro-OE on DA neuron maintenance in wild type and PINK1B9 mutant backgrounds. DA neuron number was scored in 14-day old flies. At least 5 flies were used for each genotype. (G) Mitochondrial-size distribution in DA neurons of the various genotyped flies. Data are presented as mean ± s.e.m.
Figure 2 Genetic interaction between PINK1 and Miro in DA neurons. (A–E) Mitochondrial morphology in DA neurons of different genotyped flies visualized with mitoGFP expressed under TH-Gal4 control. Red: TH, green: GFP. DA neurons of the protocerebral posterior lateral 1 (PPL1) cluster were shown. Scale bar: 5 µm. (F) Effects of Miro-RNAi and Miro-OE on DA neuron maintenance in wild type and PINK1B9 mutant backgrounds. DA neuron number was scored in 14-day old flies. At least 5 flies were used for each genotype. (G) Mitochondrial-size distribution in DA neurons of the various genotyped flies. Data are presented as mean ± s.e.m.CC BY · PubMed Central
Figure 3 PINK1 regulates mitochondrial motility in Drosophila larval motor neurons. (A) An image of a third instar larva expressing mitoGFP in the motor neurons. For time-lapse imaging, mitochondrial movement in segmental nerves (SN) was recorded near the ventral ganglion (VG) (indicated by the white box). (B) Representative kymographs of mitochondrial movement in the different genetic backgrounds. Each panel is a kymograph representation of the position of fluorescently labeled mitochondria as a function of time. Anterograde mitochondrial movements have positive slopes, whereas retrograde mitochondrial movements have negative slopes. Stationary mitochondria appear as vertical streaks. (C, D) Mitochondrial flux (C) and net velocity (D) in different genetic backgrounds. (E, F) Average mitochondrial length (E) and mitochondrial length distribution (F) in the various genetic backgrounds. Data are presented as mean ± s.e.m.
Figure 3 PINK1 regulates mitochondrial motility in Drosophila larval motor neurons. (A) An image of a third instar larva expressing mitoGFP in the motor neurons. For time-lapse imaging, mitochondrial movement in segmental nerves (SN) was recorded near the ventral ganglion (VG) (indicated by the white box). (B) Representative kymographs of mitochondrial movement in the different genetic backgrounds. Each panel is a kymograph representation of the position of fluorescently labeled mitochondria as a function of time. Anterograde mitochondrial movements have positive slopes, whereas retrograde mitochondrial movements have negative slopes. Stationary mitochondria appear as vertical streaks. (C, D) Mitochondrial flux (C) and net velocity (D) in different genetic backgrounds. (E, F) Average mitochondrial length (E) and mitochondrial length distribution (F) in the various genetic backgrounds. Data are presented as mean ± s.e.m.CC BY · PubMed Central
Figure 4 PINK1 affects mitochondrial distribution within motor neuron nerve terminals at the Drosophila larval neuromuscular junction. Pan-neuronal elav-Gal4 driver was used to express mitoGFP in different genetic backgrounds to examine the mitochondrial distribution at the neuromuscular junction (NMJ) of muscle 6/7 in A3-A4 segments. Anti-GFP antibody (Green) and anti-HRP-Texas Red antibody (Red) were used to visualize mitochondria and boutons, respectively. (A) Representative images of mitochondrial distribution at the NMJs of wild type, PINK1-OE, PINK1-RNAi, Miro-RNAi and Miro-OE flies were shown in the left panels. Detailed mitochondrial distribution of the boxed areas in the left panels was shown in the right panels. (B) PINK1 regulates mitochondrial distribution at the NMJ. Mitochondrial density of each bouton was estimated by its average GFP fluorescence intensity, represented as total GFP green fluorescence (anti-GFP staining) of each bouton/bouton size (HRP-Texas Red staining). Mitochondrial density ratio of the most distal bouton versus the next 4 boutons was used to quantitatively compare mitochondrial distribution at the NMJ. Compared to the control, PINK1 knockdown showed significant accumulation of mitochondria at the most distal bouton, although its effect was not as strong as Miro-OE. In contrast, Miro-RNAi and PINK1-OE both led to reduced mitochondrial mass in the most distal boutons.
Figure 4 PINK1 affects mitochondrial distribution within motor neuron nerve terminals at the Drosophila larval neuromuscular junction. Pan-neuronal elav-Gal4 driver was used to express mitoGFP in different genetic backgrounds to examine the mitochondrial distribution at the neuromuscular junction (NMJ) of muscle 6/7 in A3-A4 segments. Anti-GFP antibody (Green) and anti-HRP-Texas Red antibody (Red) were used to visualize mitochondria and boutons, respectively. (A) Representative images of mitochondrial distribution at the NMJs of wild type, PINK1-OE, PINK1-RNAi, Miro-RNAi and Miro-OE flies were shown in the left panels. Detailed mitochondrial distribution of the boxed areas in the left panels was shown in the right panels. (B) PINK1 regulates mitochondrial distribution at the NMJ. Mitochondrial density of each bouton was estimated by its average GFP fluorescence intensity, represented as total GFP green fluorescence (anti-GFP staining) of each bouton/bouton size (HRP-Texas Red staining). Mitochondrial density ratio of the most distal bouton versus the next 4 boutons was used to quantitatively compare mitochondrial distribution at the NMJ. Compared to the control, PINK1 knockdown showed significant accumulation of mitochondria at the most distal bouton, although its effect was not as strong as Miro-OE. In contrast, Miro-RNAi and PINK1-OE both led to reduced mitochondrial mass in the most distal boutons.CC BY · PubMed Central
Figure 5 PINK1/Parkin regulates the ubiquitination and stability of Miro protein. (A) Effects of hPINK1 and hParkin on the ubiquitination and protein stability of exogenous hMiro1 in HeLa cells. Cells transfected with the indicated plasmids were treated with or without 10 µM CCCP for 90 min. Arrowheads, Parkin or PINK1 co-precipitated with hMiro1; Asterisks: oligo-ubiquitinated hMiro1. The inner mitochondrial membrane protein COX I and the mitochondrial matrix protein Hsp60 are used as loading controls for mitochondrial proteins. β-actin serves as a loading control for cytosolic proteins. (B) Effects of mutant hPINK1 or hParkin on hMiro1 protein level in HeLa cells. The Parkin K211N mutation in the linker domain is reported to lack both ubiqutin-ligase (E3) and mitochondrial translocation activities, while the Parkin T415 mutation in the RNIG2 domain, which disrupts E3 activity, retains partial mitochondrial translocation activity [56]. The PINK1 L347P mutant is reported to have reduced kinase activity [57]. An OMM protein VDAC1 is shown as a loading control for mitochondrial proteins. (C, D) Time-course experiments examining the effects of hPINK1/hParkin on the stability of endogenous hMiro1/2 proteins. Specificity of the hMiro antibodies was confirmed using hMiro knockdown cells (C). (E) Endogenous dMiro level in fly brain extracts of the various genetic backgrounds. elav-Gal4 was used to drive the expression of the transgenes. Normalized dMiro level compared to wild type control was shown as mean ± s.e.m. (n = 3).
Figure 5 PINK1/Parkin regulates the ubiquitination and stability of Miro protein. (A) Effects of hPINK1 and hParkin on the ubiquitination and protein stability of exogenous hMiro1 in HeLa cells. Cells transfected with the indicated plasmids were treated with or without 10 µM CCCP for 90 min. Arrowheads, Parkin or PINK1 co-precipitated with hMiro1; Asterisks: oligo-ubiquitinated hMiro1. The inner mitochondrial membrane protein COX I and the mitochondrial matrix protein Hsp60 are used as loading controls for mitochondrial proteins. β-actin serves as a loading control for cytosolic proteins. (B) Effects of mutant hPINK1 or hParkin on hMiro1 protein level in HeLa cells. The Parkin K211N mutation in the linker domain is reported to lack both ubiqutin-ligase (E3) and mitochondrial translocation activities, while the Parkin T415 mutation in the RNIG2 domain, which disrupts E3 activity, retains partial mitochondrial translocation activity [56]. The PINK1 L347P mutant is reported to have reduced kinase activity [57]. An OMM protein VDAC1 is shown as a loading control for mitochondrial proteins. (C, D) Time-course experiments examining the effects of hPINK1/hParkin on the stability of endogenous hMiro1/2 proteins. Specificity of the hMiro antibodies was confirmed using hMiro knockdown cells (C). (E) Endogenous dMiro level in fly brain extracts of the various genetic backgrounds. elav-Gal4 was used to drive the expression of the transgenes. Normalized dMiro level compared to wild type control was shown as mean ± s.e.m. (n = 3).CC BY · PubMed Central
Figure 6 Miro knockdown facilitates the removal of damaged mitochondria by Parkin-mediated mitophagy. (A) Time course of CCCP treatment to monitor mitophagy in Hela cells stably transfected with Venus-Parkin. Cells were transfected with control siRNA or hMiro1+hMiro2 siRNAs and treated with 10 µM CCCP for the indicated time. Markers for mitochondrial outer membrane (Tom20, VDAC1), intermembrane space (HtrA2/Omi, Cyt c), inner membrane (Tim23, NDUFA9), and matrix (PDHA1, Hsp60) were examined by Western blot to determine the elimination of damaged mitochondria. β-actin was used as a loading control. (B) Immunofluorescence staining showing the formation of perinuclear mitochondrial aggregates after siRNA-mediated knockdown of hMiro1 or hMiro2. HeLa cells transfected with control siRNA, hMiro1 siRNA, hMiro2 siRNA, or hMiro1+hMiro2 siRNAs were stained for Tom20 and α-tubulin. Merged images are shown to the right. Insets show enlarged view of mitochondrial morphology in the boxed areas. Arrowheads indicate ring-like or round-shaped mitochondria. (C) Miro knockdown facilitates the early stage of mitophagy in venus-Parkin transfected HeLa cells treated with CCCP. HeLa cells stably transfected with venus-Parkin were co-transfected with control siRNA or hMiro1+hMiro2 siRNAs and then treated with the DMSO vehicle or CCCP for 90 mins or 3 hrs. Venus-Parkin (blue), Tom20 (green), and Hsp60 (red) were visualized by immunostaining. Merged Tom20/Hsp60 signals are shown to the right. Cells of interest are outlined.
Figure 6 Miro knockdown facilitates the removal of damaged mitochondria by Parkin-mediated mitophagy. (A) Time course of CCCP treatment to monitor mitophagy in Hela cells stably transfected with Venus-Parkin. Cells were transfected with control siRNA or hMiro1+hMiro2 siRNAs and treated with 10 µM CCCP for the indicated time. Markers for mitochondrial outer membrane (Tom20, VDAC1), intermembrane space (HtrA2/Omi, Cyt c), inner membrane (Tim23, NDUFA9), and matrix (PDHA1, Hsp60) were examined by Western blot to determine the elimination of damaged mitochondria. β-actin was used as a loading control. (B) Immunofluorescence staining showing the formation of perinuclear mitochondrial aggregates after siRNA-mediated knockdown of hMiro1 or hMiro2. HeLa cells transfected with control siRNA, hMiro1 siRNA, hMiro2 siRNA, or hMiro1+hMiro2 siRNAs were stained for Tom20 and α-tubulin. Merged images are shown to the right. Insets show enlarged view of mitochondrial morphology in the boxed areas. Arrowheads indicate ring-like or round-shaped mitochondria. (C) Miro knockdown facilitates the early stage of mitophagy in venus-Parkin transfected HeLa cells treated with CCCP. HeLa cells stably transfected with venus-Parkin were co-transfected with control siRNA or hMiro1+hMiro2 siRNAs and then treated with the DMSO vehicle or CCCP for 90 mins or 3 hrs. Venus-Parkin (blue), Tom20 (green), and Hsp60 (red) were visualized by immunostaining. Merged Tom20/Hsp60 signals are shown to the right. Cells of interest are outlined.CC BY · PubMed Central
Figure 7 Effects of PINK1 loss of function on endogenous Miro protein level in mammalian cells and testing potential direct phosphorylation of Miro by PINK1. (A) Lack of direct phosphorylation of GST-dMiroΔTM by 2× GST-dPINK1. Autoradiography (P32) and Coomassie Brilliant Blue (CBB) staining of the same SDS-PAGE gel of the in vitro kinase reactions were shown. The presence of the GST-dMiroΔTM and 2× GST-dPINK1 proteins was further confirmed by Western blot with anti-GST. The detected P32 signals represent PINK1 autophosphorylation. (B) Western blot analysis comparing the stability of transfected Myc-hMiro1-wild type (wt) and Myc-hMiro1-S156A in HeLa cells with or without CCCP treatment and in the presence or absence of hPINK1+hParkin co-expression. (C) Western blot analysis of HeLa cells or Venus-Parkin stably transfected HeLa cells with or without CCCP treatment under control RNAi or PINK1 RNAi conditions. (D) Western blot analysis of mouse Miro1 and Miro2 levels in wild type or PINK1 knockout (KO) MEF cells. Extracts were treated with or without lambda phosphatase before Western blot analysis. Note that whereas the signals for a control phsopho-ATF1 (p-ATF1) protein disappeared after lambda phosphatase treatment, neither Miro1 nor Miro 2 showed mobility or intensity change after the same treatment.
Figure 7 Effects of PINK1 loss of function on endogenous Miro protein level in mammalian cells and testing potential direct phosphorylation of Miro by PINK1. (A) Lack of direct phosphorylation of GST-dMiroΔTM by 2× GST-dPINK1. Autoradiography (P32) and Coomassie Brilliant Blue (CBB) staining of the same SDS-PAGE gel of the in vitro kinase reactions were shown. The presence of the GST-dMiroΔTM and 2× GST-dPINK1 proteins was further confirmed by Western blot with anti-GST. The detected P32 signals represent PINK1 autophosphorylation. (B) Western blot analysis comparing the stability of transfected Myc-hMiro1-wild type (wt) and Myc-hMiro1-S156A in HeLa cells with or without CCCP treatment and in the presence or absence of hPINK1+hParkin co-expression. (C) Western blot analysis of HeLa cells or Venus-Parkin stably transfected HeLa cells with or without CCCP treatment under control RNAi or PINK1 RNAi conditions. (D) Western blot analysis of mouse Miro1 and Miro2 levels in wild type or PINK1 knockout (KO) MEF cells. Extracts were treated with or without lambda phosphatase before Western blot analysis. Note that whereas the signals for a control phsopho-ATF1 (p-ATF1) protein disappeared after lambda phosphatase treatment, neither Miro1 nor Miro 2 showed mobility or intensity change after the same treatment.CC BY · PubMed Central

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Metadata

Authors
Liu, S., Sawada, T., Lee, S., Yu, W., Silverio, G., Alapatt, P., Millan, I., Shen, A., Saxton, W., Kanao, T., Takahashi, R., Hattori, N., Imai, Y., & Lu, B.
Affiliations
  • Stanford Medicine (US)
  • Kyoto University (JP)
  • Washington University in St. Louis (US)
  • University of California, Santa Cruz (US)
  • Juntendo University (JP)
Corresponding author
  • Imai, Yuzuru
  • Lu, Bingwei
ORCID
Venue
PLoS Genetics
Publisher
Public Library of Science (PLoS)
Published
2012-03-01
Volume
8
Issue
3
Pages
e1002537
ISSN
  • 1553-7390 (ISSN-L)
  • 1553-7404
DOI
10.1371/journal.pgen.1002537
PMC
PMC3291531
OpenAlex
W2148819754
Cited by
371
References
59
Citations by year
2026: 11 · 2025: 10 · 2024: 10 · 2023: 11 · 2022: 18 · 2021: 35 · 2020: 29 · 2019: 22 · 2018: 28 · 2017: 30 · 2016: 36 · 2015: 32 · 2014: 42 · 2013: 33 · 2012: 23
FWCI
27.18
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Genetics (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Ministry of Education, Culture, Sports, Science and Technology (CREST)
  • Dainippon Sumitomo Pharma
  • Ministry of Health, Labour and Welfare
  • National Institutes of Health
  • Japan Society for the Promotion of Science (23390232, 23111002, 23890206, 23790973)
  • National Institute of Mental Health (R01MH080378)
  • National Institute of General Medical Sciences (R01GM084947, GM084947)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR054926)
  • Core Research for Evolutional Science and Technology
Topics
  • Health Sciences › Medicine › Clinical Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
Keywords
PINK1, Miro1, Miro2, Parkinson's disease, mitochondrial motility, Parkin, mitophagy, mitochondrial quality control, dopaminergic neurons, motor neurons, ubiquitin-mediated proteolysis, peripheral neuropathy, Drosophila
MeSH terms
PTEN-Induced Putative Kinase, Animals, Autophagy, Axonal Transport, Carbonyl Cyanide m-Chlorophenyl Hydrazone, Disease Models, Animal, Drosophila, Gene Expression Regulation, HeLa Cells, Humans, Mitochondria, Motor Neurons, Parkinson Disease, Protein Serine-Threonine Kinases, Mice, Knockout, rho GTP-Binding Proteins, Drosophila Proteins, Ubiquitin-Protein Ligases, Mutant Proteins, Mice, Dopaminergic Neurons, Proton Ionophores

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