Recognizing the cooperative and independent mitochondrial functions of Parkin and PINK1

Comment on: Johnson BN, et al. Proc Natl Acad Sci USA 2012; 109:6283-8.     

PINK1 controls mitochondrial localization of Parkin through direct phosphorylation

PTEN-induced putative kinase 1 (PINK1) and Parkin, encoded by their respective genes associated with Parkinson’s disease (PD), are linked in a common pathway involved in the protection of mitochondrial integrity and function. However, the mechanism of their interaction at the biochemical level has not been investigated yet. Using both mammalian and Drosophila systems, we here demonstrate that the PINK1 kinase activity is required for its function in mitochondria. PINK1 regulates the localization of Parkin to the mitochondria in its kinase activity-dependent manner. In detail, Parkin phosphorylation by PINK1 on its linker region promotes its mitochondrial translocation, and the RING1 domain of Parkin is critical for this occurrence. These results demonstrate the biochemical relationship between PINK1, Parkin, and the mitochondria and thereby suggest the possible mechanism of PINK-Parkin-associated PD pathogenesis.     

PINK1 kinase dysfunction triggers neurodegeneration in the primate brain without impacting mitochondrial homeostasis

In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson's disease(PD).However,difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1.Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains.CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology.Importantly,PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival.Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.     

Structural Mechanisms of Mitochondrial Quality Control Mediated by PINK1 and Parkin

Parkinson's disease (PD) is the second most common neurodegenerative disease and represents a looming public health crisis as the global population ages. While the etiology of the more common, idiopathic form of the disease remains unknown, the last ten years have seen a breakthrough in our understanding of the genetic forms related to two proteins that regulate a quality control system for the removal of damaged or non-functional mitochondria. Here, we review the structure of these proteins, PINK1, a protein kinase, and parkin, a ubiquitin ligase with an emphasis on the molecular mechanisms responsible for their recognition of dysfunctional mitochondria and control of the subsequent ubiquitination cascade. Recent atomic structures have revealed the basis of PINK1 substrate specificity and the conformational changes responsible for activation of PINK1 and parkin catalytic activity. Progress in understanding the molecular basis of mitochondrial quality control promises to open new avenues for therapeutic interventions in PD.     

Structural basis for feedforward control in the PINK1/Parkin pathway

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson’s disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin‐like (Ubl) domain of parkin. Here, we observed that phospho‐ubiquitin can bind to two distinct sites on parkin, a high‐affinity site on RING1 that controls parkin localization and a low‐affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho‐ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra‐phospho‐ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1‐parkin pathway and likely represents an intermediate step in its evolutionary development.     

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 stabilizes PINK1 through direct interaction

Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic; however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson’s disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD.     

PINK1 protects against cell death induced by mitochondrial depolarization,by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage

Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson''s disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1–Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer.     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment

Mutations in phosphatase and tensin homologue-induced kinase 1 (PINK1) cause recessively inherited Parkinson's disease (PD), a neurodegenerative disorder linked to mitochondrial dysfunction. In healthy mitochondria, PINK1 is rapidly degraded in a process involving both mitochondrial proteases and the proteasome. However, when mitochondrial import is compromised by depolarization, PINK1 accumulates on the mitochondrial surface where it recruits the PD-linked E3 ubiquitin ligase Parkin from the cytosol, which in turn mediates the autophagic destruction of the dysfunctional organelles. Using an unbiased RNA-mediated interference (RNAi)-based screen, we identified four mitochondrial proteases, mitochondrial processing peptidase (MPP), presenilin-associated rhomboid-like protease (PARL), m-AAA and ClpXP, involved in PINK1 degradation. We find that PINK1 turnover is particularly sensitive to even modest reductions in MPP levels. Moreover, PINK1 cleavage by MPP is coupled to import such that reducing MPP activity induces PINK1 accumulation at the mitochondrial surface, leading to Parkin recruitment and mitophagy. These results highlight a new role for MPP in PINK1 import and mitochondrial quality control via the PINK1–Parkin pathway.     

Genetic Variants in Diseases of the Extrapyramidal System

Knowledge on the genetics of movement disorders has advanced significantly in recent years. It is now recognized that disorders of the basal ganglia have genetic basis and it is suggested that molecular genetic data will provide clues to the pathophysiology of normal and abnormal motor control. Progress in molecular genetic studies, leading to the detection of genetic mutations and loci, has contributed to the understanding of mechanisms of neurodegeneration and has helped clarify the pathogenesis of some neurodegenerative diseases. Molecular studies have also found application in the diagnosis of neurodegenerative diseases, increasing the range of genetic counseling and enabling a more accurate diagno-sis. It seems that understanding pathogenic processes and the significant role of genetics has led to many experiments that may in the future will result in more effective treatment of such diseases as Parkinson’s or Huntington’s. Currently used molecular diagnostics based on DNA analysis can identify 9 neurodegenerative diseases, including spinal cerebellar ataxia inherited in an autosomal dominant manner, dentate-rubro-pallido-luysian atrophy, Friedreich’s disease, ataxia with ocu-lomotorapraxia, Huntington''s disease, dystonia type 1, Wilson’s disease, and some cases of Parkinson''s disease.     

PINK1 as a molecular checkpoint in the maintenance of mitochondrial function and integrity

Parkinson's disease (PD), the most prevalent neurodegenerative movement disorder, is characterized by an age-dependent selective loss of dopaminergic (DA) neurons. Although most PD cases are sporadic, more than 20 responsible genes in familial cases were identified recently. Genetic studies using Drosophila models demonstrate that PINK1, a mitochondrial kinase encoded by a PD-linked gene PINK1, is critical for maintaining mitochondrial function and integrity. This suggests that mitochondrial dysfunction is the main cause of PD pathogenesis. Further genetic and cell biological studies revealed that PINK1 recruits Parkin, an E3 ubiquitin ligase encoded by another PD-linked gene parkin, to mitochondria and regulates the mitochondrial remodeling process via the Parkin-mediated ubiquitination of various mitochondrial proteins. PINK1 also directly phosphorylates the mitochondrial proteins Miro and TRAP1, subsequently inhibiting mitochondrial transport and mitochondrial oxidative damage, respectively. Moreover, recent Drosophila genetic analyses demonstrate that the neuroprotective molecules Sir2 and FOXO specifically complement mitochondrial dysfunction and DA neuron loss in PINK1 null mutants, suggesting that Sir2 and FOXO protect mitochondria and DA neurons downstream of PINK1. Collectively, these recent results suggest that PINK1 plays multiple roles in mitochondrial quality control by regulating its mitochondrial, cytosolic, and nuclear targets.     

PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.     

Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

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Mechanistic insights into mitochondrial tRNAAla 3’-end metabolism deficiency

Mitochondrial tRNA 3’-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3’-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber’s hereditary optic neuropathy (LHON)-associated tRNA^Ala 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3’-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNA^Ala 3’-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3’-end processing of tRNA^Ala precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNA^Ala aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNA^Ala in these cells. The impact of m.5587A>G mutation on tRNA^Ala function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNA^Ala metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3’-end metabolism deficiency.     

The Mechanistic Links Between Proteasome Activity,Aging and Age-related Diseases

Damaged and misfolded proteins accumulate during the aging process, impairing cell function and tissue homeostasis. These perturbations to protein homeostasis (proteostasis) are hallmarks of age-related neurodegenerative disorders such as Alzheimer’s, Parkinson’s or Huntington’s disease. Damaged proteins are degraded by cellular clearance mechanisms such as the proteasome, a key component of the proteostasis network. Proteasome activity declines during aging, and proteasomal dysfunction is associated with late-onset disorders. Modulation of proteasome activity extends lifespan and protects organisms from symptoms associated with proteostasis disorders. Here we review the links between proteasome activity, aging and neurodegeneration. Additionally, strategies to modulate proteasome activity and delay the onset of diseases associated to proteasomal dysfunction are discussed herein.     

Dammarane triterpenes targeting α-synuclein: biological activity and evaluation of binding sites by molecular docking

Parkinson''s disease (PD) is a neurodegenerative disorder that affects adult people whose treatment is palliative. Thus, we decided to test three dammarane triterpenes 1, 1a, 1b, and we determined that 1 and 1a inhibit β-aggregation through thioflavine T rather than 1b. Since compound 1 was most active, we determined the interaction between α-synuclein and 1 at 50 µM (Kd) through microscale thermophoresis. Also, we observed differences in height and diameter of aggregates, and α-synuclein remains unfolded in the presence of 1. Also, aggregates treated with 1 do not provoke neurites'' retraction in N2a cells previously induced by retinoic acid. Finally, we studied the potential sites of interaction between 1 with α-synuclein fibrils using molecular modelling. Docking experiments suggest that 1 preferably interact with the site 2 of α-synuclein through hydrogen bonds with residues Y39 and T44.     

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild‐type but not PD‐linked mutant parkin supports the biogenesis of a population of mitochondria‐derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin‐ and PINK1‐dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.     

Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease

Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson''s disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.     

Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1

The mitochondrial chaperone mortalin was implicated in Parkinson''s disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes.