Ubiquitin-proteasome system and Parkinson's diseases

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by nigrostriatal dopaminergic degeneration and development of cytoplasmic inclusions known as Lewy bodies. To date, the mechanisms involved in PD pathogenesis are not clearly understood. Clues from genetic studies including identification of mutations in genes for alpha-synuclein, parkin, and ubiquitin carboxy hydrolase L1 associated with familial PD and the presence of proteinaceous cytoplasmic inclusions in spared dopaminergic nigral neurons in sporadic cases of PD have suggested an important role for ubiquitin-proteasome system (UPS) and aberrant protein degradation. In vivo and in vitro studies have linked parkin, alpha-synuclein, and oxidative stress to a compromised UPS and PD pathogenesis suggesting novel therapeutic targets.     

Mutation analysis and association studies of the UCHL1 gene in German Parkinson's disease patients

Recently, an Ile93Met substitution has been identified in the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene in a single German PD family with autosomal dominant inheritance. To determine whether mutations in the UCHL1 gene are causative for Parkinson's disease (PD) a detailed mutation analysis was performed in a large sample of German sporadic and familial PD patients. We found no disease-causing mutation in the coding region of the UCHL1 gene. Direct sequencing revealed six intronic polymorphisms in the UCHL1 gene. Analysis of an S18Y polymorphism in exon 3 of the UCHL1 gene in sporadic PD patients and controls showed carriers of allele 2 (tyrosine) significantly less frequent in patients with a reduced risk of 0.57 (CI = 0.36-0.88; p = 0.012, p(c) = 0.047, chi2 = 6.31). Our study shows that sequence variations in the coding region of UCHL1 are a rare event. A protective effect of a certain UCHL1 variant in the pathogenesis of sporadic PD is suggested, underlining the relevance of UCHL1 in neurodegeneration.     

Pathological proteins in Parkinson’s disease

Parkinson's disease (PD) is a multifactorial disease that appears to arise from the effects of both genetic and environmental influences. Pesticides and heavy metals are the principle environmental factors that appear to impact on PD. The known genetic factors include multiple genes that have been identified in related parkinsonian syndromes, as well as alpha-synuclein. Genes associated with either PD or Parkinson-related disorders include parkin, DJ-1, ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1), nuclear receptor-related factor 1, and alpha-synuclein. Alpha-synuclein is particularly notable because it aggregates readily and is the main component of Lewy bodies (LBs). Aggregated alpha-synuclein binds the proteasome and potently inhibits proteasomal activity. Because ubiquitin accumulates in LBs, and parkin and UCH-L1 also interact with the ubiquitin proteasomal system, proteasomal dysfunction is thought to contribute to the pathophysiology of PD. Increasing numbers of experiments suggest that neurotoxins might interact with alpha-synuclein or other Parkinson-related proteins to contribute to the pathophysiology of PD. Transgenic animal models overexpressing alpha-synuclein develop age-dependent motor dysfunction and inclusions in the brain stem that contain alpha-synuclein. These models are very helpful in elucidating the pathophysiology of PD but do not completely recapitulate the disease process. The relationship between these transgenic models and PD is a subject of intense investigation.     

Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies

Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.     

UCHL1 is a Parkinson's disease susceptibility gene

The reported inverse association between the S18Y variant of the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene and Parkinson's disease (PD) has strong biological plausibility. If confirmed, genetic association of this variant with PD may support molecular targeting of the UCHL1 gene and its product as a therapeutic strategy for PD. In this light, we performed a collaborative pooled analysis of individual-level data from all 11 published studies of the UCHL1 S18Y gene variant and PD. There were 1,970 cases and 2,224 unrelated controls. We found a statistically significant inverse association of S18Y with PD. Carriers of the variant allele (Y/Y plus Y/S vs S/S) had an odds ratio (OR) of 0.84 (95% confidence interval [CI], 0.73-0.95) and homozygotes for the variant allele (Y/Y vs S/S plus Y/S) had an OR of 0.71 (95% CI, 0.57-0.88). There was a linear trend in the log OR consistent with a gene dose effect (p = 0.01). The inverse association was most apparent for young cases compared with young controls. There was no evidence for publication bias and the associations remained significant after excluding the first published, hypothesis-generating study. These findings confirm that UCHL1 is a susceptibility gene for PD and a potential target for disease-modifying therapies.     

Case-control study of UCHL1 S18Y variant in Parkinson's disease.

A recent meta-analysis observed a greater significant inverse association of the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) S18Y variant with Parkinson's disease (PD) for Asian (predominantly Japanese) populations compared with Caucasian populations. We performed an independent case-control study in 335 PD and 341 control subjects with data from a Chinese population to investigate the age-of-onset effect of the UCHL1 variant in PD. The Y/Y and Y/S genotypes were less frequent in the PD young-onset group than in controls and the frequency of the Y alleles was higher in young controls compared to young-onset PD (age at examination 相似文献   

Ubiquitin proteasome system in Parkinson's disease: A keeper or a witness?

ObjectiveThe aim of this work was to evaluate the role of ubiquitin–proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models.MethodWe used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations.ResultsWe found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC.We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients.InterpretationaSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC.     

Alterations of ubiquitin related proteins in the pathology and development of schizophrenia: Evidence from human and animal studies

Gene expression analyses in post-mortem schizophrenia brains suggest that a number of ubiquitin proteasome system (UPS) genes are associated with schizophrenia; however the status of UPS proteins in the schizophrenia brain is largely unknown. Ubiquitin related proteins are inherently involved in memory, neuronal survival and morphology, which are processes implicated in neurodevelopmental disorders such as schizophrenia. We examined levels of five UPS proteins (Protein Inhibitor of Activated STAT2 [PIAS2], F-Box and Leucine rich repeat protein 21 [FBXL21], Mouse Double Minute 2 homolog [MDM2], Ubiquitin Carboxyl-Terminal Hydrolase-L1 [UCHL1] and Ubiquitin Conjugating Enzyme E2D1 [UBE2D1]) involved in these neuronal processes, within the dorsolateral prefrontal cortex of post-mortem schizophrenia subjects and matched controls (n = 30/group), in addition to across neurodevelopmental time-points (juvenile, adolescent and adult stages of life), utilizing a well-established neurodevelopmental phencyclidine (PCP) animal model of schizophrenia. We observed significant reductions in PIAS2, FBXL21 and MDM2 in schizophrenia subjects compared to controls (p-values ranging from 0.002 to 0.004). In our developmental PCP model, MDM2 protein was significantly reduced in adult PCP-treated rats compared to controls (p = 0.034). Additionally, FBXL21 (p = 0.022) and UCHL1 (p = 0.022) were significantly decreased, whilst UBE2D1 was increased (p = 0.022), in juvenile phencyclidine-treated rats compared to controls. This is the first study reporting alterations of UPS proteins in post-mortem human schizophrenia subjects and in a neurodevelopmental model of schizophrenia. The findings from this study provide strong support for a role of these UPS proteins in the pathology and development of schizophrenia.     

Parkin and defective ubiquitination in Parkinson's disease

Parkinson's Disease (PD) is a common neurodegenerative disorder that is characterized by the progressive loss of dopamine (DA) neurons. Accompanying the loss the of DA neurons is the accumulation of Lewy bodies and neurites, intracytoplasmic proteinaceous inclusions that contain alpha-synuclein, synphilin-1, components of the ubiquitin proteasomal pathway and parkin. Recent advances indicate that PD is due in some individuals to genetic mutations in alpha-synuclein, DJ-1, PINK-1, LRRK2, and parkin. Understanding the molecular mechanisms by which mutations in familial-linked genes cause PD holds great promise for unraveling the mechanisms by which DA neurons degenerate in PD. Parkin is E3-ubiquitin-protein ligase that ubiquitinates itself and promotes its own degradation. Familial associated mutations of parkin have impaired ubiquitin ligase function suggesting that this may be the cause of familial autosomal recessive PD. Parkin might be required for formation of Lewy bodies as Lewy bodies are absent in patients with parkin mutations. Parkin interacts with and ubiquitinates the alpha-synuclein interacting protein, synphilin-1. Formation of Lewy-body-like ubiquitin-positive cytosolic inclusions occurs upon coexpression of alpha-synuclein, synphilin-1 and parkin. Nitric oxide inhibits Parkin's E-3 ligase activity and its protective function by nitric oxide through S-nitrosylation both in vitro and in vivo. Nitrosative and oxidative stress link parkin function with the more common sporadic form of Parkinson's disease and the related alpha-synucleinopathy, DLBD. Development of new therapies for PD and other disorders associated with nitrosative and oxidative stress may follow the elucidation of the pathways by which NO S-nitrosylates and inhibits parkin. Moreover, parkin and alpha-synuclein are linked in common pathogenic mechanism through their interaction with synphilin-1 and parkin may be important for the formation of Lewy bodies.     

Recent advances in the genetics and pathogenesis of Parkinson disease.

The identification of three genes and several additional loci associated with inherited forms of levodopa-responsive PD has confirmed that this is not a single disorder. Yet, analyses of the structure and function of these gene products point to the critical role of protein aggregation in dopaminergic neurons of the substantia nigra as the common mechanism leading to neurodegeneration in all known forms of this disease. The three specific genes identified to date--alpha-synuclein, Parkin, and ubiquitin C terminal hydrolase L1--are either closely involved in the proper functioning of the ubiquitin-proteasome pathway or are degraded by this protein-clearing machinery of cells. Knowledge gained from genetically transmitted PD also has clear implications for nonfamilial forms of the disease. Lewy bodies, even in sporadic PD, contain these three gene products, particularly abundant amounts of fibrillar alpha-synuclein. Increased aggregation of alpha-synuclein by oxidative stress, as well as oxidant-induced proteasomal dysfunction, link genetic and potential environmental factors in the onset and progression of the disease. The biochemical and molecular cascades elucidated from genetic studies in PD can provide novel targets for curative therapies.     

Mitochondrial Dysfunction Precedes Other Sub-Cellular Abnormalities in an In Vitro Model Linked with Cell Death in Parkinson’s Disease

Dysfunction of mitochondria, the ubiquitin proteasome system (UPS), and lysosomes are believed to contribute to the pathogenesis of Parkinson's disease (PD). If it were possible to rescue functionally compromised, but still viable neurons early in the disease process, this would slow the rate of neurodegeneration. Here, we used a catecholaminergic neuroblastoma cell line (SH-SY5Y) as a model of susceptible neurons in PD. To identify a target early in the cell death process that was common to all neurodegenerative processes linked with PD, cells were exposed to toxins that mimic cell death mechanisms associated with PD. The sub-cellular abnormalities that occur shortly after toxin exposure were determined. 3?h of exposure to either naphthazarin, to inhibit lysosomal function, Z-Ile-Glu(OBu(t))-Ala-Leu-H (PSI), to inhibit the UPS, or rotenone, to inhibit mitochondrial complex I, caused depolarisation of the mitochondrial membrane potential (2.5-fold, twofold, and 4.6-fold change, respectively compared to vehicle), suggesting impaired mitochondrial function. Following 24?h exposure to the same toxins, UPS and lysosomal function were also impaired, and ubiquitin levels were increased. Thus, following exposure to toxins that mimic three important, but disparate cell death mechanisms associated with PD, catecholaminergic cells initially experience mitochondrial dysfunction, which is then followed by abnormalities in UPS and lysosomal function. Thus, mitochondrial dysfunction is an early event in cell stress. We suggest that, in patients with PD, the surviving cells of the substantia nigra pars compacta are most susceptible to mitochondrial impairment. Thus, targeting the mitochondria may be useful for slowing the progression of neurodegeneration in PD.     

Mutant and wild-type alpha-synuclein interact with mitochondrial cytochrome C oxidase

Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson's disease (PD). Furthermore, rare mutations of alpha-synuclein cause autosomal-dominant PD. However, it is unknown how alpha-synuclein is involved in the pathogenesis of nigral degeneration in PD. In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) a-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique. We found that both normal and mutant alpha-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX). Wild-type and mutant alpha-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate. Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant alpha-synuclein, coimmunoprecipitated with COX. We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death. In conclusion, we found specific protein-protein interactions of alpha-synuclein, a major LB protein, to COX, a key enzyme of the mithochondrial respiratory system. This interaction suggests that alpha-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.     

Interaction of alpha-synuclein and tau genotypes in Parkinson's disease

To determine whether the microtubule-associated protein tau (MAPT) and alpha-synuclein (SNCA) genes interact to confer Parkinson's disease (PD) susceptibility, we conducted a study of 557 case-control pairs. There was an increased risk of PD for persons with either SNCA 261/261 or MAPT H1/H1 genotypes as compared with persons with neither (odds ratio, 1.96; 95% confidence interval, 1.34-2.86; p = 0.0003). However, the combined effect of the two genotypes was the same as for either of the genotypes alone (separate and equal). These findings are consistent with in vitro experiments that revealed tau-mediated fibrillization of alpha-synuclein protein at low concentrations (dose threshold effect).     

The UCHL1 S18Y polymorphism and Parkinson's disease in a Japanese population

UCHL1 plays an important role in the ubiquitin-proteasome system and is a biologically plausible candidate gene for Parkinson's disease (PD). However, results from genetic association studies of the UCHL1 S18Y polymorphism have been equivocal. Meta-analyses indicate that the polymorphism's risk effect might be restricted to Asian populations and early-onset disease. To further explore the role of UCHL1 in PD, we genotyped S18Y in 605 PD patients and 1620 controls of Japanese ancestry. We did not find evidence of an association in the overall sample (SY vs. SS: adjusted OR=1.11, P=0.37; YY vs. SS: adjusted OR=1.01, P=0.94). In the early-onset stratum, however, we observed a trend toward a reduction in risk for those with the Y allele (SY vs. SS, adjusted OR, 0.75; 95% CI, 0.47-1.20; YY vs. SS, OR, 0.64; 95% CI, 0.36-1.14; trend test, P=0.12). These results indicate that, if involved in PD, the S18Y variant is not a major determinant of risk and its effect might be restricted to early-onset disease.     

The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity.

Parkinson's disease (PD) is the most common motor disorder affecting the elderly. PD is characterized by the formation of Lewy bodies and death of dopaminergic neurons. The mechanisms underlying PD are unknown, but the discoveries that mutations in alpha-synuclein can cause familial PD and that alpha-synuclein accumulates in Lewy bodies suggest that alpha-synuclein participates in the pathophysiology of PD. Using human BE-M17 neuroblastoma cells overexpressing wild-type, A53T, or A30P alpha-synuclein, we now show that iron and free radical generators, such as dopamine or hydrogen peroxide, stimulate the production of intracellular aggregates that contain alpha-synuclein and ubiquitin. The aggregates can be identified by immunocytochemistry, electron microscopy, or the histochemical stain thioflavine S. The amount of aggregation occurring in the cells is dependent on the amount of alpha-synuclein expressed and the type of alpha-synuclein expressed, with the amount of alpha-synuclein aggregation following a rank order of A53T > A30P > wild-type > untransfected. In addition to stimulating aggregate formation, alpha-synuclein also appears to induce toxicity. BE-M17 neuroblastoma cells overexpressing alpha-synuclein show up to a fourfold increase in vulnerability to toxicity induced by iron. The vulnerability follows the same rank order as for aggregation. These data raise the possibility that alpha-synuclein acts in concert with iron and dopamine to induce formation of Lewy body pathology in PD and cell death in PD.     

Molecular genetic analysis of the α-synuclein and the parkin gene in Parkinson''s disease in Finland

Two mutations in the alpha-synuclein gene and various mutations in the parkin gene are associated with familial Parkinson's disease (PD). The present study was performed to analyse if mutations in these genes could be detected in Finnish patients with familial PD. The subjects comprised 22 unrelated patients with familial PD. The molecular genetic analysis consisted of sequence analysis of the non-coding and coding exons of the alpha-synuclein gene and screening of eight point mutations in the parkin gene. In addition, a total of 67 controls and 45 patients with sporadic PD were included in the association analysis on polymorphism of the alpha-synuclein gene. Screened point mutations in the parkin gene were not detected. Sequencing of the coding exons 2-6 of the alpha-synuclein gene did not reveal any mutations or polymorphisms. However, three novel alterations in the T10A7 sequence at the 5' end of the non-coding exon 1' of the alpha-synuclein gene were found. The frequencies of the exon 1' polymorphic genotypes or alleles between familial PD patients and control subjects revealed no statistically significant differences. No association for sporadic PD was observed. The results do not support a role for the alpha-synuclein gene or point mutations of the parkin gene in familial PD in our sample.     

Familial Parkinson's disease: a hint to elucidate the mechanisms of nigral degeneration

In the majority of patients with Parkinson's disease (PD), it is now clear that genetic factors contribute to the pathogenesis of PD, although the contribution of genetic and environmental factors remains to be elucidated. The contribution of genetic factors to the pathogenesis of PD is supported by the demonstration of the high concordance in twins, increased risk among relatives of PD patients in case control and family studies, and the existence of familial PD based on single gene defects. Recently, several genes have been mapped and identified in patients with familial PD (FPD). alpha-Synuclein is involved in a rare dominant form of familial PD with dopa responsive parkinsonian features and Lewy body positive pathology. In contrast, parkin is responsible for autosomal recessive form of earlyonset PD with Lewy body-negative pathology. This form is identified with world-wide distribution among patients with young-onset PD. Furthermore, ubiquitin carboxy terminal hydrolase L1 (UCHL1) gene is responsible for an autosomal dominant form of typical PD, although only a single family has so far been identified with a mutation of this gene. In addition, DJ-1 has been identified as a causative gene for PARK7, a recessive form of familial PD. Now, a total of five causative genes including NR4A2 have been identified, and others such as PARK3, -4, -6, -8, -9, -10 have been mapped as hereditary forms of familial PD. The presence of different loci or different causative genes indicates that PD is not a single entity but a highly heterogeneous disorder. However, the functions of causative genes may share a common pathway such as an ubiquitin-proteasome pathway. Thus, identification and elucidation of the causative genes should enhance our understanding of the pathogenesis of not only familial PD, but also sporadic PD.     

Toward identification of susceptibility genes for sporadic Parkinson's disease

To identify susceptibility genes for Parkinson's disease (PD) and to establish tailor-made medicine for PD, we studied 20 single nucleotide polymorphisms (SNPs) in 18 candidate genes for association with PD. We found that homozygosity for the V66M polymorphism of the BDNF gene occurs more frequently in PD patients than in unaffected controls and confirmed an association with the S18Y polymorphism of the UCHL1 gene. We further started microsatellite marker-based genome-wide association studies by using the pooled DNA method. We have finished checking approximately 6800 markers and found some significant associations (p=3.9 x 10(-6)) on chromosome 1 where other studies showed a linkage. Genes in linkage disequilibrium with these markers may be associated with pathogenesis of PD.