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Dissection of the function of two Parkinson's disease-linked genes encoding the protein kinase, PTEN-induced kinase 1 (PINK1) and ubiquitin E3 ligase, Parkin, has illuminated a highly conserved mitochondrial quality control pathway found in nearly every cell type including neurons. Mitochondrial damage-induced activation of PINK1 stimulates phosphorylation-dependent activation of Parkin and ubiquitin-dependent elimination of mitochondria by autophagy (mitophagy). Structural, cell biological and neuronal studies are unravelling the key steps of PINK1/Parkin-dependent mitophagy and uncovering new insights into how the pathway is regulated. The emerging role for aberrant immune activation as a driver of dopaminergic neuron degeneration after loss of PINK1 and Parkin poses new exciting questions on cell-autonomous and noncell-autonomous mechanisms of PINK1/Parkin signalling in vivo.

Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein–sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets.

The discovery of rare familial monogenic forms of early-onset Parkinson's disease has led to the identification of a mitochondrial quality control process as a key player in this disease. Loss-of-function mutations in the genes encoding PINK1 or Parkin result in insufficient removal of dysfunctional mitochondria through autophagy, a process termed mitophagy. Understanding the mechanism of this process and the function of its two key players, PINK1 and Parkin, has led to the discovery of new therapeutic approaches. Small molecule activators of mitophagy, either activating PINK1 or Parkin directly or inhibiting Parkin's counterplayer, the ubiquitin-specific protease USP30, are in preclinical development. To enable clinical success of future small molecule mitophagy enhancers, biomarkers for mitochondrial integrity and mitophagy are being developed. Only a few years after the discovery of mitophagy deficits in Parkinson's disease, research of the underlying mechanisms, drug discovery of modulators for this mechanism and identification of biomarkers provide new avenues towards the development of disease-modifying therapies.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in Parkin S65A/S65A neurons. Phenotypically, Parkin S65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN ( PARK2 ) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the Parkin S65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

The discovery of mutations in genes encoding protein kinase PTEN-induced kinase 1 (PINK1) and E3 ubiquitin ligase Parkin in familial Parkinson's disease and their association with mitochondria provides compelling evidence that mitochondrial dysfunction is a major contributor to neurodegeneration in Parkinson's disease. In recent years, tremendous progress has been made in the understanding of how PINK1 and Parkin enzymes are regulated and how they influence downstream mitochondrial signalling processes. We provide a critical overview of the key advances in the field and also discuss the outstanding questions, including novel ways in which this knowledge could be exploited to develop therapies against Parkinson's disease.

Objective: We performed a mutation screen of NR4A2 (also known as NURR1) in 409 Parkinson's disease (PD) patients. We identified a novel single base substitution in the 5'UTR of the NR4A2 (also known as NURR1) gene (c.-309C > T). Results: We have performed expression studies in neuronal cell lines showing that the c.-309C > T mutation reduces NR4A2 mRNA expression in vitro. We have confirmed this finding in vivo by performing allele specific real-time PCR from brain tissue harbouring the 309C > T mutation and show a 3.48 +/- 1.62 fold reduction in mRNA expression of the mutant allele compared to wild-type. In addition we have undertaken genome wide expression analysis of the mutant NR4A2 brain and shown underexpressed genes were significantly enriched for gene ontology categories in nervous system development and synaptic transmission and overexpressed genes were enriched for unfolded protein response and morphogenesis. Lastly we have shown that the c.-309C > T mutation abrogates the protective effect of wild-type NR4A2 against apoptopic stress. Conclusions: Our findings indicate the c.-309C > T mutation reduces NR4A2 expression resulting in the downregulation of genes involved in the development and maintenance of the nervous system and synaptic transmission. These downregulated pathways contained genes known to be transactivated by NR4A2 and were not disrupted in idiopathic PD brain suggesting causality of the mutation. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Objective To investigate the significance of PINK1 mutations in sporadic Parkinson's disease (PD). Methods We determined the frequency of PINK1 mutations by direct sequencing in a large series of PD patients with apparently sporadic disease (n = 768). Results Twelve heterozygous mutations were identified, nine in PD patients and three in control subjects. Interpretation Given the difficulty in interpreting the pathogenic significance of the heterozygous mutations that have already been reported in parkin and DJ-1, we first determined the frequency of heterozygous PINK1 mutations in the general population by sequencing a large number of control subjects (n = 768), then subsequently assessed their functional significance by examining their effects on stress-induced alterations to the mitochondrial membrane potential (ΔΨm). We demonstrate an enrichment of heterozygous mutations in sporadic PD patients compared with matched control subjects (1.2% in PD vs 0.4% in control subjects). Furthermore, we show that they adversely affect ΔΨm in a similar way to the familial G309D mutation. Although it remains difficult to conclusively demonstrate the pathogenicity of heterozygous mutations, the results of this study and the previously reported subclinical nigrostriatal dysfunction in carriers of heterozygous PINK1 mutations suggest the possibility that these heterozygous mutations are a significant risk factor in the development of later onset PD. Ann Neurol 2006

Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.

Parkinson's disease is a common incurable neurodegenerative disease whose molecular aetiology remains unclear. The identification of Mendelian genes causing rare familial forms of Parkinson's disease has revealed novel proteins and pathways that are likely to be relevant in the pathogenesis of sporadic Parkinson's disease. Recently, mutations in a novel gene, PINK1, encoding a 581 amino acid protein with both mitochondrial targeting and serine/threonine kinase domains, were identified as a cause of autosomal recessive parkinsonism. This provided important evidence for the role of the mitochondrial dysfunction and kinase pathways in neurodegeneration. In this study, we report the first characterization of the PINK1 protein in normal human and sporadic Parkinson's brains, in addition to Parkinson's cases with heterozygous PINK1 mutations. The possible role of the PINK1 protein was also assessed in a number of neurodegenerative diseases characterized by proteinaceous inclusions. For these studies, rabbit polyclonal antibodies were raised against two peptide sequences within the N-terminal hydrophilic loops of PINK1 protein. Using immunohistochemistry and western blotting we were able to demonstrate that PINK1 is a ubiquitous protein expressed throughout the human brain and it is found in all cell types showing a punctate cytoplasmic staining pattern consistent with mitochondrial localization. Fractionation studies of human and rat brain confirm that PINK1 is localized to the mitochondrial membranes. In addition, we show that PINK1 is detected in a proportion of Lewy bodies in cases of sporadic Parkinson's disease and Parkinson's disease associated with heterozygous mutations in the PINK1 gene, which are clinically and pathologically indistinguishable from the sporadic cases. PINK1 was absent in cortical Lewy bodies, in neurofibrillary tangles in Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, and in the glial and neuronal alpha-synuclein positive inclusions in multiple system atrophy. These studies provide for the first time in vivo morphological and biochemical evidence to support a mitochondrial localization of PINK1 and underpin the significance of mitochondrial dysfunction in the pathogenesis of nigral cell degeneration in Parkinson's disease.

The NR4A2 gene, which may cause autosomal dominant Parkinson's disease (PD), has also been reported to be a susceptibility factor for sporadic PD. Here, we use a haplotype-tagging approach in 802 PD patients and 784 controls and demonstrate that common genetic variation, including NR4A2 haplotypes, does not influence the risk of PD in the Caucasian population.

Lewy bodies (LBs) are the characteristic inclusions of Parkinson's disease brain but the mechanism responsible for their formation is obscure. Lewy bodies (LBs) are composed of a number of proteins of which alpha-synuclein (alpha-SYN) is a major constituent. In this study, we have investigated the distribution patterns of synphilin-1 and parkin proteins in control and sporadic PD brain tissue by immunohistochemistry (IH), immunoblotting, and immunoelectron microscopy (IEM). We demonstrate the presence of synphilin-1 and parkin in the central core of a majority of LBs using IH and IEM. Using IH, we show an overlapping distribution profile of the two proteins in central neurons. Additionally, we show sensitivity of both endogenous synphilin-1 and parkin to proteolytic dysfunction and their co-localization in aggresomes formed in response to the proteasome inhibitor MG-132. We confirm that synphilin-1 and parkin are components of majority of LBs in Parkinson's disease and that both proteins are susceptible to proteasomal degradation.

Mutations in the PINK1 gene (PARK6), a putative serine-threonine kinase, cause autosomal recessive Parkinson's disease. PINK1 functions as a protein kinase and confers protective effects in the mitochondria, where it is primarily located. We assessed in a population of European ancestry whether common genetic variation in this novel gene influences nonmendelian forms of Parkinson's disease. We defined the linkage disequilibrium structure of PINK1 and used this to identify a set of tagging single nucleotide polymorphisms that we estimate will efficiently represent all of the common DNA variation in the entire gene. Genotyping these tags in a set of 576 Parkinson's disease patients and 514 controls did not demonstrate a case-control partition for allele or for haplotype and thus provides evidence against the existence of a common functional variants in PINK1 that has a strong influence on PD risk.

Summary 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.

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro . We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated—namely Drosophila melanogaster (dPINK1) , Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)—are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.

Parkinson disease is a devastating neurodegenerative disorder with no known cure. Impairment in mitochondrial dysfunction is thought to play a major role in the pathogenesis. Recent genetic advances suggest that mitochondrial dysfunction may be the primary defect. Drugs that target the mitochondria may therefore represent the best hope for disease-modifying therapies in Parkinson disease.

The quest to disentangle the aetiopathogenesis of Parkinson's disease has been heavily influenced by the genes associated with the disease. The alpha-synuclein-centric theory of protein aggregation with the adjunct of parkin-driven proteasome deregulation has, in recent years, been complemented by the discovery and increasing knowledge of the functions of DJ1, PINK1 and OMI/HTRA2, which are all associated with the mitochondria and have been implicated in cellular protection against oxidative damage. We critically review how these genes fit into and enhance our understanding of the role of mitochondrial dysfunction in Parkinson's disease, and consider how oxidative stress might be a potential unifying factor in the aetiopathogenesis of the disease.

OBJECTIVE: The UCHL-1 gene is widely cited as a susceptibility factor for sporadic Parkinson's disease (PD). The strongest evidence comes from a meta-analysis of small studies that reported the S18Y polymorphism as protective against PD, after pooling studies of white and Asian subjects. Here, we present data that challenge this association. METHODS: In a new large case-control study in white individuals (3,023 subjects), the S18Y variant was not protective against PD under any genetic model of inheritance. Similarly, a more powerful haplotype-tagging approach did not detect other associated variants. RESULTS: Finally, in an updated S18Y-PD meta-analysis (6,594 subjects), no significant association was observed under additive, recessive, or dominant models (odds ratio = 1.00 [95% confidence interval: 0.74-1.33]; odds ratio = 1.01 [95% confidence interval: 0.76-1.35]; and odds ratio = 0.96 [95% confidence interval: 0.86-1.08], respectively), and a cumulative meta-analysis showed a trend toward a null effect. INTERPRETATION: Based on the current evidence, the UCHL-1 gene does not exhibit a protective effect in PD.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.

Human neurodegenerative diseases are characterized by the progressive loss of specific neuronal populations, resulting in substantial disability and early death. The identification of causative single-gene mutations in families with inherited neurodegenerative disorders has facilitated the modelling of these diseases in experimental organisms, including the fruitfly Drosophila melanogaster. Many neurodegenerative diseases have now been successfully modelled in Drosophila, and genetic analysis is under way in each of these models. Using fruitfly genetics to define the molecular pathways that underlie the neurodegenerative process is likely to improve substantially our understanding of the pathogenesis of the human diseases, and to provide new therapeutic targets.