Accumulation Of PINK1 Research Articles

Exploring the Impact of PARK2 Mutations on the Total and Mitochondrial Proteome of Human Skin Fibroblasts.

Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson’s disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.

BHRF1, a BCL2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction

ABSTRACT Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-β induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle’s balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Cadmium induces mitophagy via AMP-activated protein kinases activation in a PINK1/Parkin-dependent manner in PC12 cells.

ObjectivesCadmium (Cd) induces mitophagy in neuronal cells, but the underlying mechanisms remain unknown. In this study, we aimed to investigate these mechanisms.Materials and methodsThe effects of Cd on the mitophagy in rat pheochromocytoma PC12 cells were detected, and the role of PINK1/Parkin pathway in Cd‐induced mitophagy was also analysed by using PINK1 siRNA. In order to explore the relationship between AMPK and PINK1/Parkin in Cd‐induced mitophagy in PC12 cells, the CRISPR‐Cas9 system was used to knock down AMPK expression.ResultsThe results showed that Cd treatment triggered a significant increase in mitophagosome formation and the colocalization of mitochondria and lysosomes, which was further proved by the colocalization of LC3 puncta and its receptors NDP52 or P62 with mitochondria in PC12 cells. Moreover, an accumulation of PINK1 and Parkin was found in mitochondria. Additionally, upon PINK1 knock‐down using PINK1 siRNA, Cd‐induced mitophagy was efficiently suppressed. Interestingly, chemical or genetic reversal of AMPK activation: (a) significantly inhibited the activation of mitophagy and (b) promoted NLRP3 activation by inhibiting PINK/Parkin translocation.ConclusionsThese results suggest that Cd induces mitophagy via the PINK/Parkin pathway following AMPK activation in PC12 cells. Targeting the balanced activity of AMPK/PINK1/Parkin‐mediated mitophagy signalling may be a potential therapeutic approach to treat Cd‐induced neurotoxicity.

Activation of autophagic flux via LKB1/AMPK/mTOR axis against xenoestrogen Bisphenol-A exposure in primary rat hepatocytes

Bisphenol-A, an endocrine disruptive chemical widely used to manufacture polycarbonate plastics and epoxy resins, acts via multiple mechanisms that perturb cellular and molecular functions. BPA has the potential to induce hepatotoxicity via generation of ROS and oxidative stress. However, the mechanism of BPA induced oxidative stress and autophagy is still ambiguous at molecular and cellular levels. This study aims to elucidate the impact of BPA exposure (50 and 100 μM) in primary rat hepatocytes. AMP kinase, an intracellular energy sensor and key regulator in cellular signaling were found to be activated during BPA exposure. The increased AMP/ATP ratio and subsequent phosphorylation by its upstream mediator Liver Kinase B1 (LKB1) activates AMPK. BPA down-regulated AMPK downstream molecule i.e. mammalian target of rapamycin (mTOR) by inhibiting its phosphorylation, eventually enhances expression of autophagic markers LC3B, Beclin-1 while lowers p62. Results also revealed that BPA induces mitophagy by promoting accumulation of PINK1 and translocation of Parkin to damaged mitochondria culminating in decreased mitochondrial mass. Ultra-structural changes also confirmed mitochondrial disintegration, enhanced autophagic induction as evident from autophagosome formation. Findings confirm that BPA caused oxidative stress which eventually triggered LKB1/AMPK mediated autophagy and maintains cellular energy balance by mitophagic removal of unhealthy mitochondria in primary rat hepatocytes.

A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling.

SummaryEarly-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health. Therapeutic approaches for enhancing PINK1-Parkin signaling present a potential strategy for treating various diseases caused by mitochondrial dysfunction. We report two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system. These small molecules, T0466 and T0467, activate Parkin mitochondrial translocation in dopaminergic neurons and myoblasts at low doses that do not induce mitochondrial accumulation of PINK1. Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling. These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca2+ response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila. Our results suggested that T0466 and T0467 may hold promise as therapeutic reagents in Parkinson's disease and related disorders.

PINK1 phosphorylates ubiquitin predominantly in astrocytes

Loss-of-function mutations in PINK1 are causally linked to recessively inherited Parkinson’s disease (PD), with marked loss of dopaminergic neurons in the substantia nigra that are required for normal movement. PINK1 is a nuclear-encoded mitochondrial-targeted kinase that phosphorylates a conserved serine at amino acid 65 (pS65) in ubiquitin as well as Parkin, another gene with loss-of-function mutations linked to recessive parkinsonism. The steady-state levels of PINK1 protein are very low, even in cells that express PINK1, because PINK1 is normally targeted for degradation after mitochondrial import by a process that is dependent upon mitochondrial membrane potential. Dissipation of the mitochondrial membrane potential with ionophores, such as CCCP and valinomycin, causes the accumulation of PINK1 on the outer mitochondrial membrane, a marked increase of pS65-ubiquitin and the recruitment of Parkin, which targets dysfunctional mitochondria for degradation by autophagy. While the high penetrance of PINK1 mutations establish its critical function for maintaining neurons, the activity of PINK1 in primary neurons has been difficult to detect. Mounting evidence implicates non-neuronal cells, including astrocytes and microglia, in the pathogenesis of both idiopathic and inherited PD. Herein we used both western analysis and immunofluorescence of pS65-ubiquitin to directly compare the activity of PINK1 in primary neurons, astrocytes, microglia, and oligodendrocyte progenitor cells cultured from the brains of wild-type (WT) and PINK1 knockout (KO) rat pups. Our findings that PINK1-dependent ubiquitin phosphorylation is predominantly in astrocytes supports increased priority for research on the function of PINK1 in astrocytes and the contribution of astrocyte dysfunction to PD pathogenesis.

Dissociation of mitochondrial HK-II elicits mitophagy and confers cardioprotection against ischemia

Preservation of mitochondrial integrity is critical for maintaining cellular homeostasis. Mitophagy is a mitochondria-specific type of autophagy which eliminates damaged mitochondria thereby contributing to mitochondrial quality control. Depolarization of the mitochondrial membrane potential is an established mechanism for inducing mitophagy, mediated through PINK1 stabilization and Parkin recruitment to mitochondria. Hexokinase-II (HK-II) which catalyzes the first step in glucose metabolism, also functions as a signaling molecule to regulate cell survival, and a significant fraction of cellular HK-II is associated with mitochondria (mitoHK-II). We demonstrate here that pharmacological interventions and adenoviral expression of a mitoHK-II dissociating peptide which reduce mitoHK-II levels lead to robust increases in mitochondrial Parkin and ubiquitination of mitochondrial proteins in cardiomyocytes and in a human glioblastoma cell line 1321N1, independent of mitochondrial membrane depolarization or PINK1 accumulation. MitoHK-II dissociation-induced mitophagy was demonstrated using Mito-Keima in cardiomyocytes and in 1321N1 cells. Subjecting cardiomyocytes or the in vivo heart to ischemia leads to modest dissociation of mitoHK-II. This response is potentiated by expression of the mitoHK-II dissociating peptide, which increases Parkin recruitment to mitochondria and, importantly, provides cardioprotection against ischemic stress. These results suggest that mitoHK-II dissociation is a physiologically relevant cellular event that is induced by ischemic stress, the enhancement of which protects against ischemic damage. The mechanism which underlies the effects of mitoHK-II dissociation can be attributed to the ability of Bcl2-associated athanogene 5 (BAG5), an inhibitor of Parkin, to localize to mitochondria and form a molecular complex with HK-II. Overexpression of BAG5 attenuates while knockdown of BAG5 sensitizes the effect of mitoHK-II dissociation on mitophagy. We suggest that HK-II, a glycolytic molecule, can function as a sensor for metabolic derangements at mitochondria to trigger mitophagy, and modulating the intracellular localization of HK-II could be a novel way of regulating mitophagy to prevent cell death induced by ischemic stress.

Temporal integration of mitochondrial stress signals by the PINK1:Parkin pathway

BackgroundThe PINK1:Parkin pathway regulates the autophagic removal of damaged and dysfunctional mitochondria. While the response of this pathway to complete loss of ΔΨm, as caused by high concentrations of mitochondrial ionophores, has been well characterized, it remains unclear how the pathway makes coherent decisions about whether to keep or purge mitochondria in situations where ΔΨm is only partially lost or exhibits fluctuations, as has been observed in response to certain types of cellular stress.ResultsTo investigate the responses of the PINK1:Parkin pathway to mitochondrial insults of different magnitude and duration, controlled titration of the mitochondrial protonophore, CCCP, was used to manipulate ΔΨm in live cells, and the dynamics of PINK1 and Parkin recruitment was measured by fluorescence microscopy. In contrast to the stable accumulation of PINK1 and Parkin seen at completely depolarized mitochondria, partial depolarization produced a transient pulse of PINK1 stabilization and rapid loss, which was driven by small fluctuations in ΔΨm. As the rate of Parkin dissociation from the mitochondria and phospho-polyubiquitin chain removal was comparatively slow, repetitive pulses of PINK1 were able to drive a slow step-wise accumulation of Parkin and phospho-polyubiquitin leading to deferred mitophagy.ConclusionThese data suggest that the PINK1:Parkin mitophagy pathway is able to exhibit distinct dynamic responses to complete and partial mitochondrial depolarization. In this way, the pathway is able to differentiate between irretrievably damaged mitochondria and those showing signs of dysfunction, promoting either rapid or delayed autophagy, respectively.

Activation of PINK1-Parkin–Mediated Mitophagy Degrades Mitochondrial Quality Control Proteins in Fuchs Endothelial Corneal Dystrophy

Corneal endothelium (CE) is a monolayer of mitochondria-rich cells, critical for maintaining corneal transparency compatible with clear vision. Fuchs endothelial corneal dystrophy (FECD) is a heterogeneous, genetically complex disorder, where oxidative stress plays a key role in the rosette formation during the degenerative loss of CE. Increased mitochondrial fragmentation along with excessive mitophagy activation has been detected in FECD; however, the mechanism of aberrant mitochondrial dynamics in CE cell loss is poorly understood. Here, the role of oxidative stress in mitophagy activation in FECD is investigated. Immunoblotting of FECD exvivo specimens revealed an accumulation of PINK1 and phospho-Parkin (Ser65) along with loss of total Parkin and total Drp1. Similarly, modeling of rosette formation with menadione (MN), led to phospho-Parkin accumulation in fragmented mitochondria resulting in mitophagy-induced mitochondrial clearance, albeit possibly in a PINK1-independent manner. Loss of PINK1, phospho-Drp1, and total Drp1 was prominent after MN-induced oxidative stress, but not after mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone. Moreover, MN-induced mitophagy led to degradation of Parkin along with sequestration of Drp1 and PINK1 that was rescued by mitophagy inhibition. This study shows that in FECD, intracellular oxidative stress induces Parkin-mediated mitochondrial fragmentation where endogenous Drp1 and PINK1 are sequestered and degraded by mitophagy during degenerative loss of post-mitotic cells of ocular tissue.

Novel Role of Carbon Monoxide in Improving Neurological Outcome After Cardiac Arrest in Aged Rats: Involvement of Inducing Mitochondrial Autophagy.

BackgroundDysfunctional mitochondria are associated with neurological injury after cardiac arrest (CA). Although carbon monoxide (CO) has shown various potential therapeutic effects in preclinical tissue injury models, its mechanism of action in CA remains unclear. We sought to investigate the effects of a novel CO‐releasing molecule on cerebral mitochondrial dysfunction and neurological injury after CA.Methods and ResultsMale Sprague‐Dawley rats aged 20 to 22 months were subjected to 6‐minute asphyxia CA before receiving CO treatment. Survival, neurologic deficit scores, neuronal death, mitochondrial function, and autophagy were evaluated after the return of spontaneous circulation. Results showed that CO post‐treatment increased 3‐day survival rate from 25% to 70.83% and reduced neurologic deficit scores. CO also ameliorated CA‐induced neuronal apoptosis and necrosis in the cerebral cortex and improved cerebral mitochondrial function by reducing reactive oxygen species, reversing mitochondrial membrane potential depolarization, and preventing cytochrome C release. Furthermore, CO increased mitochondrial autophagy by inducing mitochondrial accumulation of PINK1 (PTEN‐induced putative kinase 1) and Parkin. Downregulation of PINK1 with genetic silencing siRNA abolished CO‐afforded mitochondrial autophagy.ConclusionsTaken together, our results indicate, for the first time, that CO treatment confers neuroprotection against ischemic neurological injury after CA possibly by promoting mitochondrial autophagy.

Miro proteins prime mitochondria for Parkin translocation and mitophagy

The Parkinson's disease‐associated protein kinase PINK1 and ubiquitin ligase Parkin coordinate the ubiquitination of mitochondrial proteins, which marks mitochondria for degradation. Miro1, an atypical GTPase involved in mitochondrial trafficking, is one of the substrates tagged by Parkin after mitochondrial damage. Here, we demonstrate that a small pool of Parkin interacts with Miro1 before mitochondrial damage occurs. This interaction does not require PINK1, does not involve ubiquitination of Miro1 and also does not disturb Miro1 function. However, following mitochondrial damage and PINK1 accumulation, this initial pool of Parkin becomes activated, leading to the ubiquitination and degradation of Miro1. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagic removal of mitochondria. Moreover, we demonstrate that Miro1 EF‐hand domains control Miro1's ubiquitination and Parkin recruitment to damaged mitochondria, and they protect neurons from glutamate‐induced mitophagy. Together, our results suggest that Miro1 functions as a calcium‐sensitive docking site for Parkin on mitochondria.

Ketoconazole exacerbates mitophagy to induce apoptosis by downregulating cyclooxygenase-2 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common cancer worldwide and remains a major clinical challenge. Ketoconazole, a traditional antifungal agent, has attracted considerable attention as a therapeutic option for cancer treatment. However, its mechanism of action is still not clearly defined. We aimed to evaluate the effect of ketoconazole on HCC and investigate the underlying mechanisms. We examined the antitumor effect of ketoconazole on HCC cells, cell line-derived xenografts, and a patient-derived xenograft (PDX) model. Ketoconazole-induced mitophagy was quantified by immunofluorescence, immunoblotting and transmission electron microscopy analysis. We used mitophagy inhibitors to study the role of mitophagy on HCC cell death induced by ketoconazole. The role of cyclooxygenase-2 (COX-2 [encoded by PTGS2]) on ketoconazole-induced mitophagy was evaluated using gain- and loss-of-function methods. The synergistic effect of ketoconazole with sorafenib on HCC was measured in vivo and in vitro. Ketoconazole stimulated apoptosis in HCC cells by triggering mitophagy in vitro and in vivo. Mechanistically, ketoconazole downregulated COX-2, which led to PINK1 accumulation and subsequent mitochondrial translocation of Parkin (PRKN), and thereby promoted mitophagy-mediated mitochondrial dysfunction. Inhibiting mitophagy alleviated ketoconazole-induced mitochondrial dysfunction and apoptosis, supporting a causal role for mitophagy in the antitumor effect of ketoconazole. In the HCC PDX model, ketoconazole demonstrated a marked antitumor effect characterized by COX-2 downregulation, mitophagy activation, and apoptosis induction. Moreover, ketoconazole acted synergistically with sorafenib to suppress HCC xenograft growth in vivo. Our results demonstrate a novel link between ketoconazole and mitophagy machinery, providing preclinical proof of concept for the use of ketoconazole in HCC treatment. Hepatocellular carcinoma (HCC) is a common malignancy worldwide and remains a major clinical challenge. Our study reveals that ketoconazole, a broad-spectrum antifungal agent, activates PINK1/Parkin-mediated mitophagy by downregulating COX-2, consequently resulting in the acceleration of apoptosis and thereby inhibiting the growth of HCC. Furthermore, ketoconazole acts synergistically with sorafenib in the suppression of HCC growth in vitro and in vivo.

Abstract 1342: Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells

Abstract Mitochondria-targeted drugs are designed to accumulate in the mitochondrion to modulate metabolism. Triphenylphosphonium (TPP) conjugated agents are a class of mitochondria-targeted drugs that induce mitochondrial dysfunction in cancer cells. Mitochondrial dysfunction is known factor that facilitates the onset of mitophagy. However, the activation of mitophagy to remove drug-induced mitochondrial damage is unknown. Here, we subjected breast MDA-MB-231 cancer cells and MCF-12A noncancerous cells to a series of mitochondria-targeted redox-active agents (MTA) that contain TPP to investigate the onset and activation of autophagy, mitophagy and mitochondrial dysfunction. Collectively, we identified three different MTAs from the series that sequentially damage the mitochondria, enhance autophagic flux, and activate mitophagy selectively in MDA-MB-231 cells. Furthermore, we generated stably expressing mt-mKeima MDA-MB-231 and MCF-12A cell lines to establish a mitophagy flux assay for screening new agents. FACS analysis of mt-mKeima, a pH-sensitive fluorophore, revealed MTAs induced lysosomal dependent mitochondrial degradation and the presence of bafilomycin, a lysosomal inhibitor, suppressed MTA-induced mitophagy. To confirm MTAs induced mitochondrial autophagy, we identified mitochondria located within autophagosomes using confocal microscopy, an endogenous autophagy-related protein complex by coimmunoprecipitation and mitochondrial PINK1 accumulation using immunoblot. These in vitro studies were translated into an in vivo rat SST-2 xenograph breast cancer model to demonstrate that mitochondrial PINK1 accumulates in rat tumor tissue following MitoQ treatment. These data suggest that mitochondria-targeted agents selectively caused mitochondrial depolarization, PINK1 accumulation and mitophagy in MDA-MB-231 cancer cells as compared to MCF-12A noncancerous cells. Citation Format: Thomas Biel, Ashutosh Rao. Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1342.

Sirt5 Plays a Critical Role in Mitochondrial Protein Acylation and Mitochondrial Metabolic Homeostasis in Brown Fat

Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis and glucose homeostasis. Recent studies have shown that mitochondrial activity can be regulated by protein succinylation and malonylation, processes that are in part controlled by Sirt5, the major mitochondrial desuccinylase and demalonylase. Compared to other tissues, brown fat has high levels of protein malonylation and succinylation, which were further increased in mice lacking Sirt5. BAT specific Sirt5 KO mice (Sirt5-BKO) are intolerant to acute cold. Furthermore, when stressed with high fat diet or aging, Sirt5-BKO mice develop impaired glucose homeostasis. Mass Spec analysis reveals that this correlates with an increase in succinylation of a variety of mitochondria enzymes and proteins, including succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), and UCP1. Lysine to glutamine mutations of UCP1 at the two target sites, which mimic acylation, resulted in significantly decreased UCP1 activity. Likewise, hyperacylation of SDH and GDH secondary to Sirt5 deficiency also impaired their activities. The reduced function of these and other proteins in Sirt5KO BAT resulted in decreased mitochondrial membrane potential and increased accumulation of PINK1, a mitochondrial serine/threonine kinase that protect cells from stress-induced mitochondrial dysfunction. Depolarized mitochondria are often smaller in size. Consistent with this, mitochondria from BAT of Sirt5KO mice had higher Fis1 expression during fasting, leading to increased mitochondrial fission and smaller mitochondria. Thus, succinylation and malonylation of key metabolic enzymes in mitochondria is regulated by Sirt5 and plays an important role in BAT activity. Over-acylation impairs mitochondrial protein function and results in depolarized mitochondria and disturbed systemic metabolic homeostasis. Disclosure G. Wang: None. J.G. Meyer: None. W. Cai: None. M.E. Li: None. S. Softic: None. C. Kahn: Advisory Panel; Self; CohBar, ERX Therapeutics, AntriaBio, Inc.. Board Member; Self; Kaleio Biosciences.

AKT signalling selectively regulates PINK1 mitophagy in SHSY5Y cells and human iPSC-derived neurons

The discovery of mutations within genes associated with autosomal recessive Parkinson’s disease allowed for the identification of PINK1/Parkin regulated mitophagy as an important pathway for the removal of damaged mitochondria. While recent studies suggest that AKT-dependent signalling regulates Parkin recruitment to depolarised mitochondria, little is known as to whether this can also regulate PINK1 mitochondrial accumulation and downstream mitophagy. Here, we demonstrate that inhibition of AKT signalling decreases endogenous PINK1 accumulation in response to mitochondria depolarisation, subsequent Parkin recruitment, phosphorylation of ubiquitin, and ultimately mitophagy. Conversely, we show that upon stimulation of AKT signalling via insulin, the mitophagy pathway is increased in SHSY5Y cells. These data suggest that AKT signalling is an upstream regulator of PINK1 accumulation on damaged mitochondria. Importantly, we show that the AKT pathway also regulates endogenous PINK1-dependent mitophagy in human iPSC-derived neurons.

Sam50 Regulates PINK1-Parkin-Mediated Mitophagy by Controlling PINK1 Stability and Mitochondrial Morphology

PINK1 and Parkin mediate mitophagy, the cellular process that clears dysfunctional mitochondria. Mitophagy is regulated by mitochondrial dynamics, but the molecules linking these two processes remain poorly understood. Here, we show that Sam50, the core component of the sorting and assembly machinery (SAM), is a critical regulator of mitochondrial dynamics and PINK1-Parkin-mediated mitophagy. In response to Sam50 depletion, normal tubular mitochondria are first fragmented and subsequently merged into large spheres. Sam50 interacts with PINK1 to facilitate its processing and degradation. Depletion of Sam50 results in PINK1 accumulation, Parkin recruitment, and mitophagy. Interestingly, Sam50 deficiency induces a piecemeal mode of mitophagy that eliminates mitochondria "bit by bit" but spares mtDNA. In C.elegans, the Sam50 homolog gop-3 is required for the maintenance of mitochondrial morphology and mass. Our findings reveal that Sam50 directly links mitochondrial dynamics and mitophagy and that Sam50 depletion induces elimination of mitochondria without affecting mtDNA content.

Ameliorating mitochondrial dysfunction restores carbon ion-induced cognitive deficits via co-activation of NRF2 and PINK1 signaling pathway

Carbon ion therapy is a promising modality in radiotherapy to treat tumors, however, a potential risk of induction of late normal tissue damage should still be investigated and protected. The aim of the present study was to explore the long-term cognitive deficits provoked by a high-linear energy transfer (high-LET) carbon ions in mice by targeting to hippocampus which plays a crucial role in memory and learning. Our data showed that, one month after 4 Gy carbon ion exposure, carbon ion irradiation conspicuously resulted in the impaired cognitive performance, neurodegeneration and neuronal cell death, as well as the reduced mitochondrial integrity, the disrupted activities of tricarboxylic acid cycle flux and electron transport chain, and the depressed antioxidant defense system, consequently leading to a decline of ATP production and persistent oxidative damage in the hippocampus region. Mechanistically, we demonstrated the disruptions of mitochondrial homeostasis and redox balance typically characterized by the disordered mitochondrial dynamics, mitophagy and glutathione redox couple, which is closely associated with the inhibitions of PINK1 and NRF2 signaling pathway as the key regulators of molecular responses in the context of neurotoxicity and neurodegenerative disorders. Most importantly, we found that administration with melatonin as a mitochondria-targeted antioxidant promoted the PINK1 accumulation on the mitochondrial membrane, and augmented the NRF2 accumulation and translocation. Moreover, melatonin pronouncedly enhanced the molecular interplay between NRF2 and PINK1. Furthermore, in the mouse hippocampal neuronal cells, overexpression of NRF2/PINK1 strikingly protected the hippocampal neurons from carbon ion-elicited toxic insults. Thus, these data suggest that alleviation of the sustained mitochondrial dysfunction and oxidative stress through co-modulation of NRF2 and PINK1 may be in charge of restoration of the cognitive impairments in a mouse model of high-LET carbon ion irradiation.

Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells.

Although deletion of certain autophagy-related genes has been associated with defects in hematopoiesis, it remains unclear whether hyperactivated mitophagy affects the maintenance and differentiation of hematopoietic stem cells (HSCs) and committed progenitor cells. Here we report that targeted deletion of the gene encoding the AAA+-ATPase Atad3a hyperactivated mitophagy in mouse hematopoietic cells. Affected mice showed reduced survival, severely decreased bone-marrow cellularity, erythroid anemia and B cell lymphopenia. Those phenotypes were associated with skewed differentiation of stem and progenitor cells and an enlarged HSC pool. Mechanistically, Atad3a interacted with the mitochondrial channel components Tom40 and Tim23 and served as a bridging factor to facilitate appropriate transportation and processing of the mitophagy protein Pink1. Loss of Atad3a caused accumulation of Pink1 and activated mitophagy. Notably, deletion of Pink1 in Atad3a-deficient mice significantly 'rescued' the mitophagy defect, which resulted in restoration of the progenitor and HSC pools. Our data indicate that Atad3a suppresses Pink1-dependent mitophagy and thereby serves a key role in hematopoietic homeostasis.

Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy.

Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson’s disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.

Autophagy Functions Of Genes Mutated In ALS And Parkinson’s Disease

TANK-binding kinase 1 (TBK1) phosphorylates Optineurin and mutations in either the kinase or the substrate can lead to ALS. Similarly, Pink1 phosphorylates Parkin and loss of function mutations in either can lead to Parkinson’s disease. Interestingly, all four proteins play roles in autophagy and mitophagy. PINK1 accumulates on the outer membrane of damaged mitochondria where it phosphorylates, in addition to Parkin, ubiquitin chains occurring on the outer mitochondrial membrane. These Phospho-S65 Ubiquitin chains bind to cytosolic Parkin and activate Parkin’s E3 ubiquitin ligase activity. This builds more chains on the mitochondrial surface for PINK1 to phosphorylate yielding a feedback amplification loop the drives mitophagy to completion. How ubiquitin chains or phospho-ubiquitin chains induce autophagy remains less clear. We have knocked out in HeLa cells a series of autophagy receptors including p62, NBR1, NDP52, Tax1BP1 and Optineurin revealing a hierarchy of autophagy receptors involved in mitophagy and revealing a link between them and those known to be involved in xenophagy. Interestingly, PINK1 accumulation on mitochondria alone, in the absence of Parkin, can recruit Optineurin to mitochondria and trigger mitophagy. Furthermore, in contrast to the central idea that autophagy receptors such as Optineurin bind LC3 to recruit cargo into autophagosomes, Optineurin following TBK1 phosphorylation can recruit Ulk1, DFCP1 and WIPI1 to mitochondria independently of LC3 proteins. Ubiquitin chains on mitochondrial can also activate Rab7 to promote pre-autophagosome enlargement and mitochondrial engulfment. New aspects regarding mitophagy in neurons will be presented.