PINK1- and PARK2-mediated local mitophagy in distal neuronal axons

Mutations in the PINK1 and PARK2/PARKIN genes are associated with hereditary early onset Parkinson disease (PD), and in cell lines the corresponding gene products play a critical role in mitophagic clearance of damaged mitochondria. In neurons, however, where the extraordinary cellular shapes pose particular challenges for maintaining healthy mitochondria, the pathways of mitophagy are less well understood. Both the location at which mitophagy occurs and the involvement of PINK1 and PARK2 have been controversial. Here we review our recent study where we found that selective damage to a subset of axonal mitochondria causes them to be engulfed within autophagosomes and cleared locally within the axon without the need for transport back to the soma. We also found this process to be completely dependent on neuronal PINK1 and PARK2.     

Backbone and side chain resonance assignments for a structured domain within Atg32

Autophagy is a catabolic cellular process that targets cytosolic material, including mitochondria, to the vacuole or lysosomes for degradation. The selective degradation of mitochondria by autophagy is termed mitophagy. Dysfunctional mitophagy, which leads to the accumulation of damaged mitochondria, has been implicated in Parkinson’s disease, cancer, cardiac disease and metabolic disease. In Saccharomyces cerevisiae, mitophagy is initiated by the autophagy receptor Atg32, an outer mitochondrial membrane protein. A lack of structural information for Atg32 has hindered our understanding of the molecular mechanisms of mitophagy initiation. To gain new structural insight into Atg32, we have identified the location of a structured domain within the cytosolic region of Atg32 and completed the backbone and side chain resonance assignments for this domain.     

Regulation of PINK1-Parkin-mediated mitophagy

Parkinson disease (PD) is a devastating disorder of the nervous system for which no cure exists. Although the exact mechanisms involved in the pathogenesis of PD are unclear, very recently, a novel cellular process has been identified that promises great future potential. Two PD-associated genes have been found to converge on the emerging mitophagy pathway that links the two major cellular dysfunctions implicated in the pathogenesis of PD. Thereby, PINK1 and Parkin physically associate and functionally cooperate to identify and label damaged mitochondria for selective degradation via autophagy. PD-associated mutations in both genes disrupt mitophagy although through different mechanisms, revealing a sequential multistep process. Further key players that tie into this process have been identified and provide the framework for future research aiming at a complete dissection of this neuroprotective pathway. This may not only yield novel targets for therapeutic intervention in PD, but possibly for other neurodegenerative disorders as well.     

The molecular mechanism of mitochondria autophagy in yeast

Mitochondria are critical for supplying energy to the cell, but during catabolism this organelle also produces reactive oxygen species that can cause oxidative damage. Accordingly, quality control of mitochondria is important to maintain cellular homeostasis. It has been assumed that autophagy is the pathway for mitochondrial recycling, and that the selective degradation of mitochondria via autophagy (mitophagy) is the primary mechanism for mitochondrial quality control, although there is little experimental evidence to support this idea. Recent studies in yeast identified several mitophagy‐related genes and have uncovered components involved in the molecular mechanism and regulation of mitophagy. Similarly, studies of Parkinson disease and reticulocyte maturation reveal that Parkin and Nix, respectively, are required for mitophagy in mammalian cells, and these analyses have revealed important physiological roles for mitophagy. Here, we review the current knowledge on mitophagy, in particular on the molecular mechanism and regulation of mitophagy in yeast. We also discuss some of the differences between yeast and mammalian mitophagy.     

Role of Autophagy Pathway in Parkinson’s Disease and Related Genetic Neurological Disorders

The elucidation of the function of the PINK1 protein kinase and Parkin ubiquitin E3 ligase in the elimination of damaged mitochondria by autophagy (mitophagy) has provided unprecedented understanding of the mechanistic pathways underlying Parkinson’s disease (PD). We provide a comprehensive overview of the general importance of autophagy in Parkinson’s disease and related disorders of the central nervous system. This reveals a critical link between autophagy and neurodegenerative and neurodevelopmental disorders and suggests that strategies to modulate mitophagy may have greater relevance in the CNS beyond PD.     

Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy

The knowledge gap separating the molecular and cellular underpinnings of Parkinson disease (PD) and its pathology hinders treatment innovation. Adding to this difficulty is the lack of a reliable biomarker for PD. Our previous studies identify a link of 2 PD proteins, PINK1/PRKN Parkin to a mitochondrial motor adaptor RHOT1/Miro-1, which mediates mitochondrial motility and mitophagy. Here we review our recent paper showing that a third PD protein, LRRK2, also targets RHOT1 and regulates mitophagy, and pathogenic LRRK2 disrupts this function. Notably, we discover impairments in RHOT1 and mitophagy in sporadic PD patients with no known genetic backgrounds, pointing to RHOT1-mediated mitophagy as a convergent pathway in PD. This novelty opens new doors in PD research toward RHOT1-based therapy and biomarker development.     

PINK1-parkin-dependent mitophagy involves ubiquitination of mitofusins 1 and 2: Implications for Parkinson disease pathogenesis

Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson disease (PD). Recent research has highlighted that two proteins encoded by genes linked to familial PD, PINK1 and parkin, play a role in the autophagic degradation of dysfunctional mitochondria (mitophagy). We have recently shown that mitochondrial dysfunction in PINK1-deficient human dopaminergic cells correlates with decreased autophagic flux and can be rescued by parkin expression. Further dissection of PINK1-parkin-dependent mitophagy indicates that the ubiquitination of mitofusins 1 and 2 is an early event. Here, we discuss how ubiquitination of the mitofusins might facilitate mitochondria degradation and the potential for activating mitophagy as a treatment for diseases affecting brain and muscle.     

Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells

Mitophagy, the selective removal of mitochondria through the autophagic pathway, is involved in cellular mitochondria quality control. Dysfunctional mitochondria can be selectively eliminated through Parkin-mediated mitophagy. Parkin is a ubiquitin E3 ligase that selectively translocates onto impaired mitochondria to initiate mitophagy, and mutations in Parkin have been identified in autosomal recessive forms of Parkinson disease. Here with the use of a genetically encoded, mitochondria-matrix targeting photosensitizer, we established a robust strategy that allows for spatiotemporally controlled initiation of Parkin-mediated mitophagy in single cells with light. The method can specifically target varying numbers of mitochondria into the Parkin-mediated mitophagy pathway for clearance. Combined with live cell imaging, we demonstrated that mitochondria can be cleared by Parkin-mediated mitophagy without juxtanuclear mito-aggresome formation. Autophagy proceeded with the asynchronous appearance of small LC3B-coated structures on Parkin-labeled mitochondria subsections in a nucleation-expansion manner. Our method allows for quantitative measurement on the Parkin-mediated mitophagy process, and can be multiplexed in imaging for higher throughput studies.     

Intramembrane protease PARL defines a negative regulator of PINK1- and PARK2/Parkin-dependent mitophagy

Mutations in PINK1 and PARK2/Parkin are a main risk factor for familial Parkinson disease. While the physiological mechanism of their activation is unclear, these proteins have been shown in tissue culture cells to serve as a key trigger for autophagy of depolarized mitochondria. Here we show that ablation of the mitochondrial rhomboid protease PARL leads to retrograde translocation of an intermembrane space-bridging PINK1 import intermediate. Subsequently, it is rerouted to the outer membrane in order to recruit PARK2, which phenocopies mitophagy induction by uncoupling agents. Consistent with a role of this retrograde translocation mechanism in neurodegenerative disease, we show that pathogenic PINK1 mutants which are not cleaved by PARL affect PINK1 kinase activity and the ability to induce PARK2-mediated mitophagy. Altogether we suggest that PARL is an important intrinsic player in mitochondrial quality control, a system substantially impaired in Parkinson disease as indicated by reduced removal of damaged mitochondria in affected patients.     

Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells

Mitophagy, the selective removal of mitochondria through the autophagic pathway, is involved in cellular mitochondria quality control. Dysfunctional mitochondria can be selectively eliminated through Parkin-mediated mitophagy. Parkin is a ubiquitin E3 ligase that selectively translocates onto impaired mitochondria to initiate mitophagy, and mutations in Parkin have been identified in autosomal recessive forms of Parkinson disease. Here with the use of a genetically encoded, mitochondria-matrix targeting photosensitizer, we established a robust strategy that allows for spatiotemporally controlled initiation of Parkin-mediated mitophagy in single cells with light. The method can specifically target varying numbers of mitochondria into the Parkin-mediated mitophagy pathway for clearance. Combined with live cell imaging, we demonstrated that mitochondria can be cleared by Parkin-mediated mitophagy without juxtanuclear mito-aggresome formation. Autophagy proceeded with the asynchronous appearance of small LC3B-coated structures on Parkin-labeled mitochondria subsections in a nucleation-expansion manner. Our method allows for quantitative measurement on the Parkin-mediated mitophagy process, and can be multiplexed in imaging for higher throughput studies.     

Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency: FUNCTIONAL ROLE OF NEURONAL NITRIC OXIDE SYNTHASE DURING MITOPHAGY*

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser¹⁴¹²) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.     

SREBF1 links lipogenesis to mitophagy and sporadic Parkinson disease

Mitochondrial quality control has an impact on many diseases, but intense research has focused on the action of 2 genes linked to heritable forms of Parkinson disease (PD), PINK1 and PARK2/parkin, which act in a common pathway to promote mitophagy. However, criticism has been raised that little evidence links this mechanism to sporadic PD. To gain a greater insight into the mechanisms of PINK1-PARK2 mediated mitophagy, we undertook a genome-wide RNAi screen in Drosophila and human cell models. Strikingly, we discovered several components of the lipogenesis pathway, including SREBF1, playing a conserved role in mitophagy. Our results suggest that lipids influence the stabilization of PINK1 during the initiation of mitophagy. Importantly, SREBF1 has previously been identified as a risk locus for sporadic PD, and thus implicates aberrant mitophagy as contributing to sporadic PD. Our findings suggest a role for lipid synthesis in PINK1-PARK2 mediated mitophagy, and propose a mechanistic link between familial and sporadic PD, supporting a common etiology.     

SREBF1 links lipogenesis to mitophagy and sporadic Parkinson disease

Mitochondrial quality control has an impact on many diseases, but intense research has focused on the action of 2 genes linked to heritable forms of Parkinson disease (PD), PINK1 and PARK2/parkin, which act in a common pathway to promote mitophagy. However, criticism has been raised that little evidence links this mechanism to sporadic PD. To gain a greater insight into the mechanisms of PINK1-PARK2 mediated mitophagy, we undertook a genome-wide RNAi screen in Drosophila and human cell models. Strikingly, we discovered several components of the lipogenesis pathway, including SREBF1, playing a conserved role in mitophagy. Our results suggest that lipids influence the stabilization of PINK1 during the initiation of mitophagy. Importantly, SREBF1 has previously been identified as a risk locus for sporadic PD, and thus implicates aberrant mitophagy as contributing to sporadic PD. Our findings suggest a role for lipid synthesis in PINK1-PARK2 mediated mitophagy, and propose a mechanistic link between familial and sporadic PD, supporting a common etiology.     

Defending the mitochondria: The pathways of mitophagy and mitochondrial-derived vesicles

Mitochondria are the powerhouses for the cell, consuming oxygen to generate sufficient energy for the maintenance of normal cellular processes. However, a deleterious consequence of this process are reactive oxygen species generated as side-products of these reactions. As a means to protect mitochondria from damage, cells and mitochondria have developed a wide-range of mitochondrial quality control mechanisms that remove damaged mitochondrial cargo, enabling the mitochondria to repair the damage and ultimately restore their normal function. If the damage is extensive and mitochondria can no longer be repaired, a process termed mitophagy is initiated in which the mitochondria are directed for autophagic clearance. Canonical mitophagy is regulated by two proteins, PINK1 and Parkin, which are mutated in familial forms of Parkinson’s disease. In this review, we discuss recent work elucidating the mechanism of PINK1/Parkin-mediated mitophagy, along with recently uncovered PINK1/Parkin-independent mitophagy pathways. Moreover, we describe a novel mitochondrial quality control pathway, involving mitochondrial-derived vesicles that direct distinct and damaged mitochondrial cargo for degradation in the lysosome. Finally, we discuss the association between mitochondrial quality control, cardiac, hepatic and neurodegenerative disease and discuss the possibility of targeting these pathways for therapeutic purposes.     

PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.     

Ambra1

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.     

Ambra1: a Parkin-binding protein involved in mitophagy

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.     

Short Mitochondrial ARF Triggers Parkin/PINK1-dependent Mitophagy

Parkinson disease (PD) is a complex neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra. Multiple genes have been associated with PD, including Parkin and PINK1. Recent studies have established that the Parkin and PINK1 proteins function in a common mitochondrial quality control pathway, whereby disruption of the mitochondrial membrane potential leads to PINK1 stabilization at the mitochondrial outer surface. PINK1 accumulation leads to Parkin recruitment from the cytosol, which in turn promotes the degradation of the damaged mitochondria by autophagy (mitophagy). Most studies characterizing PINK1/Parkin mitophagy have relied on high concentrations of chemical uncouplers to trigger mitochondrial depolarization, a stimulus that has been difficult to adapt to neuronal systems and one unlikely to faithfully model the mitochondrial damage that occurs in PD. Here, we report that the short mitochondrial isoform of ARF (smARF), previously identified as an alternate translation product of the tumor suppressor p19ARF, depolarizes mitochondria and promotes mitophagy in a Parkin/PINK1-dependent manner, both in cell lines and in neurons. The work positions smARF upstream of PINK1 and Parkin and demonstrates that mitophagy can be triggered by intrinsic signaling cascades.     

After the banquet

Mitochondria are highly dynamic organelles of crucial importance to the proper functioning of neuronal, cardiac and other cell types dependent upon aerobic efficiency. Mitochondrial dysfunction has been implicated in numerous human conditions, to include cancer, metabolic diseases, neurodegeneration, diabetes, and aging. In recent years, mitochondrial turnover by macroautophagy (mitophagy) has captured the limelight, due in part to discoveries that genes linked to Parkinson disease regulate this quality control process. A rapidly growing literature is clarifying effector mechanisms that underlie the process of mitophagy; however, factors that regulate positive or negative cellular outcomes have been less studied. Here, we review the literature on two major pathways that together may determine cellular adaptation vs. cell death in response to mitochondrial dysfunction. Mitochondrial biogenesis and mitophagy represent two opposing, but coordinated processes that determine mitochondrial content, structure, and function. Recent data indicate that the capacity to undergo mitochondrial biogenesis, which is dysregulated in disease states, may play a key role in determining cell survival following mitophagy-inducing injuries. The current literature on major pathways that regulate mitophagy and mitochondrial biogenesis is summarized, and mechanisms by which the interplay of these two processes may determine cell fate are discussed. We conclude that in primary neurons and other mitochondrially dependent cells, disruptions in any phase of the mitochondrial recycling process can contribute to cellular dysfunction and disease. Given the emerging importance of crosstalk among regulators of mitochondrial function, autophagy, and biogenesis, signaling pathways that coordinate these processes may contribute to therapeutic strategies that target or regulate mitochondrial turnover and regeneration.     

Mitochondrial autophagy as a compensatory response to PINK1 deficiency

Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission, and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.