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.     

Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

Although exact causes of Parkinson disease (PD) remain enigmatic, mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuron susceptibility in both familial and sporadic PD. Two genes associated with recessive, early-onset PD encode the ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN/PARK2/Parkin, which together orchestrate a protective mitochondrial quality control (mitoQC) pathway. Upon stress, both enzymes cooperatively identify and decorate damaged mitochondria with phosphorylated poly-Ub (p-S65-Ub) chains. This specific label is subsequently recognized by autophagy receptors that further facilitate mitochondrial degradation in lysosomes (mitophagy). Here, we analyzed human post-mortem brain specimens and identified distinct pools of p-S65-Ub-positive structures that partially colocalized with markers of mitochondria, autophagy, lysosomes and/or granulovacuolar degeneration bodies. We further quantified levels and distribution of the ‘mitophagy tag’ in 2 large cohorts of brain samples from normal aging and Lewy body disease (LBD) cases using unbiased digital pathology. Somatic p-S65-Ub structures independently increased with age and disease in distinct brain regions and enhanced levels in LBD brain were age- and Braak tangle stage-dependent. Additionally, we observed significant correlations of p-S65-Ub with LBs and neurofibrillary tangle levels in disease. The degree of co-existing p-S65-Ub signals and pathological PD hallmarks increased in the pre-mature stage, but decreased in the late stage of LB or tangle aggregation. Altogether, our study provides further evidence for a potential pathogenic overlap among different forms of PD and suggests that p-S65-Ub can serve as a biomarker for mitochondrial damage in aging and disease.

Abbreviations: BLBD: brainstem predominant Lewy body disease; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DLB: dementia with Lewy bodies; DLBD: diffuse neocortical Lewy body disease; EOPD: early-onset Parkinson disease; GVB: granulovacuolar degeneration body; LB: Lewy body; LBD: Lewy body disease; mitoQC: mitochondrial quality control; nbM: nucleus basalis of Meynert; PD: Parkinson disease; PDD: Parkinson disease with dementia; p-S65-Ub: PINK1-phosphorylated serine 65 ubiquitin; SN: substantia nigra; TLBD: transitional Lewy body disease; Ub: ubiquitin     

Disease relevance of phosphorylated ubiquitin (p-S65-Ub)

Here, we present a summary of our recent findings on the (patho-)physiological relevance of PINK1-phosphorylated ubiquitin (p-S65-Ub). Using novel polyclonal antibodies, we find that p-S65-Ub specifically accumulates on damaged mitochondria. Phosphorylation of ubiquitin on serine 65 depends on the activity of PINK1 and the signal is vastly amplified by the activity of the E3 ubiquitin ligase PARK2/Parkin in a feed-forward loop. The induction of p-S65-Ub in primary cells suggests a significant role of p-S65-Ub also in neurons. Consistent with a marker for damaged mitochondria that are undergoing mitophagy, we find anti-p-S65-Ub immunoreactive granules that partially colocalize with mitochondria, lysosomes and ubiquitin in human post-mortem brain. The number of p-S65-Ub positive granules increases with age and with PD, highlighting the relevance of p-S65-Ub as a potential biomarker and therapeutic target.     

PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury

Damaged or dysfunctional mitochondria are toxic to the cell by producing reactive oxygen species and releasing cell death factors. Therefore, timely removal of these organelles is critical to cellular homeostasis and viability. Mitophagy is the mechanism of selective degradation of mitochondria via autophagy. The significance of mitophagy in kidney diseases, including ischemic acute kidney injury (AKI), has yet to be established, and the involved pathway of mitophagy remains poorly understood. Here, we show that mitophagy is induced in renal proximal tubular cells in both in vitro and in vivo models of ischemic AKI. Mitophagy under these conditions is abrogated by Pink1 and Park2 deficiency, supporting a critical role of the PINK1-PARK2 pathway in tubular cell mitophagy. Moreover, ischemic AKI is aggravated in pink1 andpark2 single- as well as double-knockout mice. Mechanistically, Pink1 and Park2 deficiency enhances mitochondrial damage, reactive oxygen species production, and inflammatory response. Taken together, these results indicate that PINK1-PARK2-mediated mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function during AKI.     

MITOL promotes cell survival by degrading Parkin during mitophagy

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin‐mediated cell death through the FKBP38‐dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine‐tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin‐induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.     

The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR^mt) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.     

Elucidation of ubiquitin-conjugating enzymes that interact with RBR-type ubiquitin ligases using a liquid–liquid phase separation–based method

RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid–liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2–E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.     

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser⁶⁵ by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity.     

Loss of iron triggers PINK1/Parkin‐independent mitophagy

In this study, we develop a simple assay to identify mitophagy inducers on the basis of the use of fluorescently tagged mitochondria that undergo a colour change on lysosomal delivery. Using this assay, we identify iron chelators as a family of compounds that generate a strong mitophagy response. Iron chelation‐induced mitophagy requires that cells undergo glycolysis, but does not require PINK1 stabilization or Parkin activation, and occurs in primary human fibroblasts as well as those isolated from a Parkinson's patient with Parkin mutations. Thus, we have identified and characterized a mitophagy pathway, the induction of which could prove beneficial as a potential therapy for several neurodegenerative diseases in which mitochondrial clearance is advantageous.     

The reciprocal roles of PARK2 and mitofusins in mitophagy and mitochondrial spheroid formation

Mitochondrial homeostasis is critical to cellular homeostasis, and mitophagy is an important mechanism to eliminate mitochondria that are superfluous or damaged. Multiple events can be involved in the recognition of mitochondria by the phagophore, and the key one is the priming of the mitochondria with specific molecular signatures. PARK2/Parkin is an E3 ligase that can be recruited to depolarized mitochondria and is required for mitophagy caused by respiration uncoupling. PARK2 induces ubiquitination of mitochondrial outer membrane proteins, which are subsequently degraded by the proteasome. Why these PARK2-mediated priming events are necessary for mitophagy to occur is not clear. We propose that they are needed to prevent a default pathway that would be inhibitory to mitophagy. In the default pathway depolarized and fragmented mitochondria undergo a dramatic three-dimensional conformational change to become mitochondrial spheroids. This transformation requires mitofusins; however, PARK2 inhibits this process by causing mitofusin ubiquitination and degradation. The spherical transformation may prevent recognition of the damaged mitochondria by the autophagosome, and PARK2 ensures that no such transformation occurs in order to promote mitophagy. Whether the formed mitochondrial spheroids functionally represent an alternative mitigation to mitophagy or an adverse consequence in the absence of PARK2 has yet to be determined.     

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.     

Genome-wide RNAi screen identifies ATPase inhibitory factor 1 (ATPIF1) as essential for PARK2 recruitment and mitophagy

Mitochondrial dysfunction is a hallmark of aging and numerous human diseases, including Parkinson disease (PD). Multiple homeostatic mechanisms exist to ensure mitochondrial integrity, including the selective autophagic program mitophagy, that is activated during starvation or in response to mitochondrial dysfunction. Following prolonged loss of potential across the inner mitochondrial membrane (ΔΨ), PTEN-induced putative kinase 1 (PINK1) and the E3-ubiquitin ligase PARK2 work in the same pathway to trigger mitophagy of dysfunctional mitochondria. Mutations in PINK1 and PARK2, as well as PARK7/DJ-1, underlie autosomal recessive Parkinsonism and impair mitochondrial function and morphology. In a genome-wide RNAi screen searching for genes that are required for PARK2 translocation to the mitochondria, we identified ATPase inhibitory factor 1 (ATPIF1/IF1) as essential for PARK2 recruitment and mitophagy in cultured cells. During uncoupling, ATPIF1 promotes collapse of ΔΨ and activation of the PINK-PARK2 mitophagy pathway by blocking the ATPase activity of the F₁-F_o ATP synthase. Restoration of ATPIF1 in Rho0 cells, which lack mtDNA and a functional electron transport chain, lowers ΔΨ and triggers PARK2 recruitment. Our findings identified ATPIF1 and the ATP synthase as novel components of the PINK1-PARK2 mitophagy pathway and provide genetic evidence that loss of ΔΨ is an essential trigger for mitophagy.     

LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10

ABSTRACT

Parkinson disease (PD) is a disabling, incurable disorder with increasing prevalence in the western world. In rare cases PD is caused by mutations in the genes for PINK1 (PTEN induced kinase 1) or PRKN (parkin RBR E3 ubiquitin protein ligase), which impair the selective autophagic elimination of damaged mitochondria (mitophagy). Mutations in the gene encoding LRRK2 (leucine rich repeat kinase 2) are the most common monogenic cause of PD. Here, we report that the LRRK2 kinase substrate RAB10 accumulates on depolarized mitochondria in a PINK1- and PRKN-dependent manner. RAB10 binds the autophagy receptor OPTN (optineurin), promotes OPTN accumulation on depolarized mitochondria and facilitates mitophagy. In PD patients with the two most common LRRK2 mutations (G2019S and R1441C), RAB10 phosphorylation at threonine 73 is enhanced, while RAB10 interaction with OPTN, accumulation of RAB10 and OPTN on depolarized mitochondria, depolarization-induced mitophagy and mitochondrial function are all impaired. These defects in LRRK2 mutant patient cells are rescued by LRRK2 knockdown and LRRK2 kinase inhibition. A phosphomimetic RAB10 mutant showed less OPTN interaction and less translocation to depolarized mitochondria than wild-type RAB10, and failed to rescue mitophagy in LRRK2 mutant cells. These data connect LRRK2 with PINK1- and PRKN-mediated mitophagy via its substrate RAB10, and indicate that the pathogenic effects of mutations in LRRK2, PINK1 and PRKN may converge on a common pathway.

Abbreviations : ACTB: actin beta; ATP5F1B: ATP synthase F1 subunit beta; CALCOCO2: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; Co-IP: co-immunoprecipitation; EBSS: Earle’s balanced salt solution; GFP: green fluorescent protein; HSPD1: heat shock protein family D (Hsp60) member 1; LAMP1: lysosomal associated membrane protein 1; LRRK2: leucine rich repeat kinase 2; IF: immunofluorescence; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; OMM: outer mitochondrial membrane; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT1: ras homolog family member T1; ROS: reactive oxygen species; TBK1: TANK binding kinase 1; WB: western blot.     

The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance

Loss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants. We showed here that blockade of mitochondrial protein import triggers the recruitment of PARK2, by PINK1, to the TOMM machinery. PD-causing PARK2 mutations weakened or disrupted the molecular interaction between PARK2 and specific TOMM subunits: the surface receptor, TOMM70A, and the channel protein, TOMM40. The downregulation of TOMM40 or its associated core subunit, TOMM22, was sufficient to trigger OMM protein clearance in the absence of PINK1 or PARK2. However, PARK2 was required to promote the degradation of whole organelles by autophagy. Furthermore, the overproduction of TOMM22 or TOMM40 reversed mitochondrial clearance promoted by PINK1 and PARK2 after ΔΨ loss. These results indicated that the TOMM machinery is a key molecular switch in the mitochondrial clearance program controlled by the PINK1-PARK2 pathway. Loss of functional coupling between mitochondrial protein import and the neuroprotective degradation of dysfunctional mitochondria may therefore be a primary pathogenic mechanism in autosomal recessive PD.     

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.     

Regulation by mitophagy

Eukaryotes employ elaborate mitochondrial quality control to maintain the function of the power-generating organelle. Mitochondrial quality control is particularly important for the maintenance of neural and muscular tissues. Mitophagy is specialized version of the autophagy pathway. Mitophagy delivers damaged mitochondria to lysosomes for degradation. Recently, a series of elegant studies have demonstrated that two Parkinson's disease-associated genes PINK1 and parkin are involved in the maintenance of healthy mitochondria as mitophagy. Parkin in co-operation with PINK1 specifically recognizes damaged mitochondria with reduced mitochondrial membrane potential (Δψm), rapidly isolates them from the mitochondrial network and eliminates them through the ubiquitin–proteasome and autophagy pathways. Here we introduce and review recent studies that contribute to understanding the molecular mechanisms of mitophagy such as PINK1 and Parkin-mediated mitochondrial regulation. We also discuss how defects in the PINK1–Parkin pathway may cause neurodegeneration in Parkinson's disease.     

Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson''s disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK1_52, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.     

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin''s in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.     

Dual role of USP30 in controlling basal pexophagy and mitophagy

USP30 is an integral protein of the outer mitochondrial membrane that counteracts PINK1 and Parkin‐dependent mitophagy following acute mitochondrial depolarisation. Here, we use two distinct mitophagy reporter systems to reveal tonic suppression by USP30, of a PINK1‐dependent component of basal mitophagy in cells lacking detectable Parkin. We propose that USP30 acts upstream of PINK1 through modulation of PINK1‐substrate availability and thereby determines the potential for mitophagy initiation. We further show that a fraction of endogenous USP30 is independently targeted to peroxisomes where it regulates basal pexophagy in a PINK1‐ and Parkin‐independent manner. Thus, we reveal a critical role of USP30 in the clearance of the two major sources of ROS in mammalian cells and in the regulation of both a PINK1‐dependent and a PINK1‐independent selective autophagy pathway.     

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.