Pyruvate stimulates mitophagy via PINK1 stabilization

CLS-08484; No of Pages 7 Cellular Signalling xxx (2015) xxx–xxx

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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig

Pyruvate stimulates mitophagy via PINK1 stabilization

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Sungwoo Park 1, Seon-Guk Choi 1, Seung-Min Yoo, Jihoon Nah, Eunil Jeong, Hyunjoo Kim, Yong-Keun Jung ⁎

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Global Research Laboratory, School of Biological Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-747, South Korea

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Article history: Received 16 February 2015 Received in revised form 13 May 2015 Accepted 20 May 2015 Available online xxxx

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Keywords: PDK4 Pyruvate Mitophagy PARK2 PINK1 LC3

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Damaged mitochondria are targeted for degradation by an autophagy pathway known as mitophagy. Despite efforts to unravel the mechanisms underlying mitophagy, aspects of mitophagy regulation remain largely unknown. In this study, by using a cell-based fluorescence assay reflecting CCCP-induced mitophagy, we have screened cDNA expression library encoding mitochondrial proteins and identified PDK4 as a mitophagy regulator. Ectopic expression of PDK4 stimulated the clearance of mitochondrial proteins during CCCP-induced mitophagy and enhanced pyruvate levels in both the cytosol and mitochondria. Interestingly, mitochondrial degradation during the mitophagy was not efficient in the absence of pyruvate. Pyruvate was required for PINK1 stabilization during mitochondrial depolarization and subsequent PARK2 translocation and LC3 recruitment onto damaged mitochondria. This pyruvate-mediated mitophagy was not affected by OXPHOS or cellular ATP levels, thus independent of energy metabolism. Rather, pyruvate was required for the interaction between PINK1 and TOMM20 under CCCP condition. These results suggest that pyruvate is required for CCCP-induced PINK1/PARK2-mediated mitophagy. © 2015 Published by Elsevier Inc.

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Depolarized or impaired mitochondria are cleared by autophagic machinery during a process known as mitophagy. Mitophagy is part of a mitochondrial quality control system that coordinates mitochondrial dynamics, biogenesis, fission, and fusion; mitophagy dysfunction is thus associated with human diseases [1]. Two recessive Parkinson's disease (PD) genes, PINK1/PARK6 and PARK2/parkin, are key regulators of mitophagy. Accumulation of PINK1 on the mitochondrial outer membrane (OM) and the subsequent translocation of PARK2 to damaged mitochondria induce mitophagy [2]. While there has been a great advance to understand the machinery and underlying mechanisms involved in mitophagy [3–5], identification of regulatory molecules in this process remains to be elucidated. Pyruvate is produced by the glycolysis pathway and serves as an important intermediary metabolite for the maintenance of cellular homeostasis by acting as a “fuel” for mitochondrial respiration [6,7]. The metabolic reaction of mitochondrial pyruvate is regulated by the pyruvate dehydrogenase complex (PDC), which catalyzes the oxidation of pyruvate to acetyl CoA in the mitochondrial matrix and is responsible for many age-associated diseases [8,9]. In addition to its well-known role in the bioenergetics of the mitochondrial citric acid cycle, the protective effect of pyruvate on ischemic damage has been extensively

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1. Introduction

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⁎ Corresponding author at: School of Biological Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-747, South Korea. Tel.: + 82 28804401; fax: + 82 2737524. E-mail address: [email protected] (Y.-K. Jung). 1 These authors contributed equally to this study.

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studied over the last two decades [10–13]. Interestingly, pyruvate's role in ischemic damage is attributed to its anti-oxidative function [14, 15], which also contributes to its role in cancer progression under hypoxic conditions [16] and to its ability to mitigate myocardial infarction [17,18]. PDC activity is governed by four dedicated kinases, pyruvate dehydrogenase kinase (PDK) 1, 2, 3, and 4 [19–22]. Among these isoforms, PDK4 negatively regulates PDC activity in response to changing nutrient levels [23–25]. In agreement with this function, loss of PDK4 in pdk4−/− mice lowers glucose level and ameliorates glucose tolerance in dietinduced obese mice [26]. To identify the novel modulators of mitophagy, we performed a functional screening by using a cDNA expression library and isolated PDK4. PDK4 stimulates carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondria degradation through pyruvate, thereby presenting pyruvate as an essential factor for mitophagy.

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2. Materials and methods

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2.1. Functional screening of mitophagy regulators

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HEK293T cells grown on multi-well culture plates were cotransfected with Mito-GFP and cDNA expression vectors for 24 h and then treated with 100 μM CCCP for 2 h. The intensities of GFP signals were measured using the ENVISION Multi-label Plate Reader (Perkin Elmer). The cDNA clones showing more than 10% reduction in GFP intensity compared to that in the control were first selected. The positive clones were further analyzed for their effects on mitophagy by western blotting using mitochondrial marker proteins.

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http://dx.doi.org/10.1016/j.cellsig.2015.05.020 0898-6568/© 2015 Published by Elsevier Inc.

Please cite this article as: S. Park, et al., Pyruvate stimulates mitophagy via PINK1 stabilization, Cell. Signal. (2015), http://dx.doi.org/10.1016/ j.cellsig.2015.05.020

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2.2. Cell lines and culture

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Cells were grown in DMEM (HyClone, SH30243.01) supplemented with 10% fetal bovine serum (FBS) (HyClone, SH30919.03) and 50 U/mL each of penicillin and streptomycin, and were incubated at 37 °C in an atmosphere containing 5% CO2. For experiments testing the effects of pyruvate, pyruvate-negative DMEM (SH30022) containing the same concentrations of FBS, penicillin, and streptomycin was used. For glutamine-induced mitophagy, Leibovitz L-15 Medium (HyClone, SH30525) was modified to contain 25 mM glucose with basal 2 mM glutamine (glucose group) or no glucose with 4 mM glutamine (glutamine group).

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2.8. Measurement of pyruvate, ATP, and mitochondrial complex I enzyme 137 activity 138 139

2.3. Plasmids and transfection

2.9. Trypan blue exclusion assay

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Cells were monocellized using trypsin, resuspended in PBS, and added to a volume of 0.4% Trypan blue solution (final concentration, 0.2%) for 10 min. Stained cells were counted microscopically using a hemocytometer.

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2.10. Statistical analysis

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PDK4-HA was produced by subcloning PDK4 cDNA into pcDNA3-HA (Invitrogen) and Flag-PARK2 and FLAG-PDK1, 2, 3 and 4 isoforms were subcloned into p3XFLAG-CMV (Sigma Aldrich). GFP-LC3 has been described previously [27] and PINK1-GFP was a kind gift from Dr. J. Chung (Seoul National University, Korea). DNA transfections were performed with PolyFect reagent (Qiagen, 1015586) for 24 h, according to the manufacturer's instructions.

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2.4. Western blotting and antibodies

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Western blotting was performed, as described previously [28]. After samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation, 66485), the membranes were blocked with 5% skim milk and incubated with the following antibodies: HA (Santa Cruz, sc-805), ACTB (sc-47778), TOMM20 (sc-17764), CANX (sc-11397), GFP (sc-8334), TUBA (sc-23948), CHDH (sc-102442), LC3 (Novus Biologicals, NB100-2220), TIMM23 (BD Bioscience, 611222), SOD2 (Upstate, 06-984), COX4I1 (Abcam, ab14744-100), PINK1 (Novus Biologicals, BC100-494; Santa Cruz, sc-33796) and FLAG (Sigma Aldrich, F3165). The blots were detected using the enhanced chemiluminescence (ECL) method.

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2.5. Transmission electron microscopy

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Cells were fixed with 3% paraformaldehyde and 0.5% glutaraldehyde in phosphate-buffered saline (PBS) at 4 °C for 2 h. After serial dehydration with 70%, 95%, and 100% ethanol for 30 min twice, London Resin White (Polyscience, 17411-500) was used for infiltration for 24 h. During that time, the resin was refreshed at 8 h intervals. Polymerization was performed overnight at 50 °C. Sectioned specimens were placed on the Formvar-coated nickel grid (Polyscience, 300 meshes, 24928) and stained with 2% uranyl acetate and Reynold's lead citrate for 7 min each. Samples were washed with distilled water and then visualized under a transmission electron microscope (JEOL, JEM1010) at 80 kV.

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2.6. Mitochondrial fractionation

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Mitochondrial fractionation by differential centrifugation was performed as described previously [28].

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2.7. Immunofluorescence microscopy and determination of colocalization coefficient

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Cells grown on a coverslip were fixed with 4% paraformaldehyde and permeabilized with 0.01% digitonin. After blocking with 10% fetal bovine serum, cells were incubated with primary antibodies at dilutions ranging from 1:100 to 1:250, and subsequently with an Alexa Fluorconjugated secondary antibody (Molecular Probes). Samples were visualized with a confocal laser scanning microscope (Carl Zeiss, LSM700).

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3. Results

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3.1. Functional screening of mitochondrial proteins identified PDK4 as a 155 mitophagy stimulator 156 To discover the novel factor(s) that regulate mitophagy, we established a cell-based assay and performed a gain-of-function screening by using 437 cDNAs encoding mitochondrial proteins, as described in the Materials and methods section. HEK293T cells were cotransfected with an individual cDNA and Mito-GFP, a mito-tracker [28], and were then exposed to a high dose of CCCP (100 μM) for a short time to induce acute depolarization and rapid degradation of the mitochondria (Fig. 1A). From the screening, we isolated five putative positive clones, which significantly reduced the fluorescence of MitoGFP relative to the levels of fluorescence in control cells (Fig. 1B). Among them, PDK4 appeared to have the strongest effect on the fluorescence of Mito-GFP. To further characterize the effects of PDK4 overexpression on mitophagy, we examined LC3-II conversion in PDK4-expressing cells. Ectopic expression of PDK4 in HEK293T cells increased LC3-II conversion per se after treatment with 10 μM CCCP, while no difference was observed in untreated cells (Fig. 2A). LC3-II conversion was further enhanced in the presence of the vacuolar-type H+-ATPase inhibitor bafilomycin A1. When we incubated HeLa cells with CCCP and performed immunocytochemical analysis of the mitochondria by using the mitochondrial TOMM20 antibody, PDK4 overexpression greatly accelerated PARK2-mediated mitochondrial clearance (Fig. 2B). Western blot analysis similarly indicated accelerated degradation of the mitochondrial proteins TIMM23 and TOMM20 owing to PDK4 overexpression, while the endoplasmic reticulum-bound protein CANX/calnexin and the cytosolic protein TUBA/tubulin were unaffected (Fig. 2C). Together, these results suggest that PDK4 functions to regulate CCCPinduced mitophagy.

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Quantitative data are represented as mean ± standard deviation. 151 Statistical significance was determined using Student's t-test and 152 P-value b 0.05 was deemed statistically significant. 153

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Pyruvate and ATP levels were determined by using the Pyruvate Colorimetric/Fluorometric Assay Kit (BioVision, K609-100) and the ENLITEN ATP Assay System Bioluminescence Detection Kit (Promega, FF2000), respectively. Mitochondrial complex I enzyme activity was measured using Complex I Enzyme Activity Microplate Assay Kit (Abcam, ab109721).

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ZEN software (Carl Zeiss) and the images were subjected to the analysis 135 of the colocalization coefficient. 136

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3.2. Increased pyruvate levels owing to PDK4 overexpression enhance 185 mitophagy 186 Because PDK4 is one of the pyruvate dehydrogenase kinases known 187 to inhibit PDC activity, we thought that PDK4-regulated pyruvate levels 188 might affect mitophagy. We found that PDK4 overexpression increased 189

Please cite this article as: S. Park, et al., Pyruvate stimulates mitophagy via PINK1 stabilization, Cell. Signal. (2015), http://dx.doi.org/10.1016/ j.cellsig.2015.05.020

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3.3. Pyruvate-mediated mitophagy is independent of energy metabolism

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treatment similarly increased the colocalization between Mito-RFP and GFP-LC3 for mitophagy (Fig. S2A). Further, among the PDK isoforms, PDK4 was the most effective in enhancing the mitophagy in both the transiently transfected cells (Fig. S2B) and stable cell lines (Fig. S2C), consistent with the results from their effects on cellular pyruvate levels. These results indicated that PDK4 might function through pyruvate to regulate mitophagy. We used western blotting to monitor mitophagy in the absence or presence of pyruvate (Fig. 3B). The mitochondrial proteins TOMM20, TIMM23, SOD2, COX4I1, and CHDH, but not the cellular proteins CANX and TUBA, were degraded by CCCP treatment, and their degradation was augmented by pyruvate treatment. Similar results were observed with confocal image analysis showing that the clearance of Mito-RFP was enhanced from 15.2% to 38.2% by pyruvate treatment (Fig. 3C). Furthermore, electron microcopy revealed extensive digestion of the mitochondria in the autophagic vesicles in the presence of pyruvate, while in the absence of pyruvate, the inner membrane (IM) structure of the mitochondria and fragmented mitochondria still remained in the vesicles (Fig. 3D, E). Collectively, these results suggest that pyruvate stimulates mitophagy via a mechanism that is dependent on the ability of PDK4 to increase pyruvate levels.

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pyruvate levels by 40% in both the mitochondrial and cytosolic fractions of HEK293T cells (Fig. S1), as previously reported [29]. Because our cDNA screening indicated that both PDK3 and PDK4 could affect mitophagy in the transfected cells (Fig. 1B), we more closely examined the effects of PDK family members on pyruvate levels. Among the members of the PDK family, the overexpression of PDK4 had a significant effect on pyruvate levels in both the mitochondrial and cytosolic fractions, while PDK3 had a modest effect (Fig. 3A). In contrast, PDK1 and PDK2 did not significantly affect pyruvate levels. Interestingly, the cellular levels of pyruvate owing to PDK4 overexpression were similar to the levels resulting from pyruvate treatment of untransfected cells (Fig. 3A, + Pyr), and these levels did not increase further when PDK4-overexpressing cells were treated with pyruvate (Fig. 3A, PDK4 + Pyr). In addition, PDK4 overexpression and pyruvate

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Fig. 1. Isolation of PDK4 as a mitophagy enhancer by functional screening. (A) Schematic diagram depicting the functional screening. A detailed description of the screening is provided in the Materials and methods section. (B) Genes showing high impact on the fluorescence of Mito-GFP are represented.

Since pyruvate is the major energy source for aerobic respiration and is related to cellular energetics, we considered the mechanism linking energy metabolism to mitophagy. We first wondered whether PDK4 overexpression and pyruvate treatment might regulate ATP production. The measurement of ATP levels revealed that PDK4 overexpression lowered ATP content in both the cytosol and mitochondria, while pyruvate treatment increased it (Fig. 4A). Because PDK4 and pyruvate both stimulate mitophagy, these results suggest that neither PDK4-mediated nor pyruvate-mediated mitophagy is associated with ATP content. Recently, Melser et al. reported that glutamine-induced oxidative phosphorylation activates mitophagy with a concomitant increase in ATP content [30]. Thus, we examined the association of oxidative phosphorylation with PDK4-mediated mitophagy. As previously reported, cells grown on glutamine show more degradation of mitochondrial proteins than do cells grown on glucose; however, the overexpression of PDK4 did not affect mitochondrial protein degradation (Fig. 4B). We used the TCA cycle intermediate citrate to assess the relationship between ATP and mitophagy further (Fig. 4C, D). As expected, supplementary citrate enhanced ATP levels (Fig. 4C). Different ATP contents by PDK4 overexpression and citrate treatment were then applied to the mitophagy assay using western blot analysis (Fig. 4D). Citrate treatment did not affect mitophagy compared to that observed in citrate-lacking controls, and that PDK4 expression

Fig. 2. Overexpression of PDK4 enhances mitophagy. (A) Western blot analysis of cell lysates prepared from mock (DMSO)- or CCCP-treated HEK293T cells overexpressing PDK4-HA, either in the presence or in the absence of bafilomycin A1 (Baf). The β-actin (ACTB) served as a lane-loading control. (B) HeLa cells were transfected with PARK2-GFP and either control vector (Ctrl) or PDK4. After the treatment with 20 μM CCCP for 6 h, cells were fixed and immunostained with TOMM20 antibody. TOMM20-negative and PARK2-GFP-positive cells (n N 100) were counted and depicted. *P b 0.001. (C) HeLa cells were transfected with PARK2-GFP and PDK4-HA, and then treated with 10 μM CCCP for 30 h. Cell lysates were analyzed with western blotting. The α-tubulin (TUBA) served as a loading control.

Please cite this article as: S. Park, et al., Pyruvate stimulates mitophagy via PINK1 stabilization, Cell. Signal. (2015), http://dx.doi.org/10.1016/ j.cellsig.2015.05.020

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Fig. 3. PDK4 increases mitochondrial pyruvate levels, thereby enhancing mitophagy. (A) HEK293T cells were transfected with PDK family members and were then left untreated or incubated with 1 mM pyruvate for 4.5 h. After cell lysates were fractionated into the mitochondria and the cytosol fractions, pyruvate levels in the fractions were determined as described in the Materials and methods section. *P b 0.01, **P b 0.05. (B) HepG2 cells were left untreated or treated with 1 mM pyruvate for 2 h and then exposed to 10 μM CCCP for 24 h. Cell lysates were subjected to western blot analysis. (C) HeLa-Mito-RFP stable cells were transfected with PARK2-GFP and incubated with 1 mM pyruvate for 30 min prior to the treatment with 10 μM CCCP for 24 h. Lack of RFP signal under a fluorescence microscope is indicative of cleared mitochondria. *P b 0.05, scale bar = 10 μm. (D, E) HepG2 cells were treated with 1 mM pyruvate for 30 min and subsequently treated with 10 μM CCCP for 2 h. Cells were observed under a transmission electron microscope. Scale bar = 1 μm (upper panel) and 200 nm (zoom-in lower panel) (D). The percentage of digested mitochondria in panel D was determined (E).

accelerated mitochondria clearance (Fig. 4D). These results indicate that PDK4–pyruvate axis functions independently of OXPHOS-ATP-mediated mitophagy.

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To examine the mechanism by which pyruvate stimulates mitophagy, we tested the hypothesis that pyruvate affects the interaction between mitochondrial BNIP3L/NIX and autophagosomal LC3. BNIP3L/NIX has previously been reported to mediate CCCP-induced mitophagy via its interaction with LC3 [31,32]. Using confocal microscopy, we found that pyruvate did not significantly affect the co-localization of GFP-BNIP3L/NIX and mRFP-LC3 (Fig. S3A). Further, pyruvate did not affect TMRE intensities reflecting mitochondrial membrane potential (Fig. S3B). Since CCCP-induced mitophagy is mediated by the accumulation of PINK1 on the OM of the mitochondria and the subsequent recruitment of PARK2, we next examined the effects of pyruvate on this

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machinery in HeLa cells. Surprisingly, we observed that CCCP induced the accumulation of PINK1 in the presence, but not in the absence, of pyruvate (Fig. 5A). This effect of pyruvate on PINK1 stabilization was also observed by confocal microscopy using Mito-RFP and PINK1GFP (Fig. 5B). These data suggest that pyruvate is required for the stabilization of PINK1. This requirement for pyruvate was highly specific as the three pyruvate analogs, bromopyruvate, α-ketobutyrate, and phenylpyruvate [33], were unable to stabilize PINK1 (Fig. S4A) and was observed in HeLa and HepG2 cells, but not in HEK293T cells (Fig. S4B). The current model of PINK1 processing states that PINK1 is cleaved by mitochondrial proteases, such as PARL, on the inner membrane (IM) of the mitochondria and then degraded by proteasomes [34,35]. To address what happened to PINK1 in the absence of pyruvate under CCCP treatment, we performed western blot analysis after the treatment with MG132, a proteasome inhibitor (Fig. 5C). The result showed that in CCCP-treated cells, in the absence of pyruvate, PINK1 was cleaved and degraded rather than being accumulated on the mitochondrial OM. The

Please cite this article as: S. Park, et al., Pyruvate stimulates mitophagy via PINK1 stabilization, Cell. Signal. (2015), http://dx.doi.org/10.1016/ j.cellsig.2015.05.020

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Fig. 4. Pyruvate-mediated mitophagy is not related to energy metabolism. (A) HEK293T cells were either transfected with PDK4 for 24 h or incubated with 1 mM pyruvate for 4.5 h. Cell extracts were prepared and separated into the cytosol and mitochondria fractions, followed by ATP assay, as described in the Materials and methods section. ATP contents relative to that in the control (Ctrl) are represented. (B) HeLa cells grown in either 25 mM glucose or 4 mM glutamine-containing media were transfected with PDK4-HA. After treatment with 20 μg/mL cycloheximide (CHX) for 8 h, cell extracts were prepared and analyzed by western blotting (upper). The signals on the blots were quantified (lower). (C) HEK293T cells were transfected with PDK4 and then left untreated or treated with 1 mM citrate for 2 h. ATP content in cell extracts was measured as described in (A). *P b 0.001, **P b 0.005. (D) HEK293T cells were transfected with PDK4 and then treated with 10 μM CCCP for 24 h in the presence or absence of 1 mM citrate. Cell lysates were analyzed by western blotting.

Fig. 5. Pyruvate induces PINK1 stabilization and PINK1-TOMM20 interaction. (A) HeLa cells were either left untreated or were incubated with 1 mM pyruvate for 2 h, followed by treatment with 10 μM CCCP for 2 h. Cell extracts were analyzed by western blotting. (B) HeLa cells were transfected with PINK1-GFP and Mito-RFP, and were treated with 1 mM pyruvate for 2 h before the addition of 10 μM CCCP for 3 h. Samples were fixed and analyzed by confocal microscopy. Scale bar = 10 μm. (C) HeLa cells were incubated with 1 mM pyruvate or 10 μM MG132 for 2 h and then exposed to 10 μM CCCP for 3 h. Mitochondria were then purified and analyzed by western blotting. (D) HeLa cells were pretreated with 1 mM pyruvate for 1 h and then treated with 10 μM CCCP for 3 h. Pure mitochondria were obtained and subjected to immunoprecipitation (IP) with TOMM20 antibody.

Please cite this article as: S. Park, et al., Pyruvate stimulates mitophagy via PINK1 stabilization, Cell. Signal. (2015), http://dx.doi.org/10.1016/ j.cellsig.2015.05.020

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Next, we examined the translocation of PARK2 to the depolarized mitochondria following PINK1 stabilization. Using the mitochondria prepared from HeLa cells overexpressing FLAG-PARK2, we found that PARK2 recruitment onto the mitochondria during CCCP condition increased by 4.6 fold when cells were treated with pyruvate (Fig. 6A). Furthermore, GFP-LC3 recruitment onto the mitochondria was also significantly increased by pyruvate, as determined by the co-localization coefficient calculated using confocal microscopy (Fig. 6B, C). These results suggest that pyruvate is required for the conventional PINK1/ PARK2 pathway in the mitophagy. However, pyruvate did not affect the co-localization of LC3 and gamma-aminobutyric acid receptorassociated protein (GABARAP), which was previously reported to play a role in the mitophagy like LC3 [31], to the mitochondria (Fig. S5A). Moreover, pyruvate did not affect the mitophagy mediated by choline dehydrogenase (CHDH), an important molecule for mitophagy [37] (Fig. S5B).

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In this report, PDK4, which is known to be upregulated under fasting 337 condition [40] and regulates AMPK and mTOR [41], is regarded as a 338 potential regulator of mitophagy that affects PINK1 level. Accordingly, 339

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To assess the pathophysiological relevance of the pyruvate requirement in mitophagy, we treated SN4741 mouse dopaminergic neuronal cells [38] with MPP+ (1-methyl-4-phenylpyridinium), an active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that causes PD-like symptoms [39]. As expected, western blot analysis suggested that like CCCP, the treatment with MPP+ induced PINK1 stabilization (Fig. S6A). Unlike the treatment with CCCP, however, MPP+ did not stimulate efficient degradation of the mitochondrial proteins COX4I1 and TIMM23 until 24 h. Using cycloheximide to block de novo protein synthesis, we found that pyruvate stimulated MPP+-induced degradation of mitochondrial proteins (Fig. S6B). Under this condition, MPP + impaired the enzymatic activity of mitochondrial complex I by approximately 30%. The effect of MPP + treatment was ameliorated by the addition of pyruvate, causing the impaired enzyme activity to recover by approximately 12% (Fig. S6C). The symptoms of PD are caused by dopaminergic neuronal cell death; therefore, we examined the effect of pyruvate on the survival of SN4741 cells (Fig. S6D). Pyruvate lowered MPP+-induced dopaminergic cell death from 16.3% to 8.3%, indicating that mitochondrial quality control by pyruvate might inhibit cell death. These results suggest that pyruvate is important in the mitochondrial quality control which might be associated with the mitophagy and the inhibition of dopaminergic cell death.

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cleavage and degradation were inhibited by MG132, resulting in the accumulation of the 52 kDa PINK1 fragment in the mitochondria. In contrast, in the presence of pyruvate, PINK1 was accumulated on the mitochondrial OM, as indicated by the detection of the full-length 64 kDa protein. This result further supports the necessity of pyruvate for PINK1 stabilization. Since PINK1 binds to TOMM complex during stabilization on the OM of the mitochondria under CCCP condition [36], we further addressed whether pyruvate affected their interaction. An immunoprecipitation analysis revealed that endogenous TOMM20 interacted with PINK1 only in the presence of pyruvate during mitochondrial depolarization (Fig. 5D), suggesting that pyruvate might regulate PINK1 stability through the interaction with TOMM20.

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Fig. 6. Pyruvate increases PARK2 translocation and LC3 recruitment onto the depolarized mitochondria. (A) HeLa cells were transfected with FLAG-PARK2 and incubated with 1 mM pyruvate for 2 h before the treatment with 10 μM CCCP for 2 h. Then, the mitochondrial fraction was prepared and analyzed by western blotting. The signal of mitochondrial FLAGPARK2 on the blot was quantified and represented as a fold change relative to that of whole cell lysate (WCL). (B, C) After being transfected with FLAG-PARK2, Mito-RFP, and GFP-LC3, HeLa cells were treated with 1 mM pyruvate for 2 h prior to the treatment with 10 μM CCCP for 4 h. Cells were fixed and observed under a confocal microscope (B). Colocalization coefficient was determined from the fluorescence images (C). *P b 0.005.

Please cite this article as: S. Park, et al., Pyruvate stimulates mitophagy via PINK1 stabilization, Cell. Signal. (2015), http://dx.doi.org/10.1016/ j.cellsig.2015.05.020

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Statement of conflict of interest

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bafilomycin A1 carbonyl cyanide m-chlorophenylhydrazone cycloheximide inner membrane 1-methyl-4-phenylpyridinium outer membrane oxidative phosphorylation Parkinson's disease pyruvate dehydrogenase complex pyruvate dehydrogenase kinase PTEN-induced putative kinase 1 translocase of outer mitochondrial membrane 20 homolog

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[5] [6] [7] [8]

[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]

[30] [31]

[32] [33] [34] [35] [36] [37] [38] [39] [40]

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The authors declare that they have no conflicts of interest.

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Acknowledgments

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Mr. SM Yoo was in part supported by the Brain Korea 21 program. This work was supported by the Global Research Laboratory (NRF-

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Supplementary data to this article can be found online at http://dx. 402 doi.org/10.1016/j.cellsig.2015.05.020. 403

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Baf CCCP CHX IM MPP+ OM OXPHOS PD PDC PDK PINK1 TOMM20

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Appendix A. Supplementary data

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2010-00341) and a CRI grant (NRF-2013R1A2A1A01016896) funded 399 by the Ministry of Education, Science and Technology. 400

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a genome-wide siRNA screening study showed a possibility that PDK4 knockdown affects mitophagy in response to CCCP treatment without knowledge on the regulatory mechanism [4]. Here, we provide a piece of evidence showing molecular function of PDK4 in the mitophagy. Then, the remaining important question is how pyruvate induces the accumulation of PINK1 on damaged mitochondria. In the absence of pyruvate, PINK1 was still imported into the IM of mitochondrion in cells exposed to CCCP and degraded over there by proteasome [34–36]. Consistent to a recent report that TOMM complexes are required for PINK1 stabilization by reciprocal interaction [36], we also found that pyruvate increases the interaction during CCCP-induced mitophagy. We speculate that pyruvate may play a role in the regulated interaction between PINK1 and TOMM complexes for mitophagy. Pyruvate-mediated mitophagy belongs to canonical PINK1/PARK2 pathway by affecting PARK2 translocation onto depolarized mitochondria and subsequent recruitment of LC3. However, the stimulatory effects of pyruvate on PINK1 accumulation seem to be cell-type specific. In contrast to HeLa and HepG2 cells, pyruvate did not affect PINK1 stabilization but still stimulated mitophagy in HEK293T in which PDK4 overexpression also enhanced CCCP-induced mitophagy. This discrepancy raises another possibility that factor(s) other than pyruvate or additional factors, such as metabolic status of the cells or the distinct mitophagic components across the different cell types, are also functional to mediate the PDK4-mediated mitophagy. Identification of such additional factors remains to be further addressed. When we addressed the pathologic relevance of pyruvate to the defective mitophagy in a model of PD and other human disorders [42], pyruvate seems to play a role to recover the mitochondrial complex enzyme activity and to suppress cell death of MPP+-induced dopaminergic neurons. Refer to a previous report that pyruvate reduces H2O2induced cell death [43], these cellular protective effects of pyruvate in dopaminergic neurons might be attributable to its stimulatory activity on the mitophagy which is known to selectively eliminate damaged mitochondria and thus lowers the mitochondria-associated oxidative damages. Thus, pyruvate provides new insight into restoring the defective mitophagy. Taken together, from the screening of mitochondrial proteins, we successfully isolated a mitophagy regulator, PDK4, which affects pyruvate levels to stabilize PINK1 to mediate CCCP- or MPP+-mediated mitophagy depending on cell types.

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[41] [42] [43]

Please cite this article as: S. Park, et al., Pyruvate stimulates mitophagy via PINK1 stabilization, Cell. Signal. (2015), http://dx.doi.org/10.1016/ j.cellsig.2015.05.020

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