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Loss of Mitochondrial Function Impairs Lysosomes*

  • Julie Demers-Lamarche
    Footnotes
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada

    Centre de recherche Biomed, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Gérald Guillebaud
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada

    Centre de recherche Biomed, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Mouna Tlili
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Kiran Todkar
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada

    Centre de recherche Biomed, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Noémie Bélanger
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Martine Grondin
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Angela P. Nguyen
    Affiliations
    Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa K1H 8M5, Canada
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  • Jennifer Michel
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Marc Germain
    Correspondence
    To whom correspondence should be addressed: Département de Biologie Médicale, Université du Québec à Trois-Rivières, 3351 Blvd. des Forges, Trois-Rivières, Québec G9A 5H7, Canada.
    Affiliations
    Groupe de Recherche en Signalisation Cellulaire, Département de Biologie Médicale, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada

    Centre de recherche Biomed, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada
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  • Author Footnotes
    * This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. The authors declare that they have no conflicts of interest with the contents of this article.
    1 Recipient of graduate scholarships from the Fonds de Recherche du Québec-Santé and the Natural Sciences and Engineering Research Council of Canada.
Open AccessPublished:March 17, 2016DOI:https://doi.org/10.1074/jbc.M115.695825
      Alterations in mitochondrial function, as observed in neurodegenerative diseases, lead to disrupted energy metabolism and production of damaging reactive oxygen species. Here, we demonstrate that mitochondrial dysfunction also disrupts the structure and function of lysosomes, the main degradation and recycling organelle. Specifically, inhibition of mitochondrial function, following deletion of the mitochondrial protein AIF, OPA1, or PINK1, as well as chemical inhibition of the electron transport chain, impaired lysosomal activity and caused the appearance of large lysosomal vacuoles. Importantly, our results show that lysosomal impairment is dependent on reactive oxygen species. Given that alterations in both mitochondrial function and lysosomal activity are key features of neurodegenerative diseases, this work provides important insights into the etiology of neurodegenerative diseases.

      Introduction

      A prominent feature of neurodegenerative diseases, including Parkinson disease (PD)
      The abbreviations used are: PD
      Parkinson disease
      AKO
      AIF knockout
      ETC
      electron transport chain
      NAM
      nicotinamide
      MDV
      mitochondria-derived vesicle
      MEF
      mouse embryonic fibroblast
      NAC
      N-acetylcysteine
      ROS
      reactive oxygen species
      AIF
      apoptosis-inducing factor
      Q10
      ubiquinone
      SOD
      superoxide dismutase
      mtHSP70
      mitochondrial HSP70
      DDC
      diethyldithiocarbamate.
      and Alzheimer disease, is the accumulation of undigested protein aggregates (
      • Ben-Gedalya T.
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      Quality control compartments coming of age.
      ,
      • Tai H.C.
      • Schuman E.M.
      Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction.
      ). Although the underlying mechanisms are complex, several cellular alterations can cause aggregate accumulation, including impaired quality control pathways and the generation of reactive oxygen species (ROS) as a consequence of mitochondrial damage (
      • Ben-Gedalya T.
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      Quality control compartments coming of age.
      ,
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      • Germain M.
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      Reactive oxygen species: stuck in the middle of neurodegeneration.
      ).
      In healthy cells, protein aggregates and damaged cellular components are delivered to lysosomes to be degraded through a process termed autophagy (
      • Ben-Gedalya T.
      • Cohen E.
      Quality control compartments coming of age.
      ,
      • Tai H.C.
      • Schuman E.M.
      Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction.
      ,
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      Emerging regulation and functions of autophagy.
      ,
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      ). As such, autophagy plays an important neuroprotective role. Nevertheless, the defects in degradation pathways observed in neurodegenerative diseases extend well beyond alterations in autophagy. Specifically, disruption of lysosomal function has been linked to neuronal loss in several neurodegenerative diseases. For example, mutations in the lysosomal ATPase ATP13A2 cause PD, whereas lysosomal dysfunction in Gaucher disease leads to Parkinsonism and the appearance of Lewy bodies (
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      ). In addition, the α-synuclein-containing Lewy bodies found in PD are positive for lysosomal markers, suggesting that they represent lysosomes that failed to degrade their content (
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      ). In fact, lysosomal alterations are a common feature of PD (
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      Lewy body-like α-synuclein aggregates resist degradation and impair macroautophagy.
      ). Nevertheless, the pathological consequences of a loss of lysosomal function extend well beyond PD, because a wide variety of mutations that impair lysosomes and cause the accumulation of intracellular material (lysosomal storage diseases) show features of neurodegeneration (
      • Cox T.M.
      • Cachón-González M.B.
      The cellular pathology of lysosomal diseases.
      ).
      A second key metabolic pathway required for neuronal survival is mitochondrial activity. In fact, mitochondrial dysfunction is a common feature of neurodegenerative diseases. For example, decreased activity of complex I of the electron transport chain (ETC) is present in a number of PD cases (
      • Keeney P.M.
      • Xie J.
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      Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.
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      ), whereas amyloid β, Tau tangles, and Htt aggregates all cause mitochondrial dysfunction (
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      Tau accumulation causes mitochondrial distribution deficits in neurons in a mouse model of tauopathy and in human Alzheimer's disease brain.
      ,
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      • Reddy A.P.
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      • Tagle D.A.
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      Abnormal mitochondrial dynamics, mitochondrial loss and mutant huntingtin oligomers in Huntington's disease: implications for selective neuronal damage.
      ,
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      • Su B.
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      • Fujioka H.
      • Wang Y.
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      Amyloid-β overproduction causes abnormal mitochondrial dynamics via differential modulation of mitochondrial fission/fusion proteins.
      ). In addition, several genes mutated in PD (including PINK1, Parkin, and DJ-1) affect mitochondrial function and turnover (
      • Greene A.W.
      • Grenier K.
      • Aguileta M.A.
      • Muise S.
      • Farazifard R.
      • Haque M.E.
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      • Fon E.A.
      Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment.
      ,
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      Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.
      ,
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      • Grailhe R.
      • Dawson T.M.
      • et al.
      PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.
      ,
      • Irrcher I.
      • Aleyasin H.
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      • Chhabra S.
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      • Rizzu P.
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      • et al.
      Loss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics.
      ), whereas deregulation of mitochondrial dynamics causes progressive neuronal loss in humans (
      • Alexander C.
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      OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28.
      ,
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      • et al.
      Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.
      ,
      • Züchner S.
      • Mersiyanova I.V.
      • Muglia M.
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      • Takahashi Y.
      • Tsuji S.
      • et al.
      Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A.
      ). At the cellular level, these alterations in mitochondrial structure and function lead to ROS-dependent cellular damage and decreased ATP production, all of which contribute to neuronal loss (
      • Patten D.A.
      • Germain M.
      • Kelly M.A.
      • Slack R.S.
      Reactive oxygen species: stuck in the middle of neurodegeneration.
      ,
      • Exner N.
      • Lutz A.K.
      • Haass C.
      • Winklhofer K.F.
      Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences.
      ,
      • Raimundo N.
      Mitochondrial pathology: stress signals from the energy factory.
      ). Importantly, mitochondrial dysfunction is often observed concomitantly with alterations in autophagic/lysosomal function in neurodegenerative diseases (reviewed in Ref.
      • Patten D.A.
      • Germain M.
      • Kelly M.A.
      • Slack R.S.
      Reactive oxygen species: stuck in the middle of neurodegeneration.
      ), suggesting a link between the two. Although this is supported by the observation that loss of mitochondrial function in yeast impairs starvation-induced autophagy (
      • Graef M.
      • Nunnari J.
      Mitochondria regulate autophagy by conserved signalling pathways.
      ), the extent and physiological importance of this interaction remain unclear.
      Here, we demonstrate that loss of mitochondrial function impairs lysosomes both in cells in culture and within the brain. Mitochondrial dysfunction induced by genetic ablation of mitochondrial proteins or chemical inhibition of the electron transport chain impaired lysosomal activity and caused the appearance of large lysosomal vacuoles. These were not the consequence of changes in mitochondrial dynamics but were mediated by a ROS-dependent mechanism. Altogether, these results indicate that mitochondria are required to maintain lysosomal function, providing a direct link between these two important aspects of neurodegenerative diseases.

      Experimental Procedures

      Cell culture reagents were obtained from Wisent. Other chemicals were purchased from Sigma-Aldrich, except where indicated.

      Animals

      All experiments were approved by the Université du Québec à Trois-Rivières Animal Care Ethics Committee, adhering to the Guidelines of the Canadian Council on Animal Care. As described previously (
      • Germain M.
      • Nguyen A.P.
      • Khacho M.
      • Patten D.A.
      • Screaton R.A.
      • Park D.S.
      • Slack R.S.
      LKB1-regulated adaptive mechanisms are essential for neuronal survival following mitochondrial dysfunction.
      ), the forebrain-specific AIF KO mice was generated by crossing floxed AIF mice (
      • Joza N.
      • Oudit G.Y.
      • Brown D.
      • Bénit P.
      • Kassiri Z.
      • Vahsen N.
      • Benoit L.
      • Patel M.M.
      • Nowikovsky K.
      • Vassault A.
      • Backx P.H.
      • Wada T.
      • Kroemer G.
      • Rustin P.
      • Penninger J.M.
      Muscle-specific loss of apoptosis-inducing factor leads to mitochondrial dysfunction, skeletal muscle atrophy, and dilated cardiomyopathy.
      ) with CamKIIα-Cre mice (
      • Casanova E.
      • Fehsenfeld S.
      • Mantamadiotis T.
      • Lemberger T.
      • Greiner E.
      • Stewart A.F.
      • Schütz G.
      A CamKIIα iCre BAC allows brain-specific gene inactivation.
      ). To minimize phenotypic variations due to the mixed background, the mice were kept on this FVBN/C57Bl/6 background for at least 6 generations before generating the experimental animals. In addition, littermates were used as controls for all experiments. Animals were genotyped according to standard protocols with previously published primers for AIF and Cre (
      • Germain M.
      • Nguyen A.P.
      • Khacho M.
      • Patten D.A.
      • Screaton R.A.
      • Park D.S.
      • Slack R.S.
      LKB1-regulated adaptive mechanisms are essential for neuronal survival following mitochondrial dysfunction.
      ).

      Tissue Processing and Immunohistochemistry

      Mice were euthanized with a lethal injection of sodium pentobarbital. For immunohistochemistry, mice were perfused with 1× PBS followed by fresh cold 4% paraformaldehyde. Brains were then removed, post-fixed overnight in 4% paraformaldehyde, cryoprotected in 20% sucrose in 1× PBS, and frozen. Sections were collected as 14-μm coronal cryosections on Superfrost Plus® slides (Fisher). For Western blot analysis, brains were removed, and cortices dissected and flash-frozen in liquid nitrogen.
      Brain sections were analyzed by immunohistochemistry using AlexaFluor and Cy3 secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Images were taken using Zeiss 510 meta and Leica confocal microscopes. Aggregates were autofluorescent when imaged using the 543-nm laser line and where imaged as such. All quantification was done blind on at least 4 sections/brain. Diameter measurements and colocalization studies were quantified using ImageJ.

      Cell Culture

      WT, PINK1 KO (gift from Dr. David Park, University of Ottawa), and OPA1 KO MEFs (gift from Dr. Luca Scorrano, University of Padua) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. OPA1 KO MEFs reconstituted with GFP, WT OPA1, or OPA1(Q297V) were generated as described (
      • Patten D.A.
      • Wong J.
      • Khacho M.
      • Soubannier V.
      • Mailloux R.J.
      • Pilon-Larose K.
      • MacLaurin J.G.
      • Park D.S.
      • McBride H.M.
      • Trinkle-Mulcahy L.
      • Harper M.E.
      • Germain M.
      • Slack R.S.
      OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.
      ). Primary cortical neuron cultures were done as described (
      • Germain M.
      • Nguyen A.P.
      • Le Grand J.N.
      • Arbour N.
      • Vanderluit J.L.
      • Park D.S.
      • Opferman J.T.
      • Slack R.S.
      MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner.
      ). Neurons were treated at day 4. Where indicated, cells were treated for 4 h with oligomycin (10 μm), antimycin A (50 μm), rotenone (5 μm), sodium azide (150 μm), DDC (50 μm) in the absence or the presence of the antioxidant N-acetylcysteine (NAC) (10 mm), ubiquinone (Q10) (Cayman; 25 μg/ml), or MitoQ (Focus Biomolecules; 100 μm). Nicotinamide (NAM) treatments (10 mm) were for 2 days. For H2O2 production, cells were incubated with 50 milliunits/ml glucose oxidase for 1 h. Lysosomal activity was inhibited by incubating cells in the presence of 200 nm bafilomycin for 1 h. Where indicated, cells were incubated for 1 h with Magic Red-RR (Immunochemistry Technologies), according to the manufacturer's instructions. For the dextran experiments, MEFs were incubated 2 h in the presence of 500 μg/ml Mr 10,000 Texas Red-conjugated dextran (Life Technologies, Inc.). Cells were then washed and chased for 30 min or 4 h with normal medium before fixing. In neurons, dextran was added at the time of treatment and cells were fixed immediately after treatment. For immunofluorescence, cells were grown directly on glass coverslips, treated as indicated, washed in PBS, and fixed for 10 min with 4% paraformaldehyde. NAD/NADH ratios were measured using a fluorescent NAD detection kit (Abcam).

      Live Cell Confocal Imaging

      MEF cells were first transfected with RFP-LAMP1 construction from Addgene (plasmid 1817) and then incubated with 1 μm Lysosensor Green DND-189 (Life Technologies) for 15 min at 37 °C. The cells were then washed three times in PBS and maintained in DMEM without phenol before imaging.

      Flow Cytometry

      To measure ROS, MEFs were incubated with 5 μm MitoSOXTM Red mitochondrial superoxide indicator (Life Technologies) for 20 min at 37 °C, after which fluorescence was measured at a wavelength of 610 nm using a Cytomics FC 500 system (Beckman Coulter). Lysosomal acidity was measured using Lysosensor Green DND-189. Cells were incubated with 1 μm Lysosensor for 30 min at 37 °C, and the fluorescence was measured at 525 nm. To measure the mitochondrial membrane potential, MEFs were incubated with 50 nm tetramethylrhodamine (Life Technologies) for 30 min at 37 °C, and fluorescence was measured at 610 nm. For each experiment, a total of 10,000 events were counted.

      Electron Microscopy

      Cell pellets were fixed in 0.4 m sodium cacodylate buffer and 8% glutaraldehyde (10 ml of buffer, 10 ml of glutaraldehyde, and 20 ml of H2O) at room temperature for 1 h. The pellets were then shipped to Mount Sinai Hospital for processing. Images were acquired using an EMS 208S electron microscope (Philips).

      Antibodies and Immunoblots

      The following antibodies were used: mouse anti-actin (Sigma-Aldrich); goat anti-Oct6, rabbit anti-SOD1, rat anti-LAMP1, goat anti-AIF, rabbit anti-TOM20, and rabbit anti-cathepsin B (Santa Cruz Biotechnology, Inc.); mouse anti-NDUFA9, mouse anti-Core2, and rabbit anti LAMP2, (Invitrogen); rabbit anti-Rab5, rabbit anti-Rab7, and rabbit anti-Rab11 (Cell Signaling Technologies); mouse anti-NeuN (Chemicon); mouse anti-mtHSP70 (ABR Bioreagents); and mouse anti-OPA1 (BD Biosciences).
      Tissue was lysed in 10 mm Tris-HCl, pH 7.9, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100 supplemented with protease inhibitor mixture (Sigma-Aldrich) and phosphatase inhibitors and Triton-insoluble material pelleted at 15,000 × g for 5 min. For immunoblot analysis, proteins were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the specified antibodies. Blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (Bio-Rad). Samples were quantified using ImageJ.

      In Vitro Lysosomal Assays and ATP Measurements

      To measure cathepsin B activity, 5-μg proteins were diluted in 100 μl of 100 mm HEPES, pH 6.0, 150 mm NaCl, 2 mm DTT, and 5 mm EDTA in the presence of a 5 μm concentration of the cathepsin B substrate zRR-AMC (Sigma-Aldrich). Samples were incubated for 30 min at 37 °C, after which fluorescence was measured (excitation/emission = 360/440 nm) using a Fluostar Optima (BMG Labtech). Acid phosphatase was measured using a fluorometric acid phosphatase assay kit (Abcam). 10 μg of proteins were diluted in the provided buffer in the presence of 0.5 mm substrate (4-methylumbelliferyl phosphate disodium salt) and incubated for 30 min at 25 °C. The reaction was then stopped with 20 μl of Stop solution, and fluorescence was measured (excitation/emission = 360/440 nm). Lysosomal acid lipase activity was measured as described (
      • Dairaku T.
      • Iwamoto T.
      • Nishimura M.
      • Endo M.
      • Ohashi T.
      • Eto Y.
      A practical fluorometric assay method to measure lysosomal acid lipase activity in dried blood spots for the screening of cholesteryl ester storage disease and Wolman disease.
      ) by diluting 10 μg of proteins in 100 μl of reaction buffer (100 mm sodium acetate, pH 4.0, 1% (v/v) Triton, and 0.5% (w/v) cardiolipin) in the presence of 0.345 mm 4-methylumbelliferone (Sigma-Aldrich). Samples were incubated for 1 h at 37 °C. The reaction was then stopped with 150 mm EDTA, pH 11.5, and fluorescence was measured (excitation/emission = 360/440 nm). ATP was measured in whole cells using the Cell Titer Glow kit (Promega). Cells were grown in 96-well plates, and ATP was measured in triplicate following the protocol provided. ATP levels were normalized to the total amount of proteins in replicate wells using the DC protein assay (Bio-Rad).

      Statistical Analysis

      Statistical differences were determined using Student's t test or one-way analysis of variance. p < 0.05 was considered statistically significant.

      Discussion

      A complex relationship exists between aggregate-prone proteins, mitochondrial dysfunction, and cellular degradation pathways within the context of neurodegenerative diseases. In some cases, mutations in aggregate-prone proteins (α-synuclein in PD, huntingtin in Huntington disease) cause their accumulation and are directly linked to neuronal loss (
      • Ben-Gedalya T.
      • Cohen E.
      Quality control compartments coming of age.
      ,
      • Tai H.C.
      • Schuman E.M.
      Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction.
      ). However, in most circumstances, aggregate accumulation is secondary to other neuronal defects that are not fully defined but are associated with impaired degradation pathways, mitochondrial dysfunction, and oxidative stress (
      • Ben-Gedalya T.
      • Cohen E.
      Quality control compartments coming of age.
      ,
      • Tai H.C.
      • Schuman E.M.
      Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction.
      ,
      • Patten D.A.
      • Germain M.
      • Kelly M.A.
      • Slack R.S.
      Reactive oxygen species: stuck in the middle of neurodegeneration.
      ,
      • Exner N.
      • Lutz A.K.
      • Haass C.
      • Winklhofer K.F.
      Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences.
      ). We show here that loss of mitochondrial activity disrupts lysosomal structure and function, leading to aggregate accumulation. Our results thus provide a direct link between mitochondrial dysfunction and alterations in protein degradation pathways.
      We used several different models of mitochondrial dysfunction (AIF KO neurons, PINK1 KO MEFs, OPA1 KO MEFs, and ETC inhibition in MEFs and primary neurons) to define the role of mitochondria in the maintenance of lysosomes. Disruption of mitochondrial function inhibited lysosomal activity and triggered the accumulation of large vacuoles positive for lysosomal markers in every model examined, demonstrating that mitochondria are required to maintain proper lysosomal function.
      Loss of mitochondrial function is associated with mitochondrial fragmentation, decreased mitochondrial ATP, and increased ROS production (
      • Patten D.A.
      • Germain M.
      • Kelly M.A.
      • Slack R.S.
      Reactive oxygen species: stuck in the middle of neurodegeneration.
      ,
      • Raimundo N.
      Mitochondrial pathology: stress signals from the energy factory.
      ,
      • Mailloux R.J.
      • McBride S.L.
      • Harper M.E.
      Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics.
      ), each potentially affecting lysosomes. However, the fact that fusion-incompetent OPA1(Q297V) (
      • Patten D.A.
      • Wong J.
      • Khacho M.
      • Soubannier V.
      • Mailloux R.J.
      • Pilon-Larose K.
      • MacLaurin J.G.
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      ). However, we could not replicate this rescue in OPA1 KO MEFs. Because OPA1 interacts with several of the transporters required to shuttle metabolites across the mitochondrial inner membrane (
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      OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.
      ), other metabolites could be involved. Still, the accumulation of aggregates within AKO neurons with defective lysosomes highlights the crucial relationship between mitochondrial dysfunction, ROS, and impaired lysosomes.
      Mitochondrial dysfunction is a key feature of neurodegenerative diseases, including PD, Alzheimer disease, and Huntington disease (reviewed in Refs.
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      ). Similarly, impaired autophagy/lysosomes cause the accumulation of defective mitochondria in several models of neurodegenerative diseases (
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      • Perier C.
      • Recasens A.
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      ), and mutations in lysosomal proteins lead to neurodegeneration (
      • Osellame L.D.
      • Rahim A.A.
      • Hargreaves I.P.
      • Gegg M.E.
      • Richard-Londt A.
      • Brandner S.
      • Waddington S.N.
      • Schapira A.H.
      • Duchen M.R.
      Mitochondria and quality control defects in a mouse model of Gaucher disease: links to Parkinson's disease.
      ,
      • Schultheis P.J.
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      • Clippinger A.K.
      • Lewis J.
      • Tsunemi T.
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      • Chava A.K.
      • Howard J.
      • Gannon M.
      • Hoffman E.
      • et al.
      Atp13a2-deficient mice exhibit neuronal ceroid lipofuscinosis, limited α-synuclein accumulation and age-dependent sensorimotor deficits.
      ,
      • Cox T.M.
      • Cachón-González M.B.
      The cellular pathology of lysosomal diseases.
      ). For example, mutations in lysosomal hydrolases lead to the accumulation of intracellular material and neurodegeneration (
      • Cox T.M.
      • Cachón-González M.B.
      The cellular pathology of lysosomal diseases.
      ), a group of diseases collectively referred to as lysosomal storage diseases.
      On the other hand, although impaired lysosomal activity clearly leads to neurodegeneration, mitochondrial dysfunction and downstream oxidative stress also play a major role. In fact, mitochondrial dysfunction caused by mutations in mitochondrial proteins is the primary trigger of neuronal loss in several examples of neurodegeneration (
      • Finsterer J.
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      ). In addition, mutations in the mitochondrial quality control proteins PINK1 and Parkin cause the accumulation of defective mitochondria, leading to PD in humans (
      • Gautier C.A.
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      ). Although these mutations account for only a small subset of PD patients, sporadic cases of PD also generally show alterations in mitochondrial function, usually decreased complex I activity (
      • Exner N.
      • Lutz A.K.
      • Haass C.
      • Winklhofer K.F.
      Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences.
      ,
      • Morán M.
      • Moreno-Lastres D.
      • Marín-Buera L.
      • Arenas J.
      • Martín M.A.
      • Ugalde C.
      Mitochondrial respiratory chain dysfunction: implications in neurodegeneration.
      ). This possibly occurs through aging-related loss of mitochondrial fitness or exposure to complex I toxins, such as rotenone (
      • Exner N.
      • Lutz A.K.
      • Haass C.
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      Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences.
      ), all of which increase ROS-mediated damage and accumulation of protein aggregates (
      • Keeney P.M.
      • Xie J.
      • Capaldi R.A.
      • Bennett Jr., J.P.
      Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.
      ,
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      ,
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      ,
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      ,
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      • Negro A.
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      ).
      ROS play complex roles in cellular physiology and pathology, including neurodegenerative diseases. For example, ROS-mediated neuronal damage has been observed in PD, Alzheimer disease, and ALS (
      • Patten D.A.
      • Germain M.
      • Kelly M.A.
      • Slack R.S.
      Reactive oxygen species: stuck in the middle of neurodegeneration.
      ,
      • Morán M.
      • Moreno-Lastres D.
      • Marín-Buera L.
      • Arenas J.
      • Martín M.A.
      • Ugalde C.
      Mitochondrial respiratory chain dysfunction: implications in neurodegeneration.
      ). Consistent with the deleterious effects of oxidative stress, a large surge in ROS causes rapid cell death that can be associated with lysosomal membrane permeabilization. For example, neuronal death caused by the parkinsonian toxin MPP+ is associated with lysosomal depletion (
      • Dehay B.
      • Bové J.
      • Rodríguez-Muela N.
      • Perier C.
      • Recasens A.
      • Boya P.
      • Vila M.
      Pathogenic lysosomal depletion in Parkinson's disease.
      ), possibly as a result of Fenton-like reactions involving hydrogen peroxide within lysosomes (
      • Dodson M.
      • Darley-Usmar V.
      • Zhang J.
      Cellular metabolic and autophagic pathways: traffic control by redox signaling.
      ). In contrast, the lysosomal impairment that we observed in our models of mitochondrial dysfunction was not associated with a loss of structural proteins, such as LAMP1. This suggests that milder oxidative stress impairs lysosomes through mechanisms that are distinct from acute ROS exposure. Because oxidative damage and neuronal death accumulate over many years in human patients (
      • Exner N.
      • Lutz A.K.
      • Haass C.
      • Winklhofer K.F.
      Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences.
      ,
      • Gustaw-Rothenberg K.
      • Lerner A.
      • Bonda D.J.
      • Lee H.G.
      • Zhu X.
      • Perry G.
      • Smith M.A.
      Biomarkers in Alzheimer's disease: past, present and future.
      ,
      • Pagano G.
      • Talamanca A.A.
      • Castello G.
      • Cordero M.D.
      • d'Ischia M.
      • Gadaleta M.N.
      • Pallardó F.V.
      • Petrović S.
      • Tiano L.
      • Zatterale A.
      Oxidative stress and mitochondrial dysfunction across broad-ranging pathologies: toward mitochondria-targeted clinical strategies.
      ), this could be highly relevant to the long term changes occurring in neurons affected by neurodegenerative diseases. In that context, it is noteworthy that AIF KO neurons, which show increased oxidative damage (
      • Klein J.A.
      • Longo-Guess C.M.
      • Rossmann M.P.
      • Seburn K.L.
      • Hurd R.E.
      • Frankel W.N.
      • Bronson R.T.
      • Ackerman S.L.
      The harlequin mouse mutation downregulates apoptosis-inducing factor.
      ), had decreased lysosomal activity and accumulation of LAMP1 vacuoles over time but had limited neuronal death at 3 months (
      • Germain M.
      • Nguyen A.P.
      • Khacho M.
      • Patten D.A.
      • Screaton R.A.
      • Park D.S.
      • Slack R.S.
      LKB1-regulated adaptive mechanisms are essential for neuronal survival following mitochondrial dysfunction.
      ) and normal LAMP1 levels. This contrasts with the acute oxidative stress caused in mice by MPTP injections, which lead to rapid neuronal loss and loss of lysosomal content (
      • Dehay B.
      • Bové J.
      • Rodríguez-Muela N.
      • Perier C.
      • Recasens A.
      • Boya P.
      • Vila M.
      Pathogenic lysosomal depletion in Parkinson's disease.
      ).
      Altogether, our results indicate that loss of mitochondrial function impairs lysosomes. Because alterations in mitochondrial function and lysosomal activity are key features of neurodegenerative diseases, our work provides important insights into our understanding of the etiology of neurodegenerative diseases.

      Author Contributions

      J. D. L., A. P. N., and M. Germain designed research; J. D. L., G. G., M. T., K. T., N. B., A. P. N., M. Grondin, and M. Germain performed research; J. D. L., M. T., J. M., and M. Germain analyzed data; M. Germain wrote the paper.

      Acknowledgments

      We thank Linda Jiu for tissue preparation, Ujval Anilkumar and Jason MacLaurin for help with neuronal cultures, Agnes Lejeune for EM imaging, and Valerie Gervais for help with the MDV experiments. We also thank Ruth Slack, David Patten, and Mireille Khacho for insightful comments on the manuscript.

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