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Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells

Nature Cell Biology volume 15, pages 1197–1205 (2013)Cite this article

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Abstract

Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased the delivery of mitochondria to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1 light chain 3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modelling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an ‘eat-me’ signal for the elimination of damaged mitochondria from neuronal cells.

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Figure 1: Rotenone-induced mitophagy.
Figure 2: Analysis of mitochondrial CL distribution and externalization.
Figure 3: RNAi knockdown of CL enzymes attenuates CL exposure and mitophagy.
Figure 4: Effects of rotenone, STS or 6-OHDA on beclin 1 cleavage and distribution of parkin and p62.
Figure 5: Mitochondrial recognition for mitophagy requires N-terminal amino acids of LC3.
Figure 6: Schematic diagram summarizing the proposed role of CL in mitochondrial autophagy.

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Acknowledgements

This study was supported in part by the National Institutes of Health (AG026389 (C.T.C.), NS065789 (C.T.C.), F32AG030821 (R.K.D.), NS061817 (H.B.), HL70755 (V.E.K.), OH008282 (V.E.K.), U19AIO68021 (H.B., V.E.K.), NS076511 (H.B., V.E.K.), ES020693 (V.E.K., Y.Y.T.)). We thank many generous scientists listed in the Methods for gifts of reagents, and D. E. Winnica for assistance with primary neuron cultures.

Author information

Author notes

  1. Ruben K. Dagda, Dariush Mohammadyani & Judith Klein-Seetharaman

    Present address: Present addresses: Department of Pharmacology, University of Nevada, Reno, Nevada 89557, USA (R.K.D.); Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA (D.M.); Division of Metabolic and Vascular Health, University of Warwick, Coventry CV4 7AL, UK (J.K-S.),

  2. Jing Ji, Ruben K. Dagda and Jian Fei Jiang: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Charleen T. Chu, Ruben K. Dagda, Kent Zhi Qiang Wang, Jianhui Zhu, Aaron M. Gusdon, Erin K. Steer & Ruth Wang

  2. Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Jing Ji, Jian Fei Jiang, Yulia Y. Tyurina, Alexandr A. Kapralov, Vladimir A. Tyurin, Krishnakumar Balasubramanian, Andrew A. Amoscato, Grigory Borisenko, Zhentai Huang, Amin Cheikhi, Hülya Bayır & Valerian E. Kagan

  3. Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Jing Ji, Jian Fei Jiang, Yulia Y. Tyurina, Alexandr A. Kapralov, Vladimir A. Tyurin, Krishnakumar Balasubramanian, Andrew A. Amoscato, Grigory Borisenko, Zhentai Huang, Amin Cheikhi, Hülya Bayır & Valerian E. Kagan

  4. Department of Critical Care Medicine, University of Pittsburgh and Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Jing Ji & Hülya Bayır

  5. Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Jing Ji & Hülya Bayır

  6. Department of Structural Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Naveena Yanamala, Dariush Mohammadyani & Judith Klein-Seetharaman

  7. Department of Computational and Systems Biology, University of Pittsburgh, Pennsylvania 15213, USA

    Indira H. Shrivastava & Ivet Bahar

  8. Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Catherine Baty & Simon Watkins

  9. Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

    Catherine Baty & Simon Watkins

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  1. Charleen T. Chu
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Contributions

J.J., R.K.D. and J.F.J. conducted and analysed experiments in the primary neuron, SH-SY5Y and HeLa cell systems, respectively. A.A.A., Y.Y.T. and V.A.T. collected and analysed the LC–MS data. K.Z.Q.W. contributed to LC3 mutagenesis and PLS3 knockdown studies. A.A.K performed the liposome studies. D.M., N.Y., J.K-S., I.H.S. and I.B. performed computational modelling. K.B., G.B. and Z.H. developed and optimized specific assays for the study. C.B. and S.W. performed specialized fluorescence microscopy. J.Z., A.M.G., E.K.S., R.W. and A.C. contributed to the PINK1, parkin, p62 and flux analyses. C.T.C., H.B. and V.E.K. planned and designed the study, analysed data, developed the conceptual model and wrote the manuscript. All authors discussed the results, and contributed to writing or commenting on the manuscript.

Corresponding authors

Correspondence to Charleen T. Chu, Yulia Y. Tyurina, Hülya Bayır or Valerian E. Kagan.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Additional characterization of Rot and STS-induced mitophagy.

Primary rat cortical neuron cultures were treated with rotenone at 7–9 days in vitro (DIV) using the indicated doses and times. Cell death was analyzed by flow cytometry of Annexin V/propidium iodide stained neurons to define sub-lethal conditions (a). N = 6 independent experiments; mean +/− s.d., *p<0.05 vs. vehicle. Equivalent results were observed for caspase-3/7 activation (not shown), assayed using the luminescent Promega Caspase-Glo assay (Madison, WI). Rotenone elicited increased GFP-LC3 puncta (b) and colocalization with mitochondria (c), the latter of which was inhibited by siRNA knockdown of CLS (siCLS), but not scrambled siCtrl. For b,c: N = 3; mean +/− s.d. *p<0.05 vs. vehicle-treated neurons; †p<0.05 vs. respective Rot-treated siCtrl neurons. Primary cortical neurons treated with vehicle or the indicated concentrations of rotenone were analyzed for p62 protein levels by immunoblot. Densitometry indicates significant decreases in p62 levels, consistent with dose-dependent increases in autophagic flux (d). N = 3; mean +/− s.d. *p<0.01 vs. vehicle; **p<0.01 vs. Rot 125 nM. Densitometry of mitochondrial proteins analyzed by immunoblot (See Fig. 1g) confirmed decreased protein levels, inhibited by co-treatment with bafilomycin (e). N = 3; mean +/− s.d., *p<0.05 vs. Ctrl; †p<0.05 vs. Rot alone. Primary neurons transfected on DIV 4 with siCLS or siCtrl were treated with STS on DIV 7 and analyzed for autophagy by LC3 shift (f) and GFP-LC3 puncta (g), and for mitophagy by colocalization analysis (h) or by loss of mitochondrial proteins (f). For g,h: N = 3; mean +/− s.d. *p<0.05 vs. respective vehicle-treated neurons; †p<0.05 vs. STS-treated siCtrl neurons. STS also elicited mitophagy in SH-SY5Y cells (i), as assessed by delivery of mitochondria to LTR-labeled lysosomes, which was inhibited by siRNA knockdown of the autophagy proteins ATG7 or LC3, as well as PLS3. *p<0.05 vs Veh; †p<0.05 vs STS/siCtrl.

Supplementary Figure 2 Mitochondrial isolation and characterization of phospholipid content and accessibility.

Primary cortical neuron cultures (DIV 7 or 8) were exposed to rotenone followed by isolation of mitochondrial IMM and OMM for LC-MS analysis. Immunoblot analysis employed the following (a): COXIV - IMM marker; TOM40 - OMM marker; pan Cadherin - cellular membrane marker; Histone H3 - nuclear marker. Western blot analysis for contact site IMM and OMM proteins ANT and VDAC, respectively, reveal no significant cross-contamination of fractions (b, Rot, 250 nM×2 h, representative of 3 experiments). Neuronal cells were treated with rotenone (250 nM) or STS (100 nM), and the IMM and OMM fractions prepared from isolated mitochondria were pooled from 3 experiments for lipid extraction and LC-MS analysis as described in Methods. Representative spectra for phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA) are shown for mitochondrial membranes isolated from control, rotenone- and STS-treated neurons are shown (c). In contrast to CL (Fig. 2a), only subtle changes in peak distribution, intensity, or cluster pattern of IMM and OMM PC, PE, PI and PA were observed. Isolated liver mitochondria labeled with the indicated phospholipids were mixed with Annexin V in the presence of the indicated Ca2+ concentrations. Annexin V binding was assessed by flow cytometry, indicating specificity for CL at lower Ca2+ concentrations (d). The highest concentration shown is equivalent to 300 μM. N = 4 experiments, mean +/− s.d. STS-treated HeLa cells exhibited increased surface accessibility of CL on isolated mitochondria probed with Annexin V (e). Inset: purity of membrane fractions. *p<0.05vs. control. N = 3 experiments, mean +/− s.d.

Supplementary Figure 3 Additional characterization of phospholipid scramblase 3 (PLS3) knockdowns.

Mitochondrial and cytosolic fractions from SH-SY5Y cells were immunoblotted for human scramblase-3 (hPLS3), and for the human mitochondrial antigen of 60 kDa (mito-P60) or the cytosolic lactate dehydrogenase enzyme (LDH) as compartment markers (a). SH-SY5Y cells were treated with two siRNAs targeting base pairs 433-453 (#433) or base pairs 508-532 (#508), and the extent of knockdown assessed by immunoblot densitometry (b). n = 6, ‡p<0.05vs. siCtrl. RNAi knockdown of PLS3 (siPLS3#508) blocks the Rot-induced colocalization of mitochondria with LTR-labeled lysosomes (c). *p<0.05 vs. Veh/siCtrl; †p<0.05 vs. Rot/siCtrl. Western blot analysis of the effects of siPLS3 on Rot-induced decreases in COX IV and MnSOD (d). Effects of PLS3 knockdown on basal and 6-OHDA-elicited GFP-LC3 puncta (e). *p<0.05 vs. respective vehicle treated cells. There were no significant effects of PLS3 knockdown on baseline or rotenone-treated mitochondrial membrane potentials (f). n = 8 wells/condition, representative of two independent experiments, no significant changes by ANOVA. The two siRNAs showed equivalent effects on mitophagy elicited by Rot (g)or by 6-OHDA (Fig. 3d). *p<0.05 vs. Veh/siCtrl; †p<0.05 vs. Rot/siCtrl.

Supplementary Figure 4 Additional characterization of cardiolipin synthase (CLS) knockdowns.

Treatment of primary cortical neurons with siCLS resulted in knockdown of rat CLS without affecting expression of other mitochondrial proteins (a). N = 3, mean +/− s.d. *p<0.05 vs. siCtrl. CLS knockdown had no effects on membrane potential measured by TMRM staining, and did not affect the small, but significant decrease elicited by rotenone (b). N = 3, *p<0.05 vs. Veh. CLS knockdown suppressed the loss of mitochondrial proteins elicited by rotenone as quantified by densitometry (c). N = 3; *p<0.05 vs. vehicle; †p<0.05 vs. respective Rot/siCtrl. See also Fig. 3g–i for representative blots and additional quantification. A second independent CLS siRNA (siCLS#2) was also effective in reducing CLS expression (d) and inhibiting the loss of mitochondrial proteins (e), with no effect on baseline or rotenone-treated membrane potentials (f). N = 3, *p<0.05 vs. Veh.

Supplementary Figure 5 The potential relationship of CL to the FCCP/CCCP-PINK1-Parkin pathway.

A previously characterized stable PINK1-3xFlag expressing SH-SY5Y line (#24, Reference 10), was treated with FCCP (2 μM×4 hrs) beginning at 72 h after transfection with siCtrl or siPLS3. Immunoblot analysis confirms PLS3 knockdown, which had no effect on the well-characterized FCCP-induced accumulation of full-length PINK1 (a). SH-SY5Y cells were co-transfected with HA-Parkin and siCtrl or siPLS3, treated with FCCP 72 h later, fixed in paraformaldehyde and immunolabeled for HA (green) and mitochondrial p60 antigen (red). Scale bar: 10 μm. FCCP elicited HA-Parkin puncta, all of which colocalized with mitochondrial p60 (b, arrows). There were no effects of siPLS3 on basal or FCCP-induced Parkin puncta (c), nor were there effects on FCCP-induced mitochondrial translocation of Parkin analyzed by immunoblot (not shown). *p ¡0.05 vs. respective Veh-treated cells. While PLS3 knockdown had no effect on FCCP-induced autophagosomes (d), it significantly reduced the degree of mitophagy elicited by FCCP (e). Overexpression of Drp1 did not rescue the inhibition of mitophagy by PLS knockdown (e), despite promoting significant further mitochondrial fragmentation (not shown). * p¡0.05 vs. respective Veh-treated cells; †p<0.05 vs. FCCP/siCtrl. Similarly, in Parkin-expressing HeLa cells, knockdown of CLS (siCLS #S29308) did not block the mitochondrial translocation of Parkin (f). Nevertheless, CCCP treatment did elicit higher levels of CL at the mitochondrial surface, as assessed using both the AnnexinV assay (g) and the PLA2 hydrolysis assay (h), which was inhibited by siCLS. *p<0.05 vs. Veh/siCtrl. †p<0.05 vs. CCCP/siCLS. CCCP treatment of Parkin-expressing HeLa cells resulted in the loss of mitochondrial proteins MnSOD and TOM40, while RNAi knockdown of CLS partially reduced this loss (i). Representative of 3 independent experiments. Effective knockdown of human CLS was demonstrated in Parkin-transfected HeLa cells at 72 h after siRNA treatment (j), which was associated with a ∼ 30% decrease in the levels of total CL in these cells (k).

Supplementary Figure 6 Dynamic modeling of LC3/CL interactions.

LC3 and three closest CL molecules in simulation MD1 are shown at four successive snapshots (a). Water, ions and POPC molecules are not shown in the snapshots for clarity. Note that LC3 enters the interaction range (less than 6 Å) of CL within 10 ns in all three simulations. Time evolution of LC3 position (b), monitored as minimum distances between head group atoms on CL and residues R68-R70 (black), R10-R11 (red), K42 (green) or K5 (blue) as observed in each of the three simulations (MD1-3). Magnified snapshots illustrate LC3/CL binding poses with the indicated residues at the end of 50 ns in each of the three simulations (c). For clarity, only those CL molecules which interact with LC3 are illustrated. The LC3 residues interacting with CL are highlighted in stick representation and labeled. The number of CL, PA and LysoCL interacting with LC3 is graphed with time during CGMD simulations (d). Insert snapshots illustrate the simulated interaction of LC3 with a PA-containing bilayer. Color guide: DOPC: yellow; head groups of PA: dark blue; acyl chain of PA: light blue; LC3: purple. CGMD simulation of the interaction of CL with LC3 for 1 μs (e). Snapshots illustrate the simulated interaction of LC3 with a CL-containing membrane using elastic network to preserve the protein structure (Refs. 9-10, SI Note). Note that water, ions and DOPC molecules are not shown for clarity. Color guide: head groups of CL: dark blue sticks, acyl chain of CL: light blue sticks, LC3: cyan (transparent), N-terminus of LC3 (K5, R10-R11): blue spheres, K49, R68-R69-R70: green spheres, Glu117: red sphere). Arrow indicates the C-terminal region involved in lipidation.

Supplementary Figure 7 Uncropped western blots.

Shown in this figure are all immunoblots from which the multiblot figure panels or insets for Figs 1f,g, 3e,g, 4b–d,f, and 5k were derived. Also shown are key uncropped immunoblots comprising panels in Supplementary Fig. S1,S2,S4, and S5.

Supplementary Table 1 The Rotenone-Treated Cortical Neuron OMM Exhibits a Similar Number and Distribution of CL Species as the IMM.
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Supplementary Table 2 Residues of LC3 interacting with CL as evidenced by Coarse-Grained Molecular Dynamics simulation.
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Supplementary Table 3 Mammalian LC3B aligned with Atg8 homologs from different species.
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Chu, C., Ji, J., Dagda, R. et al. Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nat Cell Biol 15, 1197–1205 (2013). https://doi.org/10.1038/ncb2837

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