Genetics of Parkinson's disease and biochemical studies of implicated gene products

Parkinson's disease was thought, until recently, to have little or no genetic component. This notion has changed with the identification of three genes, and the mapping of five others, that are linked to rare familial forms of the disease (FPD). The products of the identified genes, alpha-synuclein (PARK 1), parkin (PARK 2), and ubiquitin-C-hydrolase-L1 (PARK 5) are the subject of intense cell-biological and biochemical studies designed to elucidate the underlying mechanism of FPD pathogenesis. In addition, the complex genetics of idiopathic PD is beginning to be unraveled. Genetic information may prove to be useful in identifying new therapeutic targets and identifying the preclinical phase of PD, allowing treatment to begin sooner.     

Parkinson's disease: a genetic perspective

Parkinson's disease (PD) is a common neurodegenerative disorder in the aging population, affecting more than 1% over the age of 65 years. Certain rare forms of the disease are monogenic, representing 5-10% of PD patients, but there is increasing evidence that multiple genetic risk factors are important also for common forms of PD. To date, 13 genetic loci, PARK1-13, have been suggested for rare forms of PD such as autosomal dominant and autosomal recessive PD. At six of these loci, genes have been identified and reported by several groups to carry mutations that are linked to affected family members. Genes in which mutations have been linked to familial PD have also been shown to be candidate genes for idiopathic forms of PD, as those same genes may also carry other mutations that merely increase the risk. Four of the PARK genes, SNCA at PARK1, UCH-L1 at PARK5, PINK1 at PARK6 and LRRK2 at PARK8, have been implicated in sporadic PD. There are indeed multiple genetic risk factors that combine in different ways to increase or decrease risk, and several of these need to be identified in order to begin unwinding the causative pathways leading to the different forms of PD. In this review, we present the molecular genetics of PD that are understood today, to help explain the pathways leading to neurodegeneration.     

Gene co-expression network analysis for identifying genetic markers in Parkinson's disease - a three-way comparative approach

Parkinson's disease (PD) is a neurodegenerative disorder involving progressive deterioration of dopaminergic neurons. Although few genetic markers for familial PD are known, the etiology of sporadic PD remains poorly understood. Microarray data was analysed for induced pluripotent stem cells (iPSCs) derived from PD patients and mature neuronal cells (mDA) differentiated from these iPSCs. Combining expression and semantic similarity, a highly-correlated PD interactome was constructed that included interactions of established Parkinson's disease marker genes. A novel three-way comparative approach was employed, delineating topologically and functionally important genes. These genes showed involvement in pathways like Parkin-ubiquitin proteosomal system (UPS), immune associated biological processes and apoptosis. Of interest are three genes, eEF1A1, CASK, and PSMD6 that are linked to PARK2 activity in the cell and thereby form attractive candidate genes for understanding PD. Network biology approach delineated in this study can be applied to other neurodegenerative disorders for identification of important genetic regulators.     

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.     

Genetic animal models for evaluating the role of autophagy in etiopathogenesis of Parkinson disease

Parkinson disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by the formation of ubiquitin and SNCA/α-synuclein-containing inclusions (Lewy bodies), dystrophic midbrain dopaminergic (DAergic) terminals, and degeneration of midbrain DAergic neurons. The vast majority of PD occurs sporadically, while approximately 5% of all PD cases are inherited. Genetic mutations of a few genes have been identified as causes of familiar PD, i.e., mutations in SNCA, PARK2/parkin, UCHL1, PARK7/DJ1, PINK1 and LRRK2, leading to DAergic cell death, but variable pathological changes. The evidence supports the hypothesis that several pathogenic mechanisms are likely involved at initial stages of the disease, and eventually they merge to cause parkinsonism. The current challenge facing PD research is to unravel the components in these pathways that contribute to the pathogenesis of PD. Accumulating evidence has implicated dysfunctional autophagy, a regulated lysosomal pathway with a capacity for clearing protein aggregates and cellular organelles, as one of the pathogenic systems contributing to the development of idiopathic PD.     

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.     

New insights into the role of mitochondrial dysfunction and protein aggregation in Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative movement disorder that affects increasing number of elderly in the world population. The disease is caused by a selective degeneration of dopaminergic neurons in the substantia nigra pars compacta with the molecular mechanism underlying this neurodegeneration still not fully understood. However, various studies have shown that mitochondrial dysfunction and abnormal protein aggregation are two of the major contributors for PD. In fact this notion has been supported by recent studies on genes that are linked to familial PD (FPD). For instance, FPD linked gene products such as PINK1 and parkin have been shown to play critical roles in the quality control of mitochondria, whereas α-synuclein has been found to be the major protein aggregates accumulated in PD patients. These findings suggest that further understanding of how dysfunction of these pathways in PD will help develop new approaches for the treatment of this neurodegenerative disorder.     

Genetic animal models for evaluating the role of autophagy in etiopathogenesis of Parkinson disease

Parkinson disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by the formation of ubiquitin and SNCA/α-synuclein-containing inclusions (Lewy bodies), dystrophic midbrain dopaminergic (DAergic) terminals, and degeneration of midbrain DAergic neurons. The vast majority of PD occurs sporadically, while approximately 5% of all PD cases are inherited. Genetic mutations of a few genes have been identified as causes of familiar PD, i.e., mutations in SNCA, PARK2/parkin, UCHL1, PARK7/DJ1, PINK1 and LRRK2, leading to DAergic cell death, but variable pathological changes. The evidence supports the hypothesis that several pathogenic mechanisms are likely involved at initial stages of the disease, and eventually they merge to cause parkinsonism. The current challenge facing PD research is to unravel the components in these pathways that contribute to the pathogenesis of PD. Accumulating evidence has implicated dysfunctional autophagy, a regulated lysosomal pathway with a capacity for clearing protein aggregates and cellular organelles, as one of the pathogenic systems contributing to the development of idiopathic PD.     

Genetic perspective on the role of the autophagy-lysosome pathway in Parkinson disease

Parkinson disease (PD), once considered as a prototype of a sporadic disease, is now known to be considerably affected by various genetic factors, which interact with environmental factors and the normal process of aging, leading to PD. Large studies determined that the hereditary component of PD is at least 27%, and in some populations, single genetic factors are responsible for more than 33% of PD patients. Interestingly, many of these genetic factors, such as LRRK2, GBA, SMPD1, SNCA, PARK2, PINK1, PARK7, SCARB2, and others, are involved in the autophagy-lysosome pathway (ALP). Some of these genes encode lysosomal enzymes, whereas others correspond to proteins that are involved in transport to the lysosome, mitophagy, or other autophagic-related functions. Is it possible that all these factors converge into a single pathway that causes PD? In this review, we will discuss these genetic findings and the role of the ALP in the pathogenesis of PD and will try to answer this question. We will suggest a novel hypothesis for the pathogenic mechanism of PD that involves the lysosome and the different autophagy pathways.     

Progress in the pathogenesis and genetics of Parkinson's disease

Recent progresses in the pathogenesis of sporadic Parkinson's disease (PD) and genetics of familial PD are reviewed. There are common molecular events between sporadic and familial PD, particularly between sporadic PD and PARK1-linked PD due to alpha-synuclein (SNCA) mutations. In sporadic form, interaction of genetic predisposition and environmental factors is probably a primary event inducing mitochondrial dysfunction and oxidative damage resulting in oligomer and aggregate formations of alpha-synuclein. In PARK1-linked PD, mutant alpha-synuclein proteins initiate the disease process as they have increased tendency for self-aggregation. As highly phosphorylated aggregated proteins are deposited in nigral neurons in PD, dysfunctions of proteolytic systems, i.e. the ubiquitin-proteasome system and autophagy-lysosomal pathway, seem to be contributing to the final neurodegenerative process. Studies on the molecular mechanisms of nigral neuronal death in familial forms of PD will contribute further on the understanding of the pathogenesis of sporadic PD.     

应用基因表达数量性状基因座定位（eQTL）研究PINK1表达调控

Pink 1基因编码定位于线粒体上的丝/苏氨酸激酶（即PARK6）,为常染色体隐性遗传性帕金森病（Parkinsons disease, PD）连锁的基因．该基因在遗传性和散发性PD的发病中起重要作用,但其发病机理尚未明确．本研究以近交系C57BL/6J (B6) 和DBA/2J (D2)小鼠制作MPTP诱导的PD鼠为模型,借助基因表达数量性状基因座(eQTL),结合分子生物学方法,分析Pink1的表达调控．结果显示,Pink 1基因在PD模型组中表达显著升高．区间连锁分析检测显示,引起Pink 1基因表达水平差异的染色体区域,定位于4号染色体上,距Pink 1基因自身5 Mb范围之类,属于顺式调节eQTL．Pearson相关分析表明,在BXD 基因重组近交系（recombinant inbred,RI）小鼠脑中,Camk2n等30个基因的表达与Pink 1基因高度相关,相互间可能存在一定的协同作用．Pink 1基因在行使特定生物学功能时,很可能协同这些基因一起发挥相应的作用,这部分基因是深入研究Pink 1基因在PD发病中分子机制的重要靶点．     

Copy number variation in familial Parkinson disease

Copy number variants (CNVs) are known to cause Mendelian forms of Parkinson disease (PD), most notably in SNCA and PARK2. PARK2 has a recessive mode of inheritance; however, recent evidence demonstrates that a single CNV in PARK2 (but not a single missense mutation) may increase risk for PD. We recently performed a genome-wide association study for PD that excluded individuals known to have either a LRRK2 mutation or two PARK2 mutations. Data from the Illumina370Duo arrays were re-clustered using only white individuals with high quality intensity data, and CNV calls were made using two algorithms, PennCNV and QuantiSNP. After quality assessment, the final sample included 816 cases and 856 controls. Results varied between the two CNV calling algorithms for many regions, including the PARK2 locus (genome-wide p = 0.04 for PennCNV and p = 0.13 for QuantiSNP). However, there was consistent evidence with both algorithms for two novel genes, USP32 and DOCK5 (empirical, genome-wide p-values<0.001). PARK2 CNVs tended to be larger, and all instances that were molecularly tested were validated. In contrast, the CNVs in both novel loci were smaller and failed to replicate using real-time PCR, MLPA, and gel electrophoresis. The DOCK5 variation is more akin to a VNTR than a typical CNV and the association is likely caused by artifact due to DNA source. DNA for all the cases was derived from whole blood, while the DNA for all controls was derived from lymphoblast cell lines. The USP32 locus contains many SNPs with low minor allele frequency leading to a loss of heterozygosity that may have been spuriously interpreted by the CNV calling algorithms as support for a deletion. Thus, only the CNVs within the PARK2 locus could be molecularly validated and associated with PD susceptibility.     

Identification of risk and age-at-onset genes on chromosome 1p in Parkinson disease

We previously reported a linkage region on chromosome 1p (LOD = 3.41) for genes controlling age at onset (AAO) in Parkinson disease (PD). This region overlaps with the previously reported PARK10 locus. To identify the gene(s) associated with AAO and risk of PD in this region, we first applied a genomic convergence approach that combined gene expression and linkage data. No significant results were found. Second, we performed association mapping across a 19.2-Mb region centered under the AAO linkage peak. An iterative association mapping approach was done by initially genotyping single-nucleotide polymorphisms at an average distance of 100 kb apart and then by increasing the density of markers as needed. Using the overall data set of 267 multiplex families, we identified six associated genes in the region, but further screening of a subset of 83 families linked to the chromosome 1 locus identified only two genes significantly associated with AAO in PD: the gamma subunit of the translation initiation factor EIF2B gene (EIF2B3), which was more significant in the linked subset and the ubiquitin-specific protease 24 gene (USP24). Unexpectedly, the human immunodeficiency virus enhancer-binding protein 3 gene (HIVEP3) was found to be associated with risk for susceptibility to PD. We used several criteria to define significant results in the presence of multiple testing, including criteria derived from a novel cluster approach. The known or putative functions of these genes fit well with the current suspected pathogenic mechanisms of PD and thus show great potential as candidates for the PARK10 locus.     

The regulatory role of α-synuclein and parkin in neuronal cell apoptosis; possible implications for the pathogenesis of Parkinson’s disease

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder beyond Alzheimer’s disease, affecting approximately 1% of people over the age of 65. The major pathological hallmarks of PD are significant loss of nigrostriatal dopaminergic (DA) neurons and the presence of intraneuronal protein inclusions termed Lewy bodies. Sporadic cases represent more than 90% of total patients with PD, while there exist several inherited forms caused by mutations in single genes. Identification and characterization of these causative genes and their products can help us understand the molecular mechanisms of DA neuronal cell death and design new approaches to treat both the inherited and sporadic forms of PD. Based on the finding that a point mutation in the gene encoding α-synuclein (αSyn) protein causes a rare familial form of PD, PARK1, it is now confirmed that αSyn is a major component of Lewy bodies in patients with sporadic PD. Abnormal accumulation of αSyn protein is considered a neurotoxic event in the development of PD. PARK4, another dominantly inherited form of familial PD, is caused by duplication or triplication of the αSyn gene locus. This genetic mutation results in the production of large amounts of wild-type αSyn protein, supporting the αSyn-induced neurodegeneration hypothesis. On the other hand, the recessively inherited early-onset Parkinsonism is caused in about half of the cases with loss-of-function mutations in PARK2, which encodes E3 ubiquitin ligase parkin in the ubiquitin–proteasome system. These findings have shed light on DA neurodegeneration caused by accumulation of toxic protein species that can be degraded and/or detoxicated through parkin activity. In this review, we will focus on the regulatory roles of αSyn and parkin proteins in DA neuronal cell apoptosis and provide evidence for the possible therapeutic action of parkin in sporadic patients with PD.     

PARK2 gene variants in Korean patients with Parkinson’s disease

Mutations in PARK2 are considered a common cause of Parkinson’s disease (PD). To assess the frequency of PARK2 mutations in the Korean population, we screened the PARK2 gene in 83 Korean PD patients: two young onset (YO, ≤ 49), 32 middle onset (MO, 50–69) and 49 late onset (LO, ≥ 70). Detection of the point mutations was performed by direct sequencing of the PARK2 exons, and exonic rearrangements were analyzed using multiplex ligation-dependent probe amplification. Five known PARK2 variants were identified in 53 (63.9 %) of the Korean PD patients: two missense mutations (Y267H and M458L) and three polymorphisms (S167N, L272I and V380L). We also found an increased frequency of PARK2 variants in PD patients and a lowered PD age at onset (AAO) in those having two variants, suggesting that the genetic variation in PARK2 gene might be a genetic risk factor of PD in Korean population.     

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.     

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.     

Essential control of mitochondrial morphology and function by chaperone-mediated autophagy through degradation of PARK7

As a selective degradation system, chaperone-mediated autophagy (CMA) is essential for maintaining cellular homeostasis and survival under stress conditions. Increasing evidence points to an important role for the dysfunction of CMA in the pathogenesis of Parkinson disease (PD). However, the mechanisms by which CMA regulates neuronal survival under stress and its role in neurodegenerative diseases are not fully understood. PARK7/DJ-1 is an autosomal recessive familial PD gene. PARK7 plays a critical role in antioxidative response and its dysfunction leads to mitochondrial defects. In the current study, we showed that CMA mediated the lysosome-dependent degradation of PARK7. Importantly, CMA preferentially removed the oxidatively damaged nonfunctional PARK7 protein. Furthermore, CMA protected cells from mitochondrial toxin MPP⁺-induced changes in mitochondrial morphology and function, and increased cell viability. These protective effects were lost under PARK7-deficiency conditions. Conversely, overexpression of PARK7 significantly attenuated the mitochondrial dysfunction and cell death exacerbated by blocking CMA under oxidative stress. Thus, our findings reveal a mechanism by which CMA protects mitochondrial function by degrading nonfunctional PARK7 and maintaining its homeostasis, and dysregulation of this pathway may contribute to the neuronal stress and death in PD pathogenesis.