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Targeting mitophagy in Parkinson's disease

Open AccessPublished:December 23, 2020DOI:https://doi.org/10.1074/jbc.REV120.014294
      The genetics and pathophysiology of Parkinson’s disease (PD) strongly implicate mitochondria in disease aetiology. Elegant studies over the last two decades have elucidated complex molecular signaling governing the identification and removal of dysfunctional mitochondria from the cell, a process of mitochondrial quality control known as mitophagy. Mitochondrial deficits and specifically reduced mitophagy are evident in both sporadic and familial PD. Mendelian genetics attributes loss-of-function mutations in key mitophagy regulators PINK1 and Parkin to early-onset PD. Pharmacologically enhancing mitophagy and accelerating the removal of damaged mitochondria are of interest for developing a disease-modifying PD therapeutic. However, despite significant understanding of both PINK1-Parkin-dependent and -independent mitochondrial quality control pathways, the therapeutic potential of targeting mitophagy remains to be fully explored. Here, we provide a summary of the genetic evidence supporting the role for mitophagy failure as a pathogenic mechanism in PD. We assess the tractability of mitophagy pathways and prospects for drug discovery and consider intervention points for mitophagy enhancement. We explore the numerous hit molecules beginning to emerge from high-content/high-throughput screening as well as the biochemical and phenotypic assays that enabled these screens. The chemical and biological properties of these reference compounds suggest many could be used to interrogate and perturb mitochondrial biology to validate promising drug targets. Finally, we address key considerations and challenges in achieving preclinical proof-of-concept, including in vivo mitophagy reporter methodologies and disease models, as well as patient stratification and biomarker development for mitochondrial forms of the disease.

      Keywords

      Abbreviations:

      ADP (adenosine diphosphate), AMP (adenosine monophosphate), ATP (adenosine triphosphate), cGMP (cyclic guanosine monophosphate), DA (dopaminergic), DAT (dopamine transporter), DUBs (deubiquitinases), ETC (electron transport chain), GBA (glucocerebrosidase), GFP (green fluorescent protein), HTS (high-throughput screen), IMM (inner mitochondrial membrane), IMS (intermembrane space), KR (kinetin riboside), MDVs (mitochondrially derived vesicles), MPP (mitochondrial processing peptidase), MTS (mitochondrial targeting sequence), NAD (nicotinamide adenine dinucleotide), PBMCs (peripheral blood mononuclear cells), PD (Parkinson’s disease), PET (positron emission tomography), PTMs (posttranslational modifications), SPECT (single-photon emission computed tomography), SNc (substantia nigra pars compacta), UA (Urolithin A)

      Mitochondria and Parkinson’s disease

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      Figure thumbnail gr1
      Figure 1Human genetics link mitochondria to PD. Multiple genes associated with PD play a role in aspects of mitochondrial homeostasis. LRRK2 mutations affect ER-Mitochondria tethering and Ca2+ homeostasis. α-synuclein interacts with the TOM complex, affecting mitochondrial import. Mutations in VPS35 increase mitochondrial fragmentation, while mutations in DJ-1 or CHCHD2 are associated with increased ROS production. PINK1, FBXO7, and Parkin mutations cause defective mitophagy and ATP13A2 mutations alter lysosomal function.
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      Indirect effects on mitochondria are also consequence of PD-causing mutations in genes regulating lysosomal function and the antioxidant response. Mutations in lysosomal P5 type ATPase cation transporter, ATP13A2 (encoded by PARK9), which cause autosomal recessive parkinsonism (Kufor–Rakeb syndrome), produce severe mitochondrial fragmentation and mtDNA lesions in fibroblasts, potentially due to cellular zinc dyshomeostasis (
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      • Zampese E.
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      • Jeon S.
      • Santos D.P.
      • Blanz J.
      • Obermaier C.D.
      • Strojny C.
      • Savas J.N.
      • Kiskinis E.
      • Zhuang X.
      • Kruger R.
      • Surmeier D.J.
      • et al.
      Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson's disease.
      ) and have low ETC complex I expression (
      • Pettus E.H.
      • Betarbet R.
      • Cottrell B.
      • Wallace D.C.
      • Madyastha V.
      • Greenamyre J.T.
      Immunocytochemical characterization of the mitochondrially encoded ND1 subunit of complex I (NADH : ubiquinone oxidoreductase) in rat brain.
      ). DA neurons are particularly reliant on L-type Cav1.3 Ca2+ channels to facilitate continuous rhythmic pacemaking activity and therefore subject to potentially damaging effects of large Ca2+ transients and associated oxidative stress (
      • Chan C.S.
      • Guzman J.N.
      • Ilijic E.
      • Mercer J.N.
      • Rick C.
      • Tkatch T.
      • Meredith G.E.
      • Surmeier D.J.
      Rejuvenation' protects neurons in mouse models of Parkinson's disease.
      ,
      • Guzman J.N.
      • Sanchez-Padilla J.
      • Wokosin D.
      • Kondapalli J.
      • Ilijic E.
      • Schumacker P.T.
      • Surmeier D.J.
      Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1.
      ,
      • Guzman J.N.
      • Ilijic E.
      • Yang B.
      • Sanchez-Padilla J.
      • Wokosin D.
      • Galtieri D.
      • Kondapalli J.
      • Schumacker P.T.
      • Surmeier D.J.
      Systemic isradipine treatment diminishes calcium-dependent mitochondrial oxidant stress.
      ). Accordingly, any insult leading to even modest mitochondrial impairment is particularly neurotoxic to DA neuron populations.
      Beyond genetically determined disease, mitochondrial dysfunction and reduced mitophagy are also observed in sporadic PD (
      • Bose A.
      • Beal M.F.
      Mitochondrial dysfunction in Parkinson's disease.
      ,
      • Luo Y.
      • Hoffer A.
      • Hoffer B.
      • Qi X.
      Mitochondria: a therapeutic target for Parkinson's disease?.
      ,
      • Hsieh C.H.
      • Shaltouki A.
      • Gonzalez A.E.
      • Bettencourt da Cruz A.
      • Burbulla L.F.
      • St Lawrence E.
      • Schule B.
      • Krainc D.
      • Palmer T.D.
      • Wang X.
      Functional impairment in Miro degradation and mitophagy is a shared feature in familial and sporadic Parkinson's disease.
      ,
      • Keeney P.M.
      • Xie J.
      • Capaldi R.A.
      • Bennett J.P.
      Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.
      ,
      • Ryan B.J.
      • Hoek S.
      • Fon E.A.
      • Wade-Martins R.
      Mitochondrial dysfunction and mitophagy in Parkinson's: from familial to sporadic disease.
      ,
      • Requejo-Aguilar R.
      • Bolanos J.P.
      Mitochondrial control of cell bioenergetics in Parkinson's disease.
      ). Oxidative stress and bioenergetic compromise are recognized phenotypes of PD in vivo and in vitro (
      • Keeney P.M.
      • Xie J.
      • Capaldi R.A.
      • Bennett J.P.
      Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.
      ,
      • Ryan B.J.
      • Hoek S.
      • Fon E.A.
      • Wade-Martins R.
      Mitochondrial dysfunction and mitophagy in Parkinson's: from familial to sporadic disease.
      ,
      • Requejo-Aguilar R.
      • Bolanos J.P.
      Mitochondrial control of cell bioenergetics in Parkinson's disease.
      ,
      • Briston T.
      • Hicks A.R.
      Mitochondrial dysfunction and neurodegenerative proteinopathies: mechanisms and prospects for therapeutic intervention.
      ,
      • Krige D.
      • Carroll M.T.
      • Cooper J.M.
      • Marsden C.D.
      • Schapira A.H.V.
      Platelet mitochondria function in Parkinson's disease.
      ). Mitochondrial electron transport chain (ETC) complex I deficiency and increased frequency of mtDNA mutations have been identified in sporadic PD patients (
      • Krige D.
      • Carroll M.T.
      • Cooper J.M.
      • Marsden C.D.
      • Schapira A.H.V.
      Platelet mitochondria function in Parkinson's disease.
      ,
      • Antony P.M.A.
      • Boyd O.
      • Trefois C.
      • Ammerlaan W.
      • Ostaszewski M.
      • Baumuratov A.S.
      • Longhino L.
      • Antunes L.
      • Koopman W.
      • Balling R.
      • Diederich N.J.
      Platelet mitochondrial membrane potential in Parkinson's disease.
      ), and delayed mitophagy following mitochondrial uncoupling was reported in PD patient cells (
      • Hsieh C.H.
      • Shaltouki A.
      • Gonzalez A.E.
      • Bettencourt da Cruz A.
      • Burbulla L.F.
      • St Lawrence E.
      • Schule B.
      • Krainc D.
      • Palmer T.D.
      • Wang X.
      Functional impairment in Miro degradation and mitophagy is a shared feature in familial and sporadic Parkinson's disease.
      ).

      PINK1 and Parkin

      The association between mutations in PINK1 and Parkin and the development of EOPD suggest that defective mitophagy and accumulation of damaged mitochondria are key factors involved in the etiology of disease. PINK1 and Parkin act in concert within a mitochondrial quality control system that has become well characterized over the past decade or so (Fig. 2). In healthy mitochondria, the serine/threonine kinase PINK1 is targeted to mitochondria, localizing to the translocase of the outer mitochondrial membrane (TOM) complex on the OMM. PINK1 is N-terminally translocated across the OMM to the inner mitochondrial membrane (IMM) (
      • Lazarou M.
      • Jin S.M.
      • Kane L.A.
      • Youle R.J.
      Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.
      ). Imported PINK1 is sequentially proteolytically cleaved, first by mitochondrial processing peptidase (MPP) and secondly by presenilin-associated rhomboid-like protease, PARL (
      • Jin S.M.
      • Lazarou M.
      • Wang C.
      • Kane L.A.
      • Narendra D.P.
      • Youle R.J.
      Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL.
      ,
      • Greene A.W.
      • Grenier K.
      • Aguileta M.A.
      • Muise S.
      • Farazifard R.
      • Haque M.E.
      • McBride H.M.
      • Park D.S.
      • Fon E.A.
      Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment.
      ). PINK1 is subsequently removed for degradation by the proteasome via the N-end rule, maintaining low basal levels of PINK1 protein (
      • Yamano K.
      • Youle R.J.
      PINK1 is degraded through the N-end rule pathway.
      ). Mitochondrial injury, typically presenting as reduced mitochondrial membrane potential, prohibits import of PINK1, stabilizing the active protein on the OMM. Although mitochondrial membrane potential depolarization has long been understood as the key mechanism by which PINK1 is stabilized, further methods have been used to trigger PINK1 stabilization in vitro and may perhaps represent further physiological stimuli by which mitophagy is initiated. These include initiation of the mitochondrial unfolded protein response (mtUPR) using an N-terminal deletion mutant of ornithine transcarbamylase (ΔOTC) (
      • Jin S.M.
      • Youle R.J.
      The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria.
      ) and induction of spatially restricted mitochondrial oxidative damage by using a photoreactive probe called Mito-Killer Red (
      • Ashrafi G.
      • Schlehe J.S.
      • LaVoie M.J.
      • Schwarz T.L.
      Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.
      ). Active PINK1 autophosphorylates in trans, resulting in self-amplifying activity and substrate recognition (
      • Okatsu K.
      • Oka T.
      • Iguchi M.
      • Imamura K.
      • Kosako H.
      • Tani N.
      • Kimura M.
      • Go E.
      • Koyano F.
      • Funayama M.
      • Shiba-Fukushima K.
      • Sato S.
      • Shimizu H.
      • Fukunaga Y.
      • Taniguchi H.
      • et al.
      PINK1 autophosphorylation upon membrane potential dissipation is essential for Parkin recruitment to damaged mitochondria.
      ,
      • Kondapalli C.
      • Kazlauskaite A.
      • Zhang N.
      • Woodroof H.I.
      • Campbell D.G.
      • Gourlay R.
      • Burchell L.
      • Walden H.
      • Macartney T.J.
      • Deak M.
      • Knebel A.
      • Alessi D.R.
      • Muqit M.M.
      PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65.
      ,
      • Rasool S.
      • Soya N.
      • Truong L.
      • Croteau N.
      • Lukacs G.L.
      • Trempe J.F.
      PINK1 autophosphorylation is required for ubiquitin recognition.
      ). PINK1 forms a homodimer (
      • Okatsu K.
      • Uno M.
      • Koyano F.
      • Go E.
      • Kimura M.
      • Oka T.
      • Tanaka K.
      • Matsuda N.
      A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment.
      ) and phosphorylates ubiquitin localized at the mitochondrial surface on serine 65 (Ser65) residues. March5 (also known as MITOL) is a RING finger E3-ubiquitin ligase localized to the OMM and has been proposed to catalyze formation of the initial OMM ubiquitin “seed.” The seed is proposed to serve as a substrate for PINK1-mediated phosphorylation and, subsequently, as the upstream receptor for Parkin. Thus, silencing of March5 slows Parkin recruitment to mitochondria (
      • Koyano F.
      • Yamano K.
      • Kosako H.
      • Tanaka K.
      • Matsuda N.
      Parkin recruitment to impaired mitochondria for nonselective ubiquitylation is facilitated by MITOL.
      ).
      Figure thumbnail gr2
      Figure 2PINK1-Parkin-dependent and independent mitophagy. Panel A: (1). Reduction in the mitochondrial membrane potential (Δψm) causes (2) PINK1 stabilization at the OMM where it dimerizes and autophosphorylates, resulting in activation. (3) Activated PINK1 phosphorylates Ub chains formed by E3-ubiquitin ligases such as March5 on OMM proteins such as TOM complex members. (4) Phosphorylated Ub chains (p-Ser65-Ub) allow the recruitment of Parkin from cytosol to OMM where it is phosphorylated at Ser65 by PINK1 and becomes fully activated. (5) Activated Parkin ubiquitinates OMM proteins generating a self-amplifying feedback loop with PINK1. (6) Adaptor proteins such as NDP52 or OPTN bring together p-Ser65-Ub chains with LC3-coated vesicles. Panel B: (i) Under hypoxic conditions, BNIP3, NIX, or FUNDC1 can bind ubiquitinated OMM proteins and recruit LC3 II-coated vesicles. (ii) MUL1 regulates ER-mitochondria contacts ubiquitinating MFN-2, resulting in its proteasomal degradation. (iii) HUWE1 and AMBRA1 interact to ubiquitinate proteins for proteasomal degradation or resulting in AMBRA1-mediated recruitment of LC3 II-coated vesicles. (iv) Cardiolipin externalization or IMM protein PHB2 exposure results in LC3 II-coated vesicle recruitment to the mitochondria.
      The RING/HECT hybrid E3-ubiquitin ligase Parkin translocates from the cytosol to mitochondria and is activated via two mechanisms: binding to phospho-Ser65-ubiquitin and phosphorylation by PINK1 on the homologous Ser65 residue of its own ubiquitin-like (UBL) domain (
      • Kondapalli C.
      • Kazlauskaite A.
      • Zhang N.
      • Woodroof H.I.
      • Campbell D.G.
      • Gourlay R.
      • Burchell L.
      • Walden H.
      • Macartney T.J.
      • Deak M.
      • Knebel A.
      • Alessi D.R.
      • Muqit M.M.
      PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65.
      ,
      • Kane L.A.
      • Lazarou M.
      • Fogel A.I.
      • Li Y.
      • Yamano K.
      • Sarraf S.A.
      • Banerjee S.
      • Youle R.J.
      PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.
      ,
      • Kazlauskaite A.
      • Martinez-Torres R.J.
      • Wilkie S.
      • Kumar A.
      • Peltier J.
      • Gonzalez A.
      • Johnson C.
      • Zhang J.
      • Hope A.G.
      • Peggie M.
      • Trost M.
      • van Aalten D.M.
      • Alessi D.R.
      • Prescott A.R.
      • Knebel A.
      • et al.
      Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.
      ,
      • Riley B.E.
      • Lougheed J.C.
      • Callaway K.
      • Velasquez M.
      • Brecht E.
      • Nguyen L.
      • Shaler T.
      • Walker D.
      • Yang Y.
      • Regnstrom K.
      • Diep L.
      • Zhang Z.
      • Chiou S.
      • Bova M.
      • Artis D.R.
      • et al.
      Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases.
      ). Binding of Parkin to phospho-Ser65-ubiquitin primes Parkin for phosphorylation within the UBL domain by PINK1. A key activating step of Parkin is the movement of phospho-UBL, its binding to RING0 and the release of the catalytic RING2 domain (
      • Sauvé V.
      • Sung G.
      • Soya N.
      • Kozlov G.
      • Blaimschein N.
      • Miotto L.S.
      • Trempe J.-F.
      • Lukacs G.L.
      • Gehring K.
      Mechanism of parkin activation by phosphorylation.
      ). These phosphorylation and binding events release Parkin autoinhibition to stabilize an open, active conformation capable of binding the charged E2-ubiquitin-conjugating enzyme. Active Parkin extensively ubiquitinates mitochondrial proteins (
      • Sarraf S.A.
      • Raman M.
      • Guarani-Pereira V.
      • Sowa M.E.
      • Huttlin E.L.
      • Gygi S.P.
      • Harper J.W.
      Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization.
      ), facilitating a feed-forward amplification loop of substrate ubiquitination, PINK1-mediated ubiquitin phosphorylation, and further Parkin recruitment to tag damaged mitochondria for removal by the autophagosome-lysosome system (
      • Kazlauskaite A.
      • Martinez-Torres R.J.
      • Wilkie S.
      • Kumar A.
      • Peltier J.
      • Gonzalez A.
      • Johnson C.
      • Zhang J.
      • Hope A.G.
      • Peggie M.
      • Trost M.
      • van Aalten D.M.
      • Alessi D.R.
      • Prescott A.R.
      • Knebel A.
      • et al.
      Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.
      ,
      • Wauer T.
      • Simicek M.
      • Schubert A.
      • Komander D.
      Mechanism of phospho-ubiquitin-induced PARKIN activation.
      ,
      • Narendra D.
      • Tanaka A.
      • Suen D.F.
      • Youle R.J.
      Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.
      ,
      • Ordureau A.
      • Sarraf S.A.
      • Duda D.M.
      • Heo J.M.
      • Jedrychowski M.P.
      • Sviderskiy V.O.
      • Olszewski J.L.
      • Koerber J.T.
      • Xie T.
      • Beausoleil S.A.
      • Wells J.A.
      • Gygi S.P.
      • Schulman B.A.
      • Harper J.W.
      Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis.
      ).
      Ubiquitinated proteins on the OMM act as the receptor for autophagic adaptors, including optineurin and nuclear dot protein-52 (NDP52). The subsequent recruitment and binding of the autophagy protein microtubule-associated proteins 1 light chain 3 (LC3) permit autophagosome formation (
      • Lazarou M.
      • Sliter D.A.
      • Kane L.A.
      • Sarraf S.A.
      • Wang C.
      • Burman J.L.
      • Sideris D.P.
      • Fogel A.I.
      • Youle R.J.
      The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.
      ) and lysosomal degradation. Parkin-mediated ubiquitination also targets some proteins, in particular the mitofusins, for degradation by the proteasome. Removal of these proteins early in mitophagy limits mitochondrial fusion, promoting mitochondrial fission, allowing damaged fragments of mitochondria to be sequestered from the healthy reticulum and removed (
      • Glauser L.
      • Sonnay S.
      • Stafa K.
      • Moore D.J.
      Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1.
      ).

      Alternative mitophagy pathways

      Pathways and cellular signaling events other than PINK1-Parkin can also recruit LC3 and autophagosomes to mitochondria (Fig. 2). Mitophagy is induced in response to low oxygen (hypoxia). The OMM proteins BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 (BNIP3) and Nip3-like protein X (NIX; also known as BNIP3L), members of the BCL-2 family of apoptosis regulators, have LC3-interacting domains and are upregulated during hypoxia (
      • Sowter H.M.
      • Ratcliffe P.J.
      • Watson P.
      • Greenberg A.H.
      • Harris A.L.
      HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors.
      ). NIX is also a Parkin substrate and involved in the recruitment of autophagic adaptors to mitochondria (
      • Gao F.
      • Chen D.
      • Si J.
      • Hu Q.
      • Qin Z.
      • Fang M.
      • Wang G.
      The mitochondrial protein BNIP3L is the substrate of PARK2 and mediates mitophagy in PINK1/PARK2 pathway.
      ).
      Another OMM protein, FUN14 domain-containing protein 1 (FUNDC1), is also involved in hypoxia-induced mitophagy (Fig. 2). FUNDC1 binds LC3 independently of Parkin, altering mitochondrial dynamics during mitophagy via interactions with profission protein DRP1 and mitochondrial fusion protein Dynamin-like 120 kDa protein (OPA1) (
      • Chen M.
      • Chen Z.
      • Wang Y.
      • Tan Z.
      • Zhu C.
      • Li Y.
      • Han Z.
      • Chen L.
      • Gao R.
      • Liu L.
      • Chen Q.
      Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy.
      ). FUNDC1 interacts with LC3 after dephosphorylation at Ser13 by serine/threonine-protein phosphatase PGAM5 and phosphorylation at Ser17 by the autophagy regulator kinase ULK1 in response to hypoxia (
      • Chen G.
      • Han Z.
      • Feng D.
      • Chen Y.
      • Chen L.
      • Wu H.
      • Huang L.
      • Zhou C.
      • Cai X.
      • Fu C.
      • Duan L.
      • Wang X.
      • Liu L.
      • Liu X.
      • Shen Y.
      • et al.
      A regulatory signaling loop comprising the PGAM5 phosphatase and CK2 controls receptor-mediated mitophagy.
      ). The response of FUNDC1 is fine-tuned via regulated ubiquitination and degradation by March5 in the initial stages of hypoxia (
      • Chen Z.
      • Liu L.
      • Cheng Q.
      • Li Y.
      • Wu H.
      • Zhang W.
      • Wang Y.
      • Sehgal S.A.
      • Siraj S.
      • Wang X.
      • Wang J.
      • Zhu Y.
      • Chen Q.
      Mitochondrial E3 ligase MARCH5 regulates FUNDC1 to fine-tune hypoxic mitophagy.
      ).
      HUWE1 is a HECT-type E3-ligase and promotes mitophagy via the proautophagic, LC3-interacting protein autophagy/beclin-1 regulator-1 (AMBRA1). A complex relationship exists where, upon mitochondrial stress, AMBRA1 functions as a cofactor for HUWE1, mediating both HUWE1 mitochondrial translocation and subsequent ubiquitination and proteasomal degradation of OMM proteins including MFN-2. Ubiquitination of OMM proteins is speculated to provide the signal for AMBRA1 phosphorylation at Ser1014 via IKKα and to promote AMBRA1-LC3B interaction and mitophagy (
      • Di Rita A.
      • Peschiaroli A.
      • P D.A.
      • Strobbe D.
      • Hu Z.
      • Gruber J.
      • Nygaard M.
      • Lambrughi M.
      • Melino G.
      • Papaleo E.
      • Dengjel J.
      • El Alaoui S.
      • Campanella M.
      • Dötsch V.
      • Rogov V.V.
      • et al.
      HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα.
      ). Furthermore, mitochondrial depolarization promotes a direct interaction between AMBRA1 and Parkin, activating proximal Class III PI3K, contributing to new phagophore formation (
      • Van Humbeeck C.
      • Cornelissen T.
      • Hofkens H.
      • Mandemakers W.
      • Gevaert K.
      • De Strooper B.
      • Vandenberghe W.
      Parkin interacts with Ambra1 to induce mitophagy.
      ).
      MUL1 is a multifunctional RING finger mitochondrial membrane protein with both ubiquitin and small ubiquitin-like modifier (SUMO) E3-ligase activities. MUL1 functions in parallel to the PINK1–Parkin pathway to ubiquitinate and remove MFN-2, compensating when overexpressed for the mitochondrial phenotypes associated with PINK1 and Parkin mutant Drosophila (
      • Yun J.
      • Puri R.
      • Yang H.
      • Lizzio M.A.
      • Wu C.
      • Sheng Z.-H.
      • Guo M.
      MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/Parkin.
      ). Moreover, MUL1 has recently been identified as an early checkpoint to protect mitochondria from rapid degradation under mild stress. MUL1 preserves mitochondrial morphology and mitochondria–endoplasmic reticulum (ER) contact by repressing the levels of MFN-2, to maintain Ca2+ homeostasis and metabolism. MFN-2 accumulation leads to increased cytosolic Ca2+ influx, mitochondrial fragmentation, and a decrease in mitochondrial membrane potential. It is speculated that if the MUL1-MFN-2 checkpoint fails, Parkin-mediated mitophagy will be activated (
      • Puri R.
      • Cheng X.T.
      • Lin M.Y.
      • Huang N.
      • Sheng Z.H.
      Mul1 restrains Parkin-mediated mitophagy in mature neurons by maintaining ER-mitochondrial contacts.
      ).
      Finally, externalization of cardiolipin, a unique IMM phospholipid, and exposure of IMM protein Prohibitin-2 (PHB2) following mitochondrial outer membrane rupture have each been observed to recruit LC3 and act as mitophagy receptors following mitochondrial damage (
      • Chu C.T.
      • Ji J.
      • Dagda R.K.
      • Jiang J.F.
      • Tyurina Y.Y.
      • Kapralov A.A.
      • Tyurin V.A.
      • Yanamala N.
      • Shrivastava I.H.
      • Mohammadyani D.
      • Wang K.Z.Q.
      • Zhu J.
      • Klein-Seetharaman J.
      • Balasubramanian K.
      • Amoscato A.A.
      • et al.
      Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells.
      ,
      • Wei Y.
      • Chiang W.C.
      • Sumpter Jr., R.
      • Mishra P.
      • Levine B.
      Prohibitin 2 is an inner mitochondrial membrane mitophagy receptor.
      ) (Fig. 2). By virtue of strong genetic association, the canonical PINK1–Parkin pathway remains the primary focus for PD research. However, numerous mitochondrial quality control pathways have been described and together provide additional intervention points for potential therapies to modulate mitophagy.

      Intervention points for mitophagy-based therapeutics

      PINK1-Parkin-dependent and -independent pathways provide many potential biological intervention points to enhance mitophagy. Indeed, several molecules are already available to perturb biology through inhibition or activation of a specific target (discussed in detail below). These molecules are useful to address hypothesis validity, and in some instances may provide a starting point for therapeutic development. Several additional mitophagy regulators hold potential for future therapeutic targeting.

      Protease

      OMA1

      Mitochondrial membrane depolarization induced-PINK1 import arrest is a key initiating event in mitophagy. Failure of PINK1 stabilization has been observed for several PD-linked PINK1 variants owing to inappropriate import and cleavage by the IMM-embedded metalloprotease, OMA1. Tom7, a small accessory protein of the TOM complex, facilitates PINK1 import arrest before OMA1 recognition and has been proposed to mediate the lateral release of conformationally kinase-active PINK1 from the TOM40 channel (
      • Sekine S.
      • Wang C.
      • Sideris D.P.
      • Bunker E.
      • Zhang Z.
      • Youle R.J.
      Reciprocal roles of Tom7 and OMA1 during mitochondrial import and activation of PINK1.
      ). Suppression of OMA1 restores depolarization-induced import arrest of PD-related PINK1 variants at the OMM. Additionally, mitochondrial membrane depolarization-induced PINK1 stabilization was slightly enhanced in OMA1−/− cells, independent of changes in PINK1 steady-state import (
      • Sekine S.
      • Wang C.
      • Sideris D.P.
      • Bunker E.
      • Zhang Z.
      • Youle R.J.
      Reciprocal roles of Tom7 and OMA1 during mitochondrial import and activation of PINK1.
      ). Together these data, along with observations of an in vivo protective role of OMA1−/− in a forebrain neuron-specific Phb2-deficient (Phb2NKO) neurodegenerative model (
      • Korwitz A.
      • Merkwirth C.
      • Richter-Dennerlein R.
      • Tröder S.E.
      • Sprenger H.-G.
      • Quirós P.M.
      • López-Otín C.
      • Rugarli E.I.
      • Langer T.
      Loss of OMA1 delays neurodegeneration by preventing stress-induced OPA1 processing in mitochondria.
      ), suggest inhibitors of OMA1 may be therapeutically viable.

      USP33/VDU1

      Ubiquitin chains can be removed from substrates by a family of proteins known as deubiquitinases (DUBs). USP33 is an OMM localized DUB, which antagonizes Parkin autoubiquitination mainly at Lys435. Silencing of USP33 enhances K63-linked ubiquitin chain formation on Parkin, increasing Parkin stabilization and the rate of depolarization-dependent mitochondrial translocation, accelerating mitophagy. Interestingly, USP33 is expressed at high levels in central nervous system (CNS) and PD-affected areas, including amygdala and substantia nigra (
      • Niu K.
      • Fang H.
      • Chen Z.
      • Zhu Y.
      • Tan Q.
      • Wei D.
      • Li Y.
      • Balajee A.S.
      • Zhao Y.
      USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy.
      ).

      Deubiquitinases (DUBs)

      Numerous DUBs have been shown to regulate mitophagy including USP30 (discussed below), USP15 (
      • Cornelissen T.
      • Haddad D.
      • Wauters F.
      • Van Humbeeck C.
      • Mandemakers W.
      • Koentjoro B.
      • Sue C.
      • Gevaert K.
      • De Strooper B.
      • Verstreken P.
      • Vandenberghe W.
      The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy.
      ,
      • Cornelissen T.
      • Vilain S.
      • Vints K.
      • Gounko N.
      • Verstreken P.
      • Vandenberghe W.
      Deficiency of Parkin and PINK1 impairs age-dependent mitophagy in Drosophila.
      ), USP8 (
      • Durcan T.M.
      • Tang M.Y.
      • Pérusse J.R.
      • Dashti E.A.
      • Aguileta M.A.
      • McLelland G.L.
      • Gros P.
      • Shaler T.A.
      • Faubert D.
      • Coulombe B.
      • Fon E.A.
      USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin.
      ), and a splice variant of USP35 (
      • Wang Y.
      • Serricchio M.
      • Jauregui M.
      • Shanbhag R.
      • Stoltz T.
      • Di Paolo C.T.
      • Kim P.K.
      • McQuibban G.A.
      Deubiquitinating enzymes regulate PARK2-mediated mitophagy.
      ). These have been described elsewhere (
      • Fritsch L.E.
      • Moore M.E.
      • Sarraf S.A.
      • Pickrell A.M.
      Ubiquitin and receptor-dependent mitophagy pathways and their implication in neurodegeneration.
      ).

      Phosphatase

      Protein phosphatase with EF-hand domain 2 (PPEF2)

      A phosphatase antagonistic to PINK1, which dephosphorylates ubiquitin and supresses PINK1-mediated mitophagy. Silencing of PPEF2 increases phospho-Ser65-ubiquitin and enhances basal and stress-induced mitophagy both dependent and independent of Parkin. Interestingly, numerous proteins have been identified as being inversely regulated by PPEF2 and PINK1. These observations point to potential roles in mitochondrial biogenesis, regulation of mitophagy in cells and tissues with low Parkin expression, and as an antiapoptotic phosphatase (
      • Wall C.E.
      • Rose C.M.
      • Adrian M.
      • Zeng Y.J.
      • Kirkpatrick D.S.
      • Bingol B.
      PPEF2 opposes PINK1-mediated mitochondrial quality control by dephosphorylating ubiquitin.
      ). Notably, phosphorylated ubiquitin linkages have greater resistance to DUB-mediated cleavage, suggesting that dephosphorylation of ubiquitin may be a critical regulator in controlling rates of mitophagy (
      • Wauer T.
      • Swatek K.N.
      • Wagstaff J.L.
      • Gladkova C.
      • Pruneda J.N.
      • Michel M.A.
      • Gersch M.
      • Johnson C.M.
      • Freund S.M.
      • Komander D.
      Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis.
      ).

      PTEN-L

      A translational variant of PTEN, localized to the OMM and cytosol. Comparable with PPEF2, PTEN-L antagonizes PINK1-mediated phosphorylation of ubiquitin, reducing Parkin translocation and relief of autoinhibition and thereby suppressing mitophagy. PTEN-L decreases abundance of phospho-Ser65-Parkin; however, it is unclear if PTEN-L dephosphorylates Parkin directly or whether the reduction is secondary to reduced mitochondrial translocation and proximity to PINK1 (
      • Wang L.
      • Cho Y.L.
      • Tang Y.
      • Wang J.
      • Park J.E.
      • Wu Y.
      • Wang C.
      • Tong Y.
      • Chawla R.
      • Zhang J.
      • Shi Y.
      • Deng S.
      • Lu G.
      • Wu Y.
      • Tan H.W.
      • et al.
      PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1-Parkin-mediated mitophagy.
      ).

      Mitophagy reporter assay systems

      Several mitophagy reporters have been developed to exploit the pH differential between cellular compartments and discrete organelles to discriminate stages of mitophagy. These have been effectively used to study mitophagy in vitro and in vivo.

      Mt-keima

      mt-Keima utilizes unique fluorescent properties of the coral-derived protein Keima, artificially targeted to the mitochondrial matrix using the COX8A mitochondrial targeting sequence (MTS) (
      • Sun N.
      • Yun J.
      • Liu J.
      • Malide D.
      • Liu C.
      • Rovira I.I.
      • Holmström K.M.
      • Fergusson M.M.
      • Yoo Y.H.
      • Combs C.A.
      • Finkel T.
      Measuring in vivo mitophagy.
      ). Keima has a single, pH-independent emission peak at 620 nm but a pH-dependent bimodal excitation. The excitation maximum at 440 nm in slightly alkali environments (the mitochondria) shifts to 586 nm in the acidic environment of the lysosome. Ratiometric analysis of 586 nm: 440 nm fluorescence intensity yields a “mitophagy index,” describing the relative proportion of mitochondria within acidic lysosomes (pH 4.5) to healthy mitochondria with normal matrix pH (pH 8) residing within the cytoplasm (
      • Sun N.
      • Yun J.
      • Liu J.
      • Malide D.
      • Liu C.
      • Rovira I.I.
      • Holmström K.M.
      • Fergusson M.M.
      • Yoo Y.H.
      • Combs C.A.
      • Finkel T.
      Measuring in vivo mitophagy.
      ,
      • Sun N.
      • Malide D.
      • Liu J.
      • Rovira I.I.
      • Combs C.A.
      • Finkel T.
      A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.
      ). Several mechanistic and biological properties however limit the use of mt-Keima. Keima is incompatible with immunohistochemical fixation, which dissipates proton/pH gradients, preventing delineation of mitophagy index within specific cell populations. Additionally, though studies have demonstrated that Keima is relatively insensitive to proteolytic degradation, the fate of lysosomal mt-Keima protein remains ill-defined (
      • Sun N.
      • Yun J.
      • Liu J.
      • Malide D.
      • Liu C.
      • Rovira I.I.
      • Holmström K.M.
      • Fergusson M.M.
      • Yoo Y.H.
      • Combs C.A.
      • Finkel T.
      Measuring in vivo mitophagy.
      ). The partial overlap of the 440:586 nm bimodal excitation ranges of Keima in different pH environments may also complicate ratiometric analysis (
      • Sun N.
      • Malide D.
      • Liu J.
      • Rovira I.I.
      • Combs C.A.
      • Finkel T.
      A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.
      ).

      Mito-QC (mCherry-GFP-FIS1(aa.101–152))

      Mito-QC exploits the pH-sensitive quenching of green fluorescent protein (GFP) in acidic environments. The targeting sequence from FIS1 directs a tandem GFP-mCherry protein to the OMM. Within the cytosol, mito-QC fluoresces both red and green, however, upon delivery to the lysosome GFP is quenched, allowing analysis of mCherry puncta as index of mitophagy (
      • Allen G.F.
      • Toth R.
      • James J.
      • Ganley I.G.
      Loss of iron triggers PINK1/Parkin-independent mitophagy.
      ). However, mito-QC is cytoplasmic facing, making it open to extraction and clearance by OMM proteasome-dependent pathways. Indeed recently, proteasome-sensitive Parkin-dependent clearance of mito-QC following mitochondrial uncoupling has been observed (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ). Further, much like mt-Keima, constitutive expression over the course of mouse development may produce basal signal of unknown specificity, which may mask subtle changes in mitophagic signaling. This basal signal may also reflect tissue-specific differences in lysosomal activity and clearance. Notably, inducible expression of mito-QC reduced Parkin-independent signals (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ).

      Mito-SRAI (mitochondrial matrix targeted signal-retaining autophagy indicator)

      Mito-SRAI is a tandem YPet-afCFP (a.k.a. TOLLES: TOLerance of Lysosomal EnvironmentS) construct. Acid sensitivity of YPet allows distinction of TOLLES-positive puncta in acid compartments, indicating mitophagy (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ). Unlike mito-QC, initial characterization of mito-SRAI in vitro demonstrates insensitivity to proteasomal clearance, and unlike mt-Keima, mito-SRAI is amenable to fixation.

      Pharmacological enhancement of mitophagy: from tool molecules to potential therapeutics

      The relationship between mitochondrial dysfunction and PD suggests that improving the efficiency of mitochondrial clearance by mitophagy may be a disease-modifying strategy for PD (Table 1). Increased understanding of mitophagy pathways has led to the identification of potential therapeutic targets and intervention points to positively modulate mitophagy. To date, several small molecules and natural compounds targeting mitophagy have been identified using target-based, in silico, or phenotypic screening strategies and, encouragingly, have subsequently demonstrated neuroprotection in PD models (Table 1).
      Table 1Structure and experimental summary of molecules enhancing mitophagy
      CompoundStructureTargetMechanism of actionRead outReferences
      AUTAC4TSPOPINK1–Parkin-independent mitophagyICC: LC3 II puncta colocalization with mito-EGFP-HT or K63-linked Ub, Mito-Rosella dye, MtPhagy dye(
      • Takahashi D.
      • Moriyama J.
      • Nakamura T.
      • Miki E.
      • Takahashi E.
      • Sato A.
      • Akaike T.
      • Itto-Nakama K.
      • Arimoto H.
      AUTACs: cargo-specific degraders using selective autophagy.
      )
      BC1464FBXO7PINK1-dependent mitophagyWB: Phosphorylation of Ubiquitin(
      • Liu Y.
      • Lear T.B.
      • Verma M.
      • Wang K.Z.
      • Otero P.A.
      • McKelvey A.C.
      • Dunn S.R.
      • Steer E.
      • Bateman N.W.
      • Wu C.
      • Jiang Y.
      • Weathington N.M.
      • Rojas M.
      • Chu C.T.
      • Chen B.B.
      • et al.
      Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.
      )
      Compound 3Miro1Unknown mechanism, potentially through PINK1WB: MFN-2, VDAC, LRRK2, Parkin

      ICC: TOM20, ATP5β
      (
      • Hsieh C.H.
      • Li L.
      • Vanhauwaert R.
      • Nguyen K.T.
      • Davis M.D.
      • Bu G.
      • Wszolek Z.K.
      • Wang X.
      Miro1 marks Parkinson's disease subset and Miro1 reducer rescues neuron loss in Parkinson's models.
      )
      DeferiproneIronPINK1-independent mitophagyWB: MFN-2, HSP60, TIMM50, Omi

      ICC: mCherry-GFP-FIS1(aa.101–152)



      IHC: mt-Keima
      (
      • Allen G.F.
      • Toth R.
      • James J.
      • Ganley I.G.
      Loss of iron triggers PINK1/Parkin-independent mitophagy.
      )
      FT385USP30PINK1–Parkin-dependent mitophagyWB: Ub-TOM20/TOM20, PINK1

      ICC: mCherry-GFP- FIS1(aa.101–152)
      (
      • Rusilowicz-Jones E.V.
      • Jardine J.
      • Kallinos A.
      • Pinto-Fernandez A.
      • Guenther F.
      • Giurrandino M.
      • Barone F.G.
      • McCarron K.
      • Burke C.J.
      • Murad A.
      • Martinez A.
      • Marcassa E.
      • Gersch M.
      • Buckmelter A.J.
      • Kayser-Bricker K.J.
      • et al.
      USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.
      )
      GemcitabineNot reportedMUL1, PINK1-dependent mitophagyICC: mt-Keima, high-content image analysis(
      • Igarashi R.
      • Yamashita S.I.
      • Yamashita T.
      • Inoue K.
      • Fukuda T.
      • Fukuchi T.
      • Kanki T.
      Gemcitabine induces Parkin-independent mitophagy through mitochondrial-resident E3 ligase MUL1-mediated stabilization of PINK1.
      )
      GYY4137Pleiotropic/Keap1PINK1–Parkin dependent mitophagyBiochemical: Parkin E3-ubiquitin ligase activity(
      • Vandiver M.S.
      • Paul B.D.
      • Xu R.
      • Karuppagounder S.
      • Rao F.
      • Snowman A.M.
      • Ko H.S.
      • Lee Y.I.
      • Dawson V.L.
      • Dawson T.M.
      • Sen N.
      • Snyder S.H.
      Sulfhydration mediates neuroprotective actions of parkin.
      )
      Ivermectin

      Mixture of 2 isomers
      TRAF2 proposedPINK1–Parkin-independent mitophagy

      WB: TOM20

      ICC: LC3 II and mCherry-colocalization with TOM20

      Electron microscopy: mitophagosome formation
      (
      • Zachari M.
      • Gudmundsson S.R.
      • Li Z.
      • Manifava M.
      • Shah R.
      • Smith M.
      • Stronge J.
      • Karanasios E.
      • Piunti C.
      • Kishi-Itakura C.
      • Vihinen H.
      • Jokitalo E.
      • Guan J.L.
      • Buss F.
      • Smith A.M.
      • et al.
      Selective autophagy of mitochondria on a ubiquitin-endoplasmic-reticulum platform.
      )
      KinetinPINK1PINK1-dependent mitophagyICC: Parkin–mitochondria colocalization(
      • Hertz N.T.
      • Berthet A.
      • Sos M.L.
      • Thorn K.S.
      • Burlingame A.L.
      • Nakamura K.
      • Shokat K.M.
      A neo-substrate that amplifies catalytic activity of Parkinson's-disease-related kinase PINK1.
      ,
      • Osgerby L.
      • Lai Y.C.
      • Thornton P.J.
      • Amalfitano J.
      • Le Duff C.S.
      • Jabeen I.
      • Kadri H.
      • Miccoli A.
      • Tucker J.H.R.
      • Muqit M.M.K.
      • Mehellou Y.
      Kinetin riboside and its ProTides activate the Parkinson's disease associated PTEN-induced putative kinase 1 (PINK1) independent of mitochondrial depolarization.
      ,
      • Shetty R.S.
      • Gallagher C.S.
      • Chen Y.T.
      • Hims M.M.
      • Mull J.
      • Leyne M.
      • Pickel J.
      • Kwok D.
      • Slaugenhaupt S.A.
      Specific correction of a splice defect in brain by nutritional supplementation.
      )
      Kinetin RibosidePINK1PINK1-dependent mitophagyICC: Parkin–mitochondria colocalization(
      • Osgerby L.
      • Lai Y.C.
      • Thornton P.J.
      • Amalfitano J.
      • Le Duff C.S.
      • Jabeen I.
      • Kadri H.
      • Miccoli A.
      • Tucker J.H.R.
      • Muqit M.M.K.
      • Mehellou Y.
      Kinetin riboside and its ProTides activate the Parkinson's disease associated PTEN-induced putative kinase 1 (PINK1) independent of mitochondrial depolarization.
      )
      NicotinamideNAD(+)-precursorSIRT1-PCG1α-dependent mitophagyWB: LC3, PINK1

      ICC: LC3 II, mitochondrial morphology, mitophagy dye
      (
      • Jang S.Y.
      • Kang H.T.
      • Hwang E.S.
      Nicotinamide-induced mitophagy: event mediated by high NAD+/NADH ratio and SIRT1 protein activation.
      ,
      • Fang E.F.
      • Hou Y.
      • Lautrup S.
      • Jensen M.B.
      • Yang B.
      • SenGupta T.
      • Caponio D.
      • Khezri R.
      • Demarest T.G.
      • Aman Y.
      • Figueroa D.
      • Morevati M.
      • Lee H.-J.
      • Kato H.
      • Kassahun H.
      • et al.
      NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome.
      )
      Nilotinibc-AblParkin-dependent mitophagyWB: LC3, phospho-Ser65-Ub

      ICC: phospho-c-Abl and Parkin colocalization
      (
      • Lonskaya I.
      • Hebron M.L.
      • Desforges N.M.
      • Schachter J.B.
      • Moussa C.E.
      Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance.
      )
      p62-mediated mitophagy inducer (PMI)Nrf2PINK1-independent mitophagy p62-dependent mitophagyICC: colocalization Parkin, ATP synthase β subunit(
      • East D.A.
      • Fagiani F.
      • Crosby J.
      • Georgakopoulos N.D.
      • Bertrand H.
      • Schaap M.
      • Fowkes A.
      • Wells G.
      • Campanella M.
      PMI: a ΔΨm independent pharmacological regulator of mitophagy.
      )
      MWP00839Not reportedUnknown mechanismICC: mito-Timer high-content image analysis(
      • Cerqueira F.M.
      • Kozer N.
      • Petcherski A.
      • Baranovski B.M.
      • Wolf D.
      • Assali E.A.
      • Roth Y.
      • Gazit R.
      • Barr H.
      • Lewis E.C.
      • Las G.
      • Shirihai O.S.
      MitoTimer-based high-content screen identifies two chemically-related benzothiophene derivatives that enhance basal mitophagy.
      )
      SPB08007
      SR3677ROCK2PINK1-dependent mitophagyWB: MFN-2, VDAC1

      ICC: Parkin- mito-dsRed colocalization, mito-QC

      (
      • Moskal N.
      • Riccio V.
      • Bashkurov M.
      • Taddese R.
      • Datti A.
      • Lewis P.N.
      • Angus McQuibban G.
      ROCK inhibitors upregulate the neuroprotective Parkin-mediated mitophagy pathway.
      )
      Y27632ROCK1/2PINK1-dependent mitophagyICC: Parkin–mitochondria colocalization
      SulforaphaneKeap1p62-dependent mitophagyICC: p62/SQSTM1, LC3(
      • East D.A.
      • Fagiani F.
      • Crosby J.
      • Georgakopoulos N.D.
      • Bertrand H.
      • Schaap M.
      • Fowkes A.
      • Wells G.
      • Campanella M.
      PMI: a ΔΨm independent pharmacological regulator of mitophagy.
      )
      T0466Not reportedPINK1–Parkin dependent mitophagyICC: High-content image analysis, Luciferase tagged MFN-2(
      • Shiba-Fukushima K.
      • Inoshita T.
      • Sano O.
      • Iwata H.
      • Ishikawa K.I.
      • Okano H.
      • Akamatsu W.
      • Imai Y.
      • Hattori N.
      A cell-based high-throughput screening identified two compounds that enhance PINK1-parkin signaling.
      )
      T-271Not reportedParkin-dependent mitophagyICC: High-content image analysis, mito-SRAI signal(
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      )
      USP30iUSP30PINK1-dependent mitophagyWB: Ub-TOM20(
      • Phu L.
      • Rose C.M.
      • Tea J.S.
      • Wall C.E.
      • Verschueren E.
      • Cheung T.K.
      • Kirkpatrick D.S.
      • Bingol B.
      Dynamic regulation of mitochondrial import by the ubiquitin system.
      )
      Urolithin ANot reportedUnknown mechanism, potentially through PINK1WB: PINK1, Parkin, pTBK, pULK, p62, LC3 I/II, Ub

      ICC: GFP-DsRed, MitoRosella, TOM20-LAMP2 colocalization, Mitophagy Dye, mito-GFP, LC3B-GFP, mRFP-GFP-LC3B

      Electron microscopy
      (
      • Ryu D.
      • Mouchiroud L.
      • Andreux P.A.
      • Katsyuba E.
      • Moullan N.
      • Nicolet-Dit-Félix A.A.
      • Williams E.G.
      • Jha P.
      • Lo Sasso G.
      • Huzard D.
      • Aebischer P.
      • Sandi C.
      • Rinsch C.
      • Auwerx J.
      Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents.
      ,
      • Fang E.F.
      • Hou Y.
      • Palikaras K.
      • Adriaanse B.A.
      • Kerr J.S.
      • Yang B.
      • Lautrup S.
      • Hasan-Olive M.M.
      • Caponio D.
      • Dan X.
      • Rocktäschel P.
      • Croteau D.L.
      • Akbari M.
      • Greig N.H.
      • Fladby T.
      • et al.
      Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease.
      )
      aa, amino acids; CFP, cyan fluorescent protein; ICC, immunocytochemistry; Ub, ubiquitin; WB, western blot.

      Target-based drug discovery strategies for mitophagy enhancement

      PINK1 is a mitochondrially localized serine/threonine kinase with a direct genetic relationship with PD (
      • Valente E.M.
      • Abou-Sleiman P.M.
      • Caputo V.
      • Muqit M.M.
      • Harvey K.
      • Gispert S.
      • Ali Z.
      • Del Turco D.
      • Bentivoglio A.R.
      • Healy D.G.
      • Albanese A.
      • Nussbaum R.
      • Gonzalez-Maldonado R.
      • Deller T.
      • Salvi S.
      • et al.
      Hereditary early-onset Parkinson's disease caused by mutations in PINK1.
      ), as described above. Disease-causing mutations in the kinase domain and C-terminal noncatalytic region of PINK1 suppress catalytic activity, leading to the hypothesis that restoration of kinase activity may have disease-modifying effects in PD (
      • Hertz N.T.
      • Berthet A.
      • Sos M.L.
      • Thorn K.S.
      • Burlingame A.L.
      • Nakamura K.
      • Shokat K.M.
      A neo-substrate that amplifies catalytic activity of Parkinson's-disease-related kinase PINK1.
      ). Indeed, PINK1 can induce mitophagy even in the absence of Parkin (
      • Lazarou M.
      • Sliter D.A.
      • Kane L.A.
      • Sarraf S.A.
      • Wang C.
      • Burman J.L.
      • Sideris D.P.
      • Fogel A.I.
      • Youle R.J.
      The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.
      ), suggesting PINK1 is an important and potentially druggable intervention point for therapeutic development. PINK1 is unique among kinases in its ability to accept the neo-substrate kinetin triphosphate (KTP; N6 furfuryl ATP) with greater catalytic efficiency than ATP, creating opportunities for drug development (
      • Hertz N.T.
      • Berthet A.
      • Sos M.L.
      • Thorn K.S.
      • Burlingame A.L.
      • Nakamura K.
      • Shokat K.M.
      A neo-substrate that amplifies catalytic activity of Parkinson's-disease-related kinase PINK1.
      ) (Table 1). KTP is produced by consecutive metabolic steps from kinetin or kinetin riboside (KR), once internalized into the cell (
      • Hertz N.T.
      • Berthet A.
      • Sos M.L.
      • Thorn K.S.
      • Burlingame A.L.
      • Nakamura K.
      • Shokat K.M.
      A neo-substrate that amplifies catalytic activity of Parkinson's-disease-related kinase PINK1.
      ). However, the efficiency by which cells convert kinetin and KR to KTP is low. KR monophosphate also has poor cellular stability. To overcome these limitations, KR ProTides (PROdrug + nucleoTIDE) have been developed to deliver KR into the cell (
      • Osgerby L.
      • Lai Y.C.
      • Thornton P.J.
      • Amalfitano J.
      • Le Duff C.S.
      • Jabeen I.
      • Kadri H.
      • Miccoli A.
      • Tucker J.H.R.
      • Muqit M.M.K.
      • Mehellou Y.
      Kinetin riboside and its ProTides activate the Parkinson's disease associated PTEN-induced putative kinase 1 (PINK1) independent of mitochondrial depolarization.
      ) (Table 1). Cellular studies have shown that once converted to its active form (KTP), kinetin administration can enhance PINK1 activity. Kinetin treatment increases Parkin translocation to mitochondria and reduces mitochondrial motility in neuronal axons, critical steps in removal of damaged mitochondria by mitophagy (
      • Hertz N.T.
      • Berthet A.
      • Sos M.L.
      • Thorn K.S.
      • Burlingame A.L.
      • Nakamura K.
      • Shokat K.M.
      A neo-substrate that amplifies catalytic activity of Parkinson's-disease-related kinase PINK1.
      ). Furthermore, kinetin prevents cleavage of BCL-XL to its proapoptotic form through increasing PINK1-mediated phosphorylation of BCL-XL at Ser62 (
      • Hertz N.T.
      • Berthet A.
      • Sos M.L.
      • Thorn K.S.
      • Burlingame A.L.
      • Nakamura K.
      • Shokat K.M.
      A neo-substrate that amplifies catalytic activity of Parkinson's-disease-related kinase PINK1.
      ,
      • Arena G.
      • Gelmetti V.
      • Torosantucci L.
      • Vignone D.
      • Lamorte G.
      • De Rosa P.
      • Cilia E.
      • Jonas E.A.
      • Valente E.M.
      PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage.
      ). Interestingly, in vivo pharmacokinetic studies have demonstrated that kinetin crosses the blood–brain barrier (BBB) and is well tolerated in humans (
      • Shetty R.S.
      • Gallagher C.S.
      • Chen Y.T.
      • Hims M.M.
      • Mull J.
      • Leyne M.
      • Pickel J.
      • Kwok D.
      • Slaugenhaupt S.A.
      Specific correction of a splice defect in brain by nutritional supplementation.
      ,
      • Axelrod F.B.
      • Liebes L.
      • Gold-Von Simson G.
      • Mendoza S.
      • Mull J.
      • Leyne M.
      • Norcliffe-Kaufmann L.
      • Kaufmann H.
      • Slaugenhaupt S.A.
      Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia.
      ).
      Genetic rationale supports targeting Parkin for drug development (
      • Kitada T.
      • Asakawa S.
      • Hattori N.
      • Matsumine H.
      • Yamamura Y.
      • Minoshima S.
      • Yokochi M.
      • Mizuno Y.
      • Shimizu N.
      Mutations in the Parkin gene cause autosomal recessive juvenile Parkinsonism.
      ) although complex structural biology, autoinhibition, and promiscuity among target proteins create challenges. Parkin ubiquitinates a large collection of functionally diverse proteins and is believed to have little or no requirement for defined consensus sequences to determine substrate specificity (
      • Sarraf S.A.
      • Raman M.
      • Guarani-Pereira V.
      • Sowa M.E.
      • Huttlin E.L.
      • Gygi S.P.
      • Harper J.W.
      Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization.
      ). Instead, specific PTMs or substrate conformation may be required (
      • Koyano F.
      • Yamano K.
      • Kosako H.
      • Tanaka K.
      • Matsuda N.
      Parkin recruitment to impaired mitochondria for nonselective ubiquitylation is facilitated by MITOL.
      ). Compelling structural studies have identified both naturally occurring and artificially designed activating mutations in Parkin (
      • Trempe J.F.
      • Sauvé V.
      • Grenier K.
      • Seirafi M.
      • Tang M.Y.
      • Ménade M.
      • Al-Abdul-Wahid S.
      • Krett J.
      • Wong K.
      • Kozlov G.
      • Nagar B.
      • Fon E.A.
      • Gehring K.
      Structure of parkin reveals mechanisms for ubiquitin ligase activation.
      ). Activating Parkin mutations can rescue phospho-dead and UBL-domain-deleted Parkin (
      • Tang M.Y.
      • Vranas M.
      • Krahn A.I.
      • Pundlik S.
      • Trempe J.F.
      • Fon E.A.
      Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation.
      ) as well as many pathogenic PD mutations (
      • Yi W.
      • MacDougall E.J.
      • Tang M.Y.
      • Krahn A.I.
      • Gan-Or Z.
      • Trempe J.F.
      • Fon E.A.
      The landscape of parkin variants reveals pathogenic mechanisms and therapeutic targets in Parkinson's disease.
      ). These data collectively provide proof-of-concept that recessive Parkin mutations in PD can be rescued and highlights the potential that rational drug design may produce pharmacological agents that mimic conformational changes associated with activating mutations. Furthermore, Gladkova et al. (
      • Gladkova C.
      • Maslen S.L.
      • Skehel J.M.
      • Komander D.
      Mechanism of parkin activation by PINK1.
      ) identified a small conserved helix in the Parkin UBL-RING0 linker, known as the activation element (ACT), which contributes to catalytic RING2 domain release by mimicking RING2 interactions in the RING0 domain and may potentially serve as a scaffold for the creation of a small-molecule Parkin activating compounds. Additionally, although peer-reviewed research is not available, several activators of Parkin have been described in the patent literature (US 2016/0160205A1 and WO 2018/023029). These compounds provide the first evidence of direct Parkin modulation.
      An alternative and indirect strategy is to modulate Parkin activity via endogenous regulators such as c-Abl, a tyrosine kinase with a prominent role in neurons. c-Abl regulates Parkin by phosphorylation at Tyr143, resulting in decreased Parkin activity and reduced mitophagy. Interestingly, increased levels of phosphorylated, active c-Abl have been found in PD brains (
      • Ko H.S.
      • Lee Y.
      • Shin J.-H.
      • Karuppagounder S.S.
      • Gadad B.S.
      • Koleske A.J.
      • Pletnikova O.
      • Troncoso J.C.
      • Dawson V.L.
      • Dawson T.M.
      Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function.
      ,
      • Imam S.Z.
      • Zhou Q.
      • Yamamoto A.
      • Valente A.J.
      • Ali S.F.
      • Bains M.
      • Roberts J.L.
      • Kahle P.J.
      • Clark R.A.
      • Li S.
      Novel regulation of parkin function through c-Abl-mediated tyrosine phosphorylation: implications for Parkinson's disease.
      ). Nilotinib, a c-Abl inhibitor, is currently used to treat chronic myelogenous leukaemia (CML) (Table 1). Nilotinib prevents α-synuclein accumulation and dopaminergic cell loss in an in vivo model of PD (
      • Hebron M.L.
      • Lonskaya I.
      • Moussa C.E.H.
      Nilotinib reverses loss of dopamine neurons and improves motor behavior via autophagic degradation of α-synuclein in Parkinson's disease models.
      ), and has been investigated in PD clinical trials (NCT02281474, NCT02954978). As c-Abl negatively regulates Parkin, it has been proposed that some of nilotinib’s protective effects are related to modulation of Parkin-dependent pathways (
      • Lonskaya I.
      • Hebron M.L.
      • Desforges N.M.
      • Schachter J.B.
      • Moussa C.E.
      Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance.
      ). Nonetheless, further studies are needed to determine whether Parkin activation by nilotinib can induce mitophagy as a protective approach in neurodegeneration (Table 1). Some authors have highlighted nilotinib inhibition of kinases other than c-Abl, suggesting that it may achieve neuroprotective effects via these other pathways (
      • Karuppagounder S.S.
      • Brahmachari S.
      • Lee Y.
      • Dawson V.L.
      • Dawson T.M.
      • Ko H.S.
      The c-Abl inhibitor, nilotinib, protects dopaminergic neurons in a preclinical animal model of Parkinson's disease.
      ).
      Ubiquitination of mitochondrial proteins is tightly regulated at multiple different levels. Ubiquitin specific protease 30 (USP30) is a mitochondrially localized DUB, hypothesized to oppose Parkin-mediated mitophagy by removing poly-ubiquitin chains from damaged mitochondria (
      • Marcassa E.
      • Kallinos A.
      • Jardine J.
      • Rusilowicz-Jones E.V.
      • Martinez A.
      • Kuehl S.
      • Islinger M.
      • Clague M.J.
      • Urbe S.
      Dual role of USP30 in controlling basal pexophagy and mitophagy.
      ). Loss of USP30 enhances both stress-induced and basal mitophagy. USP30 has lower activity against phosphorylated ubiquitin linkages, therefore potentially acting upstream or independently of PINK1 (
      • Wauer T.
      • Swatek K.N.
      • Wagstaff J.L.
      • Gladkova C.
      • Pruneda J.N.
      • Michel M.A.
      • Gersch M.
      • Johnson C.M.
      • Freund S.M.
      • Komander D.
      Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis.
      ,
      • Marcassa E.
      • Kallinos A.
      • Jardine J.
      • Rusilowicz-Jones E.V.
      • Martinez A.
      • Kuehl S.
      • Islinger M.
      • Clague M.J.
      • Urbe S.
      Dual role of USP30 in controlling basal pexophagy and mitophagy.
      ). This provokes the hypothesis that USP30 may act on the initial mitochondrial ubiquitin “seeds” before PINK1–Parkin activation, defining a threshold for mitophagy initiation, and therefore making it an attractive target for modulating mitophagy in PD.
      Two recent studies described potent inhibitors of USP30 (Table 1) (
      • Phu L.
      • Rose C.M.
      • Tea J.S.
      • Wall C.E.
      • Verschueren E.
      • Cheung T.K.
      • Kirkpatrick D.S.
      • Bingol B.
      Dynamic regulation of mitochondrial import by the ubiquitin system.
      ,
      • Rusilowicz-Jones E.V.
      • Jardine J.
      • Kallinos A.
      • Pinto-Fernandez A.
      • Guenther F.
      • Giurrandino M.
      • Barone F.G.
      • McCarron K.
      • Burke C.J.
      • Murad A.
      • Martinez A.
      • Marcassa E.
      • Gersch M.
      • Buckmelter A.J.
      • Kayser-Bricker K.J.
      • et al.
      USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.
      ). FT3967385 (FT385) was used as a tool to study the impact of USP30 inhibition on the total cellular ubiquitinome, identifying only subtle effects overall but a large impact on ubiquitination of mitochondrial proteins such as voltage-dependent anion channel (VDAC) and TOM components. A significant increase in phospho-Ser65-ubiquitin and mitophagy was also observed (
      • Rusilowicz-Jones E.V.
      • Jardine J.
      • Kallinos A.
      • Pinto-Fernandez A.
      • Guenther F.
      • Giurrandino M.
      • Barone F.G.
      • McCarron K.
      • Burke C.J.
      • Murad A.
      • Martinez A.
      • Marcassa E.
      • Gersch M.
      • Buckmelter A.J.
      • Kayser-Bricker K.J.
      • et al.
      USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.
      ). The authors proposed a model in which USP30 regulates the ubiquitin chains available for PINK1 phosphorylation following mitochondrial depolarization. The study concluded that USP30 plays a key role in regulating activities of PINK1 and Parkin, suggesting USP30 inhibition as a viable strategy to induce mitophagy (
      • Rusilowicz-Jones E.V.
      • Jardine J.
      • Kallinos A.
      • Pinto-Fernandez A.
      • Guenther F.
      • Giurrandino M.
      • Barone F.G.
      • McCarron K.
      • Burke C.J.
      • Murad A.
      • Martinez A.
      • Marcassa E.
      • Gersch M.
      • Buckmelter A.J.
      • Kayser-Bricker K.J.
      • et al.
      USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.
      ).
      A second study shows that USP30 also antagonizes the effect of E3-ubiquitin ligases other than Parkin to regulate distinct mitochondrial functions. Using a newly reported USP30 inhibitor, USP30i, as a tool, the authors dissected the mechanisms by which USP30 works together with March5 to regulate import of proteins into the mitochondrial matrix (
      • Phu L.
      • Rose C.M.
      • Tea J.S.
      • Wall C.E.
      • Verschueren E.
      • Cheung T.K.
      • Kirkpatrick D.S.
      • Bingol B.
      Dynamic regulation of mitochondrial import by the ubiquitin system.
      ). In agreement with the previous study, Phu et al. (
      • Phu L.
      • Rose C.M.
      • Tea J.S.
      • Wall C.E.
      • Verschueren E.
      • Cheung T.K.
      • Kirkpatrick D.S.
      • Bingol B.
      Dynamic regulation of mitochondrial import by the ubiquitin system.
      ) demonstrated that USP30 regulates TOM complex component ubiquitination, which may serve as a potential ubiquitin seed for PINK1-Parkin function. Importantly, USP30 inhibition enhanced ubiquitin phosphorylation and mitophagy even in the absence of Parkin, placing USP30 upstream of Parkin. This study supports USP30 as a possible invention strategy for mitophagy enhancement even in the context of PINK1 or Parkin dysfunction.
      Expression of several autophagy adaptor proteins involved in mitophagy, including p62, is positively regulated by the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2). Based on this mechanism, an Nrf2 pharmacological inducer, known as p62-mediated mitophagy inducer (PMI), has been developed (Table 1) (
      • East D.A.
      • Fagiani F.
      • Crosby J.
      • Georgakopoulos N.D.
      • Bertrand H.
      • Schaap M.
      • Fowkes A.
      • Wells G.
      • Campanella M.
      PMI: a ΔΨm independent pharmacological regulator of mitophagy.
      ). PMI stabilizes Nrf2, resulting in higher p62 expression and induction of mitophagy. In contrast with previously reported compounds exploiting this mechanism such as sulforaphane (Table 1), PMI does not contain a covalent-binding motif and may have less toxic potential (
      • East D.A.
      • Fagiani F.
      • Crosby J.
      • Georgakopoulos N.D.
      • Bertrand H.
      • Schaap M.
      • Fowkes A.
      • Wells G.
      • Campanella M.
      PMI: a ΔΨm independent pharmacological regulator of mitophagy.
      ).

      Targeted in silico screening to identify novel enhancers of mitophagy

      Mitochondrial motility is critical in neuronal physiology. Modulating mitochondrial dynamics to facilitate mitophagy might be a promising approach, especially in the context of highly polarized neurons where mitochondrial trafficking plays an important role in cell physiology. Miro1 is an OMM GTPase participating in mitochondrial trafficking and impairment of Miro1 clearance is associated with both familial and sporadic PD (
      • Hsieh C.H.
      • Shaltouki A.
      • Gonzalez A.E.
      • Bettencourt da Cruz A.
      • Burbulla L.F.
      • St Lawrence E.
      • Schule B.
      • Krainc D.
      • Palmer T.D.
      • Wang X.
      Functional impairment in Miro degradation and mitophagy is a shared feature in familial and sporadic Parkinson's disease.
      ). Ubiquitination and degradation of Miro1 is a critical step in mitophagy initiation (
      • Wang X.
      • Winter D.
      • Ashrafi G.
      • Schlehe J.
      • Wong Y.L.
      • Selkoe D.
      • Rice S.
      • Steen J.
      • LaVoie M.J.
      • Schwarz T.L.
      PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility.
      ). Recent efforts to target Miro1 in the context of PD have led to the characterization of Compound 3 as a mitophagy inducer (Table 1) (
      • Hsieh C.H.
      • Li L.
      • Vanhauwaert R.
      • Nguyen K.T.
      • Davis M.D.
      • Bu G.
      • Wszolek Z.K.
      • Wang X.
      Miro1 marks Parkinson's disease subset and Miro1 reducer rescues neuron loss in Parkinson's models.
      ). For compound identification, machine learning was applied to predict the docking capacity between molecules and protein structures based on available knowledge. This approach was subsequently used to virtually screen for compounds able to bind C-terminal region of Miro1, the region being the minimal element required to be recognized and ubiquitination by Parkin. Starting from a large library of over 6 million commercially available molecules, the study narrowed down the drug candidates to 11 promising compounds, which were tested in vitro, four of which showed activity. Further characterization revealed that Compound 3 (Table 1) reduced Miro1 levels after mitochondrial depolarization in patient-derived PD fibroblasts, showing a neuroprotective effect, both in vivo and in vitro models (
      • Hsieh C.H.
      • Li L.
      • Vanhauwaert R.
      • Nguyen K.T.
      • Davis M.D.
      • Bu G.
      • Wszolek Z.K.
      • Wang X.
      Miro1 marks Parkinson's disease subset and Miro1 reducer rescues neuron loss in Parkinson's models.
      ).
      Recently, in silico screening identified a novel inhibitor (BC1464) of FBXO7, an E3-ligase complex adaptor protein (described above). Interestingly, the authors identified that FBXO7 can target PINK1 for degradation (
      • Liu Y.
      • Lear T.B.
      • Verma M.
      • Wang K.Z.
      • Otero P.A.
      • McKelvey A.C.
      • Dunn S.R.
      • Steer E.
      • Bateman N.W.
      • Wu C.
      • Jiang Y.
      • Weathington N.M.
      • Rojas M.
      • Chu C.T.
      • Chen B.B.
      • et al.
      Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.
      ). The substrate-binding FP-domain within FBXO7 was interrogated as a target region for small molecule interactions. Starting from a virtual library of 3 million compounds, docking experiments on a three-dimensional (3D) model of the FP-domain allowed the authors to narrow down to 20 hit compounds to perform further biochemical studies. From these compounds, BC1464 increased PINK1 levels by preventing PINK1-FBXO7 interaction and subsequent PINK1 degradation, without loss of mitochondrial membrane potential. Importantly, BC1464 prevented MPP+-induced cell death in vitro and protected human fibroblasts derived from PD patients carrying LRRK2 mutations exposed to 6-OHDA (
      • Liu Y.
      • Lear T.B.
      • Verma M.
      • Wang K.Z.
      • Otero P.A.
      • McKelvey A.C.
      • Dunn S.R.
      • Steer E.
      • Bateman N.W.
      • Wu C.
      • Jiang Y.
      • Weathington N.M.
      • Rojas M.
      • Chu C.T.
      • Chen B.B.
      • et al.
      Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.
      ). Together, these studies highlight the impact of in silico screening and advances in machine learning, processing power, and computational biology to help yield promising hit molecules.

      Phenotypic screening strategies for mitophagy enhancement

      PD is a complex multifactorial disease and the complex nature has hampered PD drug development. One approach with potential to overcome previous methodological limitations is the application of phenotypic screening. Phenotypic screens aim to identify hit compounds by measuring the effect of the molecules on a disease-related phenotype. This screening paradigm provides an unbiased approach as it does not require a specific drug target. Output from a phenotypic assay should both reflect and be able to predict success in modulating the physiological endpoint of interest. This assumes a shared mechanistic basis between bioassay endpoint and disease phenotype, which is necessary to create robust predictive power for therapeutic development. Together these elements provide the ability to identify novel small molecules and novel biological modifiers of mitophagy.
      Moskal and coworkers (
      • Moskal N.
      • Riccio V.
      • Bashkurov M.
      • Taddese R.
      • Datti A.
      • Lewis P.N.
      • Angus McQuibban G.
      ROCK inhibitors upregulate the neuroprotective Parkin-mediated mitophagy pathway.
      ) recently applied a high-throughput screen (HTS), employing Parkin translocation into the mitochondria as a readout of mitophagy activation. The authors stably expressed GFP-tagged Parkin in HEK293 cells and, using a machine-learning approach for image analysis, identified hit compounds enhancing GFP-Parkin translocation. The study identified SR3677, a Rho-associated coiled-coil containing protein kinase 2 (ROCK-2) inhibitor, as a hit compound. Further characterization suggested that SR3677 enhances Parkin-mediated mitophagy, potentially via increased recruitment and activity of Hexokinase II, promoting Parkin translocation (
      • Moskal N.
      • Riccio V.
      • Bashkurov M.
      • Taddese R.
      • Datti A.
      • Lewis P.N.
      • Angus McQuibban G.
      ROCK inhibitors upregulate the neuroprotective Parkin-mediated mitophagy pathway.
      ). SR3677 was found to be protective in an in vivo model of Parkinsonism. The authors caution, however, that the effect of ROCK inhibition may be via destabilization of the actin cytoskeleton and encapsulation of depolarized mitochondria by F-actin cages, hence the mechanism requires confirmation (
      • Moskal N.
      • Riccio V.
      • Bashkurov M.
      • Taddese R.
      • Datti A.
      • Lewis P.N.
      • Angus McQuibban G.
      ROCK inhibitors upregulate the neuroprotective Parkin-mediated mitophagy pathway.
      ).
      Another recent HTS assessed degradation of luciferase-tagged mitofusin-1 (MFN-1) as a readout of Parkin activity. MFN-1 is an OMM GTPase mediating mitochondrial fusion (
      • Escobar-Henriques M.
      • Joaquim M.
      Mitofusins: disease gatekeepers and hubs in mitochondrial quality control by E3 ligases.
      ) and has a critical role in mitophagy. On damaged mitochondria PINK1 phosphorylates MFN-1, contributing to recruitment of Parkin, which in turns ubiquitinates MFN-1, resulting in MFN-1 degradation and mitochondrial fragmentation (
      • Rodolfo C.
      • Campello S.
      • Cecconi F.
      Mitophagy in neurodegenerative diseases.
      ). Here, mitophagy was assessed in HeLa cells stably transfected with luciferase-tagged MFN-1, in the presence and absence of Parkin expression to determine Parkin dependence. Using this approach two new molecules, T0466 and T0467, were identified (Table 1) from a library of ∼45,000 compounds. Both compounds were able to induce Parkin translocation to the mitochondria without loss of mitochondrial membrane potential or toxicity in DA neurons. Additionally, both compounds improve motor defects in the PINK1 knockout mitochondrial degeneration Drosophila model (
      • Shiba-Fukushima K.
      • Inoshita T.
      • Sano O.
      • Iwata H.
      • Ishikawa K.I.
      • Okano H.
      • Akamatsu W.
      • Imai Y.
      • Hattori N.
      A cell-based high-throughput screening identified two compounds that enhance PINK1-parkin signaling.
      ).
      Mitochondrial turnover is vital to maintain long-term mitochondrial capacity. As such, mitochondrial biogenesis is intimately linked to mitophagy. Considering this, a recent study using high-content analysis of Mito-Timer reporter was developed (
      • Cerqueira F.M.
      • Kozer N.
      • Petcherski A.
      • Baranovski B.M.
      • Wolf D.
      • Assali E.A.
      • Roth Y.
      • Gazit R.
      • Barr H.
      • Lewis E.C.
      • Las G.
      • Shirihai O.S.
      MitoTimer-based high-content screen identifies two chemically-related benzothiophene derivatives that enhance basal mitophagy.
      ). Mito-Timer is based on a mitochondrially targeted dsRed1-E5, a mutated form of dsRed fluorescent protein. dsRed1-E5 evolves from green to red fluorescence in a time of 18 to 20 h, allowing measurement of mitochondrial age and turnover (
      • Hernandez G.
      • Thornton C.
      • Stotland A.
      • Lui D.
      • Sin J.
      • Ramil J.
      • Magee N.
      • Andres A.
      • Quarato G.
      • Carreira R.S.
      • Sayen M.R.
      • Wolkowicz R.
      • Gottlieb R.A.
      MitoTimer: a novel tool for monitoring mitochondrial turnover.
      ,
      • Stotland A.
      • Gottlieb R.A.
      α-MHC MitoTimer mouse: in vivo mitochondrial turnover model reveals remarkable mitochondrial heterogeneity in the heart.
      ). Using the time-dependent fluorescent properties of Mito-Timer, a study found two new mitophagy inducers, SPB08007 and MWP00839 (Table 1), from a library of ∼15,000 molecules. Both compounds were able to increase mitochondrial turnover by stimulating mitophagy without causing reduction of mitochondrial membrane potential or increased superoxide formation (
      • Cerqueira F.M.
      • Kozer N.
      • Petcherski A.
      • Baranovski B.M.
      • Wolf D.
      • Assali E.A.
      • Roth Y.
      • Gazit R.
      • Barr H.
      • Lewis E.C.
      • Las G.
      • Shirihai O.S.
      MitoTimer-based high-content screen identifies two chemically-related benzothiophene derivatives that enhance basal mitophagy.
      ).
      HTS relies on the availability of robust, specific readouts to measure the process of interest. Mito-SRAI has been recently described as a new mitophagy reporter. Using a human glioblastoma H4 cell line stably expressing Mito-SRAI, the authors successfully applied this new probe to a large-scale high-content image analysis approach to identify novel mitophagy enhancers within a library of ∼76,000 compounds (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ). The screen focused on compounds inducing Parkin-dependent, Bafilomycin A-1 sensitive mitophagy and does not affect mitochondrial membrane potential, identifying T-271 as a novel mitophagy inducer (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ), while also validating a novel mitophagy reporter system.
      Finally, using the mt-Keima reporter in HeLa cells and the LOPAC1280 chemical library, several mitophagy-enhancing compounds have been identified. One compound, the anticancer drug gemcitabine, was identified as inducing mitophagy independent of Parkin (Table 1) but dependent on MUL1. Gemcitabine caused the stabilization of PINK1 without reducing mitochondrial membrane potential (
      • Igarashi R.
      • Yamashita S.I.
      • Yamashita T.
      • Inoue K.
      • Fukuda T.
      • Fukuchi T.
      • Kanki T.
      Gemcitabine induces Parkin-independent mitophagy through mitochondrial-resident E3 ligase MUL1-mediated stabilization of PINK1.
      ).
      Together, these studies have demonstrated the importance of high-throughput/high-content phenotypic screening and the development of innovative image analysis pipelines in the identification of novel compounds. The compounds identified are not only interesting therapeutic candidates, targeting different components of the mitophagy machinery without causing toxicity, but also provide insights into the molecular biology of mitophagy in both physiological and pathological context. However, these studies often lack information about the specific target of the newly identified molecules, and subsequent deconvolution is necessary to further understand the precise mechanism modulating the cellular functions.

      Novel modalities to enhance mitophagy: targeted protein degradation using AUTACs

      PINK1-Parkin activation generates a feed-forward loop in which poly-ubiquitin chains accumulate on OMM proteins, targeting mitochondria for degradation via autophagosomes (
      • Ordureau A.
      • Sarraf S.A.
      • Duda D.M.
      • Heo J.M.
      • Jedrychowski M.P.
      • Sviderskiy V.O.
      • Olszewski J.L.
      • Koerber J.T.
      • Xie T.
      • Beausoleil S.A.
      • Wells J.A.
      • Gygi S.P.
      • Schulman B.A.
      • Harper J.W.
      Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis.
      ). Based on this system, autophagy targeting chimera 4 (AUTAC4) has been recently described (Table 1). AUTAC4 is a bivalent chimera combining a 2-phenylindole derivative, a ligand of the OMM translocator protein (18 kDa) TSPO, and a guanine tag, to induce Lys63-linked poly-ubiquitination on the OMM, separated by a linker. S-guanylation has been identified as a moiety that can independently trigger cargo-selective autophagy (
      • Takahashi D.
      • Moriyama J.
      • Nakamura T.
      • Miki E.
      • Takahashi E.
      • Sato A.
      • Akaike T.
      • Itto-Nakama K.
      • Arimoto H.
      AUTACs: cargo-specific degraders using selective autophagy.
      ). This chemical arrangement together results in targeting of mitochondria for mitophagic degradation. Importantly, AUTAC4 specifically triggered mitophagy only with depolarized and fragmented mitochondria, as after uncoupler treatment, in HeLa cells, independently of PINK1 and Parkin. It has been hypothesized that intact mitochondria and those not excised from the mitochondrial network are too large to be degraded by a phagosome (
      • Takahashi D.
      • Moriyama J.
      • Nakamura T.
      • Miki E.
      • Takahashi E.
      • Sato A.
      • Akaike T.
      • Itto-Nakama K.
      • Arimoto H.
      AUTACs: cargo-specific degraders using selective autophagy.
      ), leading to the specificity of AUTAC4 for small, damaged mitochondrial fragments. Altogether, AUTAC4 and modulation of poly-ubiquitin chain formation are promising approaches in developing novel mitophagy inducers. However, limitations in physicochemical properties of these molecules may affect their therapeutic development potential (
      • Ermondi G.
      • Vallaro M.
      • Caron G.
      Degraders early developability assessment: face-to-face with molecular properties.
      ).

      Small molecule enhancers of mitophagy with complex or undefined mechanism

      There are an increasing number of promising mitophagy inducers with a good safety profile but without a fully elucidated mechanism of action. The elegantin Urolithin A (UA) has been extensively studied in clinical trials to prevent aging associated changes. UA has been observed to increase life span and cognition in C. elegans as well as improve muscular function in rodents (
      • Ryu D.
      • Mouchiroud L.
      • Andreux P.A.
      • Katsyuba E.
      • Moullan N.
      • Nicolet-Dit-Félix A.A.
      • Williams E.G.
      • Jha P.
      • Lo Sasso G.
      • Huzard D.
      • Aebischer P.
      • Sandi C.
      • Rinsch C.
      • Auwerx J.
      Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents.
      ). The mechanism of action is not fully understood, although beneficial effects of UA are believed, at least in part, to be due to induction of PINK1-Parkin dependent mitophagy (
      • Ryu D.
      • Mouchiroud L.
      • Andreux P.A.
      • Katsyuba E.
      • Moullan N.
      • Nicolet-Dit-Félix A.A.
      • Williams E.G.
      • Jha P.
      • Lo Sasso G.
      • Huzard D.
      • Aebischer P.
      • Sandi C.
      • Rinsch C.
      • Auwerx J.
      Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents.
      ,
      • Fang E.F.
      • Hou Y.
      • Palikaras K.
      • Adriaanse B.A.
      • Kerr J.S.
      • Yang B.
      • Lautrup S.
      • Hasan-Olive M.M.
      • Caponio D.
      • Dan X.
      • Rocktäschel P.
      • Croteau D.L.
      • Akbari M.
      • Greig N.H.
      • Fladby T.
      • et al.
      Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease.
      ) (Table 1). Nicotinamide, a precursor to nicotinamide adenine dinucleotide (NAD+), has been extensively studied in clinical trials for age-related diseases including PD and Alzheimer's disease (NCT03568968, NCT03816020). The therapeutic potential of nicotinamide has been associated with the NAD+-dependent deacetylase, SIRT1 (
      • Fang E.F.
      Mitophagy and NAD(+) inhibit Alzheimer disease.
      ,
      • Fang E.F.
      • Scheibye-Knudsen M.
      • Brace L.E.
      • Kassahun H.
      • SenGupta T.
      • Nilsen H.
      • Mitchell J.R.
      • Croteau D.L.
      • Bohr V.A.
      Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction.
      ). SIRT1 function is dependent on the intracellular NAD+ levels, and a high ratio of NAD+/NADH is associated with longevity and enhanced mitochondrial metabolism (
      • Mouchiroud L.
      • Houtkooper R.H.
      • Moullan N.
      • Katsyuba E.
      • Ryu D.
      • Cantó C.
      • Mottis A.
      • Jo Y.S.
      • Viswanathan M.
      • Schoonjans K.
      • Guarente L.
      • Auwerx J.
      The NAD(+)/Sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling.
      ). Nicotinamide has been proposed to induce mitophagy via SIRT1 activation, resulting in mitochondrial clearance independent of mitochondrial membrane potential depolarization (
      • Jang S.Y.
      • Kang H.T.
      • Hwang E.S.
      Nicotinamide-induced mitophagy: event mediated by high NAD+/NADH ratio and SIRT1 protein activation.
      ,
      • Fang E.F.
      • Hou Y.
      • Lautrup S.
      • Jensen M.B.
      • Yang B.
      • SenGupta T.
      • Caponio D.
      • Khezri R.
      • Demarest T.G.
      • Aman Y.
      • Figueroa D.
      • Morevati M.
      • Lee H.-J.
      • Kato H.
      • Kassahun H.
      • et al.
      NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome.
      ) (Table 1). Deferiprone, an iron chelator, has undergone Phase II clinical trials to evaluate this mechanism to reduce oxidative stress in the SNc of PD patients (NCT02655315, NCT01539837, NCT00943748, NCT02728843). Clinical data has emerged, demonstrating reduction in iron content in specific brain areas, a trend for improvement in motor function and improved quality of life without significant side effects (
      • Martin-Bastida A.
      • Ward R.J.
      • Newbould R.
      • Piccini P.
      • Sharp D.
      • Kabba C.
      • Patel M.C.
      • Spino M.
      • Connelly J.
      • Tricta F.
      • Crichton R.R.
      • Dexter D.T.
      Brain iron chelation by deferiprone in a phase 2 randomised double-blinded placebo controlled clinical trial in Parkinson's disease.
      ,
      • Devos D.
      • Moreau C.
      • Devedjian J.C.
      • Kluza J.
      • Petrault M.
      • Laloux C.
      • Jonneaux A.
      • Ryckewaert G.
      • Garçon G.
      • Rouaix N.
      • Duhamel A.
      • Jissendi P.
      • Dujardin K.
      • Auger F.
      • Ravasi L.
      • et al.
      Targeting chelatable iron as a therapeutic modality in Parkinson's disease.
      ). Iron chelation can trigger mitophagy in vitro and in vivo, independently of PINK1 and mitochondrial membrane potential dissipation although the exact mechanism needs to be further elucidated (
      • Allen G.F.
      • Toth R.
      • James J.
      • Ganley I.G.
      Loss of iron triggers PINK1/Parkin-independent mitophagy.
      ,
      • Schiavi A.
      • Maglioni S.
      • Palikaras K.
      • Shaik A.
      • Strappazzon F.
      • Brinkmann V.
      • Torgovnick A.
      • Castelein N.
      • De Henau S.
      • Braeckman B.P.
      • Cecconi F.
      • Tavernarakis N.
      • Ventura N.
      Iron-starvation-induced mitophagy mediates lifespan extension upon mitochondrial stress in C. elegans.
      ) (Table 1). Finally, the anthelmintic drug ivermectin has also recently been described as potent inducer of ubiquitin-dependent mitophagy (Table 1), working via a mechanism independent of PINK1 and Parkin potentially involving other E3-ligases, TRAF2, cIAP1, and cIAP2 (
      • Zachari M.
      • Gudmundsson S.R.
      • Li Z.
      • Manifava M.
      • Shah R.
      • Smith M.
      • Stronge J.
      • Karanasios E.
      • Piunti C.
      • Kishi-Itakura C.
      • Vihinen H.
      • Jokitalo E.
      • Guan J.L.
      • Buss F.
      • Smith A.M.
      • et al.
      Selective autophagy of mitochondria on a ubiquitin-endoplasmic-reticulum platform.
      ) (Table 1).
      In summary, many compounds are available to perturb mitophagy and several show efficacy in disease models. One caveat of many of the studies described above is the use of mitochondrial toxins to induce mitophagy. These compounds trigger mitophagy via mitochondrial damage, either by inhibition of the respiratory chain, ROS generation (as in the case of rotenone or antimycin A), or directly collapsing mitochondrial membrane potential (such as the ionophores CCCP, FCCP, and valinomycin). These compounds have proven invaluable in understanding the cell biology around mitophagy. However, the mechanisms to induce mitophagy in the experimental systems must be considered in wider implications of the results. A second caveat is the use of Parkin overexpression. Expressing high levels of the E3-ligase Parkin may result in supraphysiological ubiquitination of proteins. Reintroduction of Parkin into Parkin-negative cells has been used to good effect in determining compound mechanism; however, expression levels should, ideally, be carefully titrated to represent endogenous concentrations within related cell types. Nevertheless, good progress has been made over the past 5 years and therapeutic promise of this pathway continues to develop.
      Selection of an appropriate intervention point for mitophagy accelerating therapeutics must also consider the growing evidence of autophagy-independent, lysosome-dependent mitochondrial degradation through PINK1 or Parkin, outside the canonical mitophagy pathway (
      • Vincow E.S.
      • Merrihew G.
      • Thomas R.E.
      • Shulman N.J.
      • Beyer R.P.
      • MacCoss M.J.
      • Pallanck L.J.
      The PINK1–Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo.
      ). PINK1 and Parkin have been associated with formation of mitochondrially derived vesicles (MDVs), which traffic damaged mitochondrial components directly to the lysosome as a response to oxidative stress (
      • Soubannier V.
      • McLelland G.L.
      • Zunino R.
      • Braschi E.
      • Rippstein P.
      • Fon E.A.
      • McBride H.M.
      A vesicular transport pathway shuttles cargo from mitochondria to lysosomes.
      ,
      • McLelland G.L.
      • Soubannier V.
      • Chen C.X.
      • McBride H.M.
      • Fon E.A.
      Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control.
      ,
      • McLelland G.L.
      • Lee S.A.
      • McBride H.M.
      • Fon E.A.
      Syntaxin-17 delivers PINK1/parkin-dependent mitochondrial vesicles to the endolysosomal system.
      ). Furthermore, damaged respiratory chain components have been observed to be selectively eliminated from mitochondria in a PINK1- and Parkin-dependent manner, distinct from en masse mitochondrial degradation (
      • Vincow E.S.
      • Merrihew G.
      • Thomas R.E.
      • Shulman N.J.
      • Beyer R.P.
      • MacCoss M.J.
      • Pallanck L.J.
      The PINK1–Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo.
      ). These data suggest that accelerating the autophagic steps of PINK1–Parkin signaling may not be the only route to therapeutic benefit. Appropriate in vitro models and endpoints may allow identification and targeting of different aspects of the PINK1–Parkin system, though careful validation will be necessary.

      In vivo models for proof-of-mechanism and proof-of-concept studies

      To date, only a limited number of mitophagy-enhancing compounds have been assessed in vivo. However, several in vivo models exist for investigating the contribution of PINK1 and Parkin to mitophagy and disease. These models may provide a platform to assess future pharmacological enhancers of mitophagy.

      Pharmacodynamic modeling using mitophagy reporter mice

      In vivo models are required to enable analysis of the spatiotemporal dynamics of mitophagy as a pharmacodynamic endpoint for testing novel therapeutic candidates. To that end, mice ubiquitously expressing the mt-Keima reporter at the Hip11 locus and mito-QC at the Rosa26 locus have been generated (
      • Sun N.
      • Yun J.
      • Liu J.
      • Malide D.
      • Liu C.
      • Rovira I.I.
      • Holmström K.M.
      • Fergusson M.M.
      • Yoo Y.H.
      • Combs C.A.
      • Finkel T.
      Measuring in vivo mitophagy.
      • McWilliams T.G.
      • Prescott A.R.
      • Allen G.F.
      • Tamjar J.
      • Munson M.J.
      • Thomson C.
      • Muqit M.M.
      • Ganley I.G.
      mito-QC illuminates mitophagy and mitochondrial architecture in vivo.
      ). A small number of studies using either mt-Keima or mito-QC “mitophagy reporter” models have identified pervasive basal mitophagy, often with significant heterogeneity even within the same tissue type (Table 2) (
      • Sun N.
      • Malide D.
      • Liu J.
      • Rovira I.I.
      • Combs C.A.
      • Finkel T.
      A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.
      ,
      • McWilliams T.G.
      • Prescott A.R.
      • Allen G.F.
      • Tamjar J.
      • Munson M.J.
      • Thomson C.
      • Muqit M.M.
      • Ganley I.G.
      mito-QC illuminates mitophagy and mitochondrial architecture in vivo.
      ). Numerous metabolic and pathogenic insults, including hypoxia, expression of mutant huntingtin (HTT), and accumulation of mtDNA mutations have been demonstrated to perturb mitophagy (
      • Sun N.
      • Malide D.
      • Liu J.
      • Rovira I.I.
      • Combs C.A.
      • Finkel T.
      A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.
      • McWilliams T.G.
      • Prescott A.R.
      • Montava-Garriga L.
      • Ball G.
      • Singh F.
      • Barini E.
      • Muqit M.M.K.
      • Brooks S.P.
      • Ganley I.G.
      Basal mitophagy occurs independently of PINK1 in mouse tissues of high metabolic demand.
      ). Interestingly, global knockout of the key mitophagy gene Pink1 failed to modulate basal mitophagy in any tissue analyzed in mice (mito-QC; Pink1−/− mouse) (
      • McWilliams T.G.
      • Prescott A.R.
      • Montava-Garriga L.
      • Ball G.
      • Singh F.
      • Barini E.
      • Muqit M.M.K.
      • Brooks S.P.
      • Ganley I.G.
      Basal mitophagy occurs independently of PINK1 in mouse tissues of high metabolic demand.
      ). However, following exhaustive exercise-induced metabolic stress, PINK1-dependent mitophagy was observed in the heart, and a reduced mt-Keima signal observed following Pink1 knockout (mt-Keima; Pink1−/− mouse) (
      • Sliter D.A.
      • Martinez J.
      • Hao L.
      • Chen X.
      • Sun N.
      • Fischer T.D.
      • Burman J.L.
      • Li Y.
      • Zhang Z.
      • Narendra D.P.
      • Cai H.
      • Borsche M.
      • Klein C.
      • Youle R.J.
      Parkin and PINK1 mitigate STING-induced inflammation.
      ). Recently, a novel FRET mitophagy reporter has been described: Mito-SRAI (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ). Although full in vivo analysis has yet to be completed, AAV-expressed mito-SRAI within SNc has been assessed in mice. Unilateral 6-hydroxydopamine (6-OHDA) administration produced a mitophagy signal in numerous mito-SRAI infected neurons. Interestingly despite loss of midbrain DA neurons following administration of 6-OHDA, the mitophagy signal originated only from tyrosine hydroxylase (TH)-negative (non-DA) neurons (
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      ).
      Table 2In vivo mitophagy reporters and mitochondrial dysfunction-induced neurodegeneration models
      In vivo modelPromoter and expression patternModel descriptionCharacteristics and phenotypeReferences
      Mitophagy Reporter Models
       mt-KeimaHip11 locus (ubiquitous expression)

      CAG-promoter driven expression
      Mitophagy reporter

      Not amenable to chemical fixation
      Mitophagy phenotype [whole body]: considerable heterogeneity in mitophagy within the same tissue. Low levels of mitophagy in the thymus, high in the heart.

      Mitophagy phenotype [brain]: high anatomic variation. Cortex, striatum, and substantia nigra exhibit modest levels of basal mitophagy. Mitophagy greater in dentate gyrus, lateral ventricle, and Purkinje cell layer within the cerebellum. Reduced mitophagy in dentate gyrus of aged mice (3 versus 21 months; 70% reduction).

      Pathological insult: age-related decline in mitophagy in dentate gyrus. Expression of mutant human Huntingtin’s transgene reduced mitophagy in dentate gyrus. Low oxygen (10% oxygen) significantly increased hepatic mitophagy.
      (
      • Sun N.
      • Yun J.
      • Liu J.
      • Malide D.
      • Liu C.
      • Rovira I.I.
      • Holmström K.M.
      • Fergusson M.M.
      • Yoo Y.H.
      • Combs C.A.
      • Finkel T.
      Measuring in vivo mitophagy.
      )
       Mito-QC (mCherry-GFP- FIS1(aa.101–152)Rosa26 locus (ubiquitous expression)

      CAG-promoter driven expression
      Mitophagy reporter

      Amenable to chemical fixation
      Mitophagy phenotype [whole body]: considerable heterogeneity of mitophagy within the same tissue. High levels within cortex of adult kidney, differential mitophagy between proximal (high mitophagy) and distal (low mitophagy) convoluted tubules within kidney.

      Mitophagy phenotype [brain]: pronounced mito-lysosomes within Purkinje cell layer. Significant mitochondrial turnover in the Purkinje somata.

      Pathological insult: no change in mitophagy in any tissue analyzed with Pink1 knock-out.
      (
      • McWilliams T.G.
      • Prescott A.R.
      • Allen G.F.
      • Tamjar J.
      • Munson M.J.
      • Thomson C.
      • Muqit M.M.
      • Ganley I.G.
      mito-QC illuminates mitophagy and mitochondrial architecture in vivo.
      ,
      • McWilliams T.G.
      • Prescott A.R.
      • Montava-Garriga L.
      • Ball G.
      • Singh F.
      • Barini E.
      • Muqit M.M.K.
      • Brooks S.P.
      • Ganley I.G.
      Basal mitophagy occurs independently of PINK1 in mouse tissues of high metabolic demand.
      )
       mt-SRAI-CL1-PESTAAV- expression into right SNcMitophagy reporter

      Amenable to chemical fixation
      Mitophagy phenotype [Brain]: numerous infected neurons positive for mitophagy signal in 6-OHDA-injected mice (same route as for viral infection); mitophagy signal in TH-negative (non-DA) neurons only.(
      • Katayama H.
      • Hama H.
      • Nagasawa K.
      • Kurokawa H.
      • Sugiyama M.
      • Ando R.
      • Funata M.
      • Yoshida N.
      • Homma M.
      • Nishimura T.
      • Takahashi M.
      • Ishida Y.
      • Hioki H.
      • Tsujihata Y.
      • Miyawaki A.
      Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration.
      )
      Genetic Neurodegenerative Models of Mitochondrial Origin
      Disruption of mtDNA Homeostasis
      Mutator (POLγAD257A)PolγA locus; ubiquitous expressionHomozygous knock-in mutant of PolγA (nucleus-encoded catalytic subunit of mtDNA polymerase)

      D257 A mutation causes loss of 3′-5′ exonuclease activity necessary for proof-reading newly synthesized mtDNA
      Aging phenotype: decreased lifespan and premature onset of age-associated phenotypes (weight loss, reduced subcutaneous fat, alopecia, kyphosis, osteoporosis, anemia, reduced fertility, and cardiac hypertrophy).

      Neuronal phenotype: No neurodegeneration up to 12 months. Intact nigralstriatal pathway, no astrogliosis.

      POLγAD257A (Mutator); Parkin−/−: large reduction in TH-positive (DA) neurons in midbrain. Reduced striatal dopamine, decreased DA metabolites. L-DOPA responsive motor phenotype. No neuroinflammation or Lewy body formation.

      Mitochondrial phenotype: 3-5x increase in mtDNA point mutations, increased mtDNA deletions. Reduced mtDNA copy number. Random point mutations in genes for respiratory chain subunits. Increased apoptosis. Little age-related decline in cardiac mitochondrial fitness. Increased megamitochondria in aged hearts (6 months).

      Mitophagy phenotype: Increased phospho-Ser65-ubiquitin in cortex (not liver), increased hepatic mitophagy (POLγAD257A; mt-Keima), reduced Parkin protein expression.

      POLγAD257A (Mutator); Parkin−/−mice: strong inflammatory phenotype (high serum IL-6, IFNβ1, TNF, IL-1β, CCL2, IL-12(p70), IL-13, IL-17, CXCL1 and CCL4). No change in mtDNA mutation frequency compared with POLγAD257A; Parkin+/+, but reduced mtDNA pathogenicity. Reduced ETC complex activity (complex I and III).

      POLγAD257A (Mutator); Parkin-Tg and POLγAD257A (Mutator); Parkin−/−: Parkin fails to prevent accelerated cardiac aging.
      (
      • Sun N.
      • Yun J.
      • Liu J.
      • Malide D.
      • Liu C.
      • Rovira I.I.
      • Holmström K.M.
      • Fergusson M.M.
      • Yoo Y.H.
      • Combs C.A.
      • Finkel T.
      Measuring in vivo mitophagy.
      ,
      • Sliter D.A.
      • Martinez J.
      • Hao L.
      • Chen X.
      • Sun N.
      • Fischer T.D.
      • Burman J.L.
      • Li Y.
      • Zhang Z.
      • Narendra D.P.
      • Cai H.
      • Borsche M.
      • Klein C.
      • Youle R.J.
      Parkin and PINK1 mitigate STING-induced inflammation.
      ,
      • Trifunovic A.
      • Wredenberg A.
      • Falkenberg M.
      • Spelbrink J.N.
      • Rovio A.T.
      • Bruder C.E.
      • Bohlooly Y.M.
      • Gidlöf S.
      • Oldfors A.
      • Wibom R.
      • Törnell J.
      • Jacobs H.T.
      • Larsson N.G.
      Premature ageing in mice expressing defective mitochondrial DNA polymerase.
      ,
      • Hauser D.N.
      • Primiani C.T.
      • Langston R.G.
      • Kumaran R.
      • Cookson M.R.
      The Polg mutator phenotype does not cause dopaminergic neurodegeneration in DJ-1-deficient mice.
      ,
      • Pickrell A.M.
      • Huang C.H.
      • Kennedy S.R.
      • Ordureau A.
      • Sideris D.P.
      • Hoekstra J.G.
      • Harper J.W.
      • Youle R.J.
      Endogenous parkin preserves dopaminergic substantia nigral neurons following mitochondrial DNA mutagenic stress.
      ,
      • Woodall B.P.
      • Orogo A.M.
      • Najor R.H.
      • Cortez M.Q.
      • Moreno E.R.
      • Wang H.
      • Divakaruni A.S.
      • Murphy A.N.
      • Gustafsson Å B.
      Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice.
      ,
      • Kujoth G.C.
      • Hiona A.
      • Pugh T.D.
      • Someya S.
      • Panzer K.
      • Wohlgemuth S.E.
      • Hofer T.
      • Seo A.Y.
      • Sullivan R.
      • Jobling W.A.
      • Morrow J.D.
      • Van Remmen H.
      • Sedivy J.M.
      • Yamasoba T.
      • Tanokura M.
      • et al.
      Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.
      )
      mitoPARK (DAT-cre x TfamloxP)DAT promoter: DA neuron expression

      Homozygous deletion of mitochondrial transcription factor A (Tfam)
      TFAM knockout in midbrain DA neurons

      TFAM knockout leads to mtDNA depletion and abolishes mtDNA expression.
      Neuronal phenotype: adult onset of slowly progressive motor impairment, loss of TH-positive neurons and TH-positive terminals in striatum; depletion of nigral and striatal dopamine, age-dependent reduction in soma size and neurite branching in DA neurons. Loss of dopamine in olfactory bulb, intraneuronal inclusions, cognitive dysfunction (preceding motor dysfunction). Gastrointestinal dysfunction, gut inflammation, and gut-microbiome changes. Age-dependent L-DOPA responsive motor phenotype.

      Mitochondrial phenotype: Severe respiratory chain deficiency, reduced cytochrome oxidase subunit I expression and activity in midbrain DA neurons, fragmentation, large mitochondrial aggregates. Reduced distal axonal mitochondria [dysfunctional axonal mitochondrial transport].

      Mitophagy phenotype: Endogenous Parkin recruitment not detected [potential technical limitations or low expression].

      TfamloxP/loxP; DAT-cre; AAV-Parkin-mCherry: no Parkin colocalization with mitochondria.

      TfamloxP/loxP; DAT-cre; Parkin−/−: no Parkin-dependent effect on mitochondrial aggregates, mitochondrial morphology, locomotion, or TH-positive cell loss in SNc.
      (
      • Ekstrand M.I.
      • Terzioglu M.
      • Galter D.
      • Zhu S.
      • Hofstetter C.
      • Lindqvist E.
      • Thams S.
      • Bergstrand A.
      • Hansson F.S.
      • Trifunovic A.
      • Hoffer B.
      • Cullheim S.
      • Mohammed A.H.
      • Olson L.
      • Larsson N.G.
      Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons.
      ,
      • Galter D.
      • Pernold K.
      • Yoshitake T.
      • Lindqvist E.
      • Hoffer B.
      • Kehr J.
      • Larsson N.G.
      • Olson L.
      MitoPark mice mirror the slow progression of key symptoms and L-DOPA response in Parkinson's disease.
      ,
      • Sterky F.H.
      • Lee S.
      • Wibom R.
      • Olson L.
      • Larsson N.-G.
      Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo.
      ,
      • Ghaisas S.
      • Langley M.R.
      • Palanisamy B.N.
      • Dutta S.
      • Narayanaswamy K.
      • Plummer P.J.
      • Sarkar S.
      • Ay M.
      • Jin H.
      • Anantharam V.
      • Kanthasamy A.
      • Kanthasamy A.G.
      MitoPark transgenic mouse model recapitulates the gastrointestinal dysfunction and gut-microbiome changes of Parkinson's disease.
      ,
      • Lynch W.B.
      • Tschumi C.W.
      • Sharpe A.L.
      • Branch S.Y.
      • Chen C.
      • Ge G.
      • Li S.
      • Beckstead M.J.
      Progressively disrupted somatodendritic morphology in dopamine neurons in a mouse Parkinson's model.
      )
      PD-mito-PstIDAT promoter-driven tetracycline transactivator protein (tTA)

      Inducible mito-PstI exclusively in DA neurons
      Expression of tetracycline-sensitive mitochondria-targeted restriction enzyme, PstI, in DA neurons

      Mitochondrial matrix localization—COX8A MTS

      Mito-targeted restriction enzyme damages mtDNA in DA neurons
      Neuronal phenotype: progressive degeneration of the DA population within SNc, striatal dopamine depletion, age-dependent loss of TH-positive neurons, L-DOPA reversible motor deficit. Locomotor deficits precede TH-positive cell loss. Absence of inclusions. Motor phenotypes initially arise from a striatal dysfunction.

      Mitochondrial phenotype: double strand breaks in mtDNA, mtDNA depletion, mtDNA deletions, ETC dysfunction.

      Mitophagy phenotype:

      PD-mito-PstI; Parkin−/−: mild acceleration, but no worsening of motor dysfunction and neuronal degeneration.
      (
      • Pickrell A.M.
      • Pinto M.
      • Hida A.
      • Moraes C.T.
      Striatal dysfunctions associated with mitochondrial DNA damage in dopaminergic neurons in a mouse model of Parkinsons disease.
      ,
      • Pinto M.
      • Nissanka N.
      • Moraes C.T.
      Lack of parkin anticipates the phenotype and affects mitochondrial morphology and mtDNA levels in a mouse model of Parkinson's disease.
      )
      Twinkle-duplication (Twinkle-Tg)Transgenic expression of Twinkle (in-frame duplication of aa. 353–365)

      TH promoter: DA neuron expression

      4x Twinkle [mRNA] increase in Twinkle-Tg
      In-frame duplication of the mitochondrial DNA helicase, Twinkle

      Disruption of mtDNA replication
      Neuronal phenotype: motor impairment, decreased TH-positive neurons, age-dependent neurobehavioral deficits.

      Mitochondrial phenotype: Age-dependent increase in mtDNA deletions, reduced mtDNA copy number, mild bioenergetic defects.

      Mitophagy phenotype: reduced Parkin protein expression, increased LC3 protein expression.

      Twinkledup/+; Parkin−/− (TwinkPark): increased mtDNA deletions, reduced mitochondrial function (complex II activity) and compromised bioenergetics. Reduced membrane potential, neurobehavioral deficits, reduced striatal dopamine and increased TH-positive cell loss by 19 months.
      (
      • Song L.
      • Shan Y.
      • Lloyd K.C.K.
      • Cortopassi G.A.
      Mutant Twinkle increases dopaminergic neurodegeneration, mtDNA deletions and modulates Parkin expression.
      ,
      • Song L.
      • McMackin M.
      • Nguyen A.
      • Cortopassi G.
      Parkin deficiency accelerates consequences of mitochondrial DNA deletions and Parkinsonism.
      )
       Disruption of Key Mitochondrial Processes
      ΔOTCTH-Cre; ΔOTC

      Cre-mediated recombination in DA neurons
      Exogenous expression of ΔOTC proposed to induce mitochondrial unfolded protein response (mtUPR) in vivo

      Ornithine transcarbamylase enzyme transgene (OTC normally restricted to liver) – Deletion mutant Δ30–114

      Mitochondrially localized enzyme. Cre-recombination induces mitochondrial unfolded protein response in TH-positive neurons
      Neuronal phenotype: mildly reduced motor function, reduced SNc dopamine content, decreased TH-positive neurons, L-DOPA responsive motor phenotype.

      Mitophagy phenotype:

      Pink1−/− versus Pink1−/−; ΔOTC: reduced DA neurons or reduction in DA content following ΔOTC expression [additive effects of PINK1 loss unknown—no comparison of ΔOTC alone versus Pink1−/−; ΔOTC], no L-DOPA responsive motor phenotype.
      (
      • Moisoi N.
      • Fedele V.
      • Edwards J.
      • Martins L.M.
      Loss of PINK1 enhances neurodegeneration in a mouse model of Parkinson's disease triggered by mitochondrial stress.
      )
      Ndufs4−/−Mox2-cre: Ndufs4LoxP: Ubiquitous expression

      DAT-cre; Ndufs4LoxP: DA neuron expression
      Conditional knockout of ETC complex I subunit, NDUFS4

      Mitochondrial ETC complex I deficiency (activity and expression)
      Neuronal phenotype:

      Ndufs4−/−: TH-positive cell loss, motor deficits, reduced striatal dopamine. Decreased 20S proteasome activity in SNc, decreased neurofilaments in SNc, increased ubiquitinated protein levels in DA neurons in SNc.

      DAT-cre; Ndufs4loxP: no motor deficits, slight decrease in TH-positive neurons at 24 months, no loss of DA nerve terminals, no overt neurodegeneration. Slight decrease in dopamine content and alterations to dopamine homeostasis in striatum. Reduced dopamine release. [Conflicting data around TH-positive cell loss, motor deficits, reduced striatal dopamine].

      Mitochondrial phenotype: reduced complex I expression and activity.

      Mitophagy phenotype: small reduction in PINK1 expression, no change in Parkin expression
      (
      • Kruse S.E.
      • Watt W.C.
      • Marcinek D.J.
      • Kapur R.P.
      • Schenkman K.A.
      • Palmiter R.D.
      Mice with mitochondrial complex I deficiency develop a fatal encephalomyopathy.
      ,
      • Song L.
      • Cortopassi G.
      Mitochondrial complex I defects increase ubiquitin in substantia nigra.
      ,
      • Sterky F.H.
      • Hoffman A.F.
      • Milenkovic D.
      • Bao B.
      • Paganelli A.
      • Edgar D.
      • Wibom R.
      • Lupica C.R.
      • Olson L.
      • Larsson N.G.
      Altered dopamine metabolism and increased vulnerability to MPTP in mice with partial deficiency of mitochondrial complex I in dopamine neurons.
      )
      AAV, adeno-associated virus; CFP, cyan fluorescent protein; DA, dopaminergic; DAT, dopamine transporter; FRET, Förster resonance energy transfer; GFP, green fluorescent protein; mtDNA, mitochondrial DNA; MTS, mitochondrial targeting sequence; mtUPR, mitochondrial unfolded protein response; OTC, ornithine transcarbamylase; SNc, substantia nigra pars compacta; Tg, transgenic; TH, tyrosine hydroxylase; TOLLES, TOLerance of Lysosomal EnvironmentS; VTA, ventral tegmental area.
      Despite limitations associated with each reporter (highlighted above), analysis of mitophagy dynamics in vivo has provided meaningful insight into mitochondrial quality control at an organismal level and holds significant potential for future study. Mitophagy reporters described to date cannot directly distinguish between PINK1–Parkin-dependent and -independent pathways. Full characterization and systematic comparison of loss-of-function (partial or full) or pathogenic mutations within key mitophagy regulators, including but not limited to Pink1 and Parkin, are necessary in each model, especially given differences in reporter localization and behavior. Close examination of PD-related cells and tissues (DA neurons, astrocytes, gastrointestinal tissue), in combination with the correct “pathophysiological trigger” (see below), may be required to validate roles of mitophagy regulators in vivo.

      Disease modeling by genetically-induced mitochondrial dysfunction

      Achievement of preclinical proof-of-concept for a potential therapy relies on the specific, mechanism-of-action (MoA)-governed efficacy in human biology-relevant disease models, such as transgenic, knock-in or knockout animals. Interestingly, there has been a striking failure to recapitulate neurodegeneration in vivo with Pink1−/− and Parkin−/−, with a few exceptions (
      • Lu X.H.
      • Fleming S.M.
      • Meurers B.
      • Ackerson L.C.
      • Mortazavi F.
      • Lo V.
      • Hernandez D.
      • Sulzer D.
      • Jackson G.R.
      • Maidment N.T.
      • Chesselet M.F.
      • Yang X.W.
      Bacterial artificial chromosome transgenic mice expressing a truncated mutant parkin exhibit age-dependent hypokinetic motor deficits, dopaminergic neuron degeneration, and accumulation of proteinase K-resistant alpha-synuclein.
      ,
      • Dave K.D.
      • De Silva S.
      • Sheth N.P.
      • Ramboz S.
      • Beck M.J.
      • Quang C.
      • Switzer 3rd, R.C.
      • Ahmad S.O.
      • Sunkin S.M.
      • Walker D.
      • Cui X.
      • Fisher D.A.
      • McCoy A.M.
      • Gamber K.
      • Ding X.
      • et al.
      Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.
      ). Hypotheses as to these failures are discussed further below and include species differences, developmental adaptation due to germline ablation, or mitophagy-independent roles of these proteins. Pink1−/− and Parkin−/− knockout models themselves are not highly relevant in the search for novel enhancers of mitophagy. PINK1 and Parkin proteins are key targets and their complete loss of protein is not representative of most cases of human disease, though they will be of use to demonstrate target engagement or MoA. Instead, in vivo models with primary mitochondrial dysfunction may be of use. Genetic perturbation within key mitochondrial processes can serve as the pathological trigger to produce a neurodegenerative or aging phenotype and demonstrable alterations in mitophagy. These observations not only support a key role for mitochondrial dysfunction in aging and PD etiology but provide a model in which future therapeutics could be tested. However, given the complexity of PD pathophysiology, it may be unrealistic to expect one in vivo model to accurately recapitulate all elements of PD. Instead, recognition of an appropriate pathophysiological trigger may allow direct analysis of one or more factors contributing to the PD syndrome, whereby pharmacodynamic endpoints for compound-driven disease modification via mitophagy enhancement could be derived (Table 2).

      Disruption of mtDNA homeostasis triggers aging and neurodegenerative phenotypes in vivo

      Disturbed mtDNA homeostasis is frequently observed in sporadic PD. By genetically perturbing key cellular regulators of mtDNA maintenance and transcription, either aging (a highly significant PD risk factor) or neurodegenerative phenotypes (characteristic of PD pathology) can be accurately recapitulated in vivo, depending on the mutation (Table 2). The Mutator mouse, a model in which a proof-reading deficient mitochondrial DNA polymerase γ (POLγAD257A) has been knocked-in, spontaneously accumulates mtDNA mutations (
      • Trifunovic A.
      • Wredenberg A.
      • Falkenberg M.
      • Spelbrink J.N.
      • Rovio A.T.
      • Bruder C.E.
      • Bohlooly Y.M.
      • Gidlöf S.
      • Oldfors A.
      • Wibom R.
      • Törnell J.
      • Jacobs H.T.
      • Larsson N.G.
      Premature ageing in mice expressing defective mitochondrial DNA polymerase.
      ). Although these mice exhibit no neurodegeneration and the nigrostriatal pathway remains intact up to 1 year (
      • Hauser D.N.
      • Primiani C.T.
      • Langston R.G.
      • Kumaran R.
      • Cookson M.R.
      The Polg mutator phenotype does not cause dopaminergic neurodegeneration in DJ-1-deficient mice.
      ), they display a dramatic aging phenotype. Aging is a significant risk factor for PD, and age-related decline in respiratory function and accumulation of mtDNA mutations, often observed in PD pathophysiology, is paralleled in the Mutator mice (
      • Trifunovic A.
      • Wredenberg A.
      • Falkenberg M.
      • Spelbrink J.N.
      • Rovio A.T.
      • Bruder C.E.
      • Bohlooly Y.M.
      • Gidlöf S.
      • Oldfors A.
      • Wibom R.
      • Törnell J.
      • Jacobs H.T.
      • Larsson N.G.
      Premature ageing in mice expressing defective mitochondrial DNA polymerase.
      ) (Table 2). Numerous studies have addressed mitophagy and the role of Parkin in this model (Table 2). Interestingly, neurodegeneration becomes evident by 12 months in POLγAD257A (Mutator); Parkin−/− mice, with evidence of midbrain DA cell loss and disrupted DA signaling and metabolism (
      • Pickrell A.M.
      • Huang C.H.
      • Kennedy S.R.
      • Ordureau A.
      • Sideris D.P.
      • Hoekstra J.G.
      • Harper J.W.
      • Youle R.J.
      Endogenous parkin preserves dopaminergic substantia nigral neurons following mitochondrial DNA mutagenic stress.
      ). Here, the appearance of neurodegeneration coincides with mitochondrial dysfunction, suggesting both a role of mitochondria in the degenerative phenotype and of Parkin in neuroprotection (
      • Pickrell A.M.
      • Huang C.H.
      • Kennedy S.R.
      • Ordureau A.
      • Sideris D.P.
      • Hoekstra J.G.
      • Harper J.W.
      • Youle R.J.
      Endogenous parkin preserves dopaminergic substantia nigral neurons following mitochondrial DNA mutagenic stress.
      ). No difference in mutational frequency is identified following Parkin knockout; however, a small but significant effect on mutation pathogenicity (i.e., biological impact of mutations) was observed, suggesting Parkin selectively limits mitochondrial mutagenic stress. In addition, POLγAD257A (Mutator); Parkin−/− mice display a strong STING-dependent type 1 interferon inflammatory response, as measured by serum cytokines. This STING-dependent response is a cellular innate immune response to cytosolic DNA in hematopoietic and epithelial cells, hypothesized to be triggered through released mtDNA and sensed by cyclic guanosine monophosphate (cGMP)–adenosine monophosphate (AMP) synthase (cGAS) (
      • Sliter D.A.
      • Martinez J.
      • Hao L.
      • Chen X.
      • Sun N.
      • Fischer T.D.
      • Burman J.L.
      • Li Y.
      • Zhang Z.
      • Narendra D.P.
      • Cai H.
      • Borsche M.
      • Klein C.
      • Youle R.J.
      Parkin and PINK1 mitigate STING-induced inflammation.
      ). In a cardiac model, restoring Parkin-mediated mitophagy in the Mutator hearts does not rescue the cardiac hypertrophy that develops with age in these mice, suggesting Parkin plays a minimal role in mtDNA mutation-induced cardiac aging (
      • Woodall B.P.
      • Orogo A.M.
      • Najor R.H.
      • Cortez M.Q.
      • Moreno E.R.
      • Wang H.
      • Divakaruni A.S.
      • Murphy A.N.
      • Gustafsson Å B.
      Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice.