Identification of brain proteins that interact with 2-methylnorharman. An analog of the parkinsonian-inducing toxin,MPP+

N-Methylated beta-carbolines, including 2-methylnorharman, are structural and functional analogs of the parkinsonian-inducing toxin, MPP+. We are investigating N-methylated beta-carbolines, including 2-methylnorharman, as possible etiologic factors in the pathogenesis of Parkinson's disease. The cellular targets of N-methylated beta-carboline-mediated cytotoxicity are unknown; therefore, we used the T7Select Phage Display System in a novel approach to identify brain proteins that bind to 2-methylnorharman. We incubated (biopanned) immobilized 2-methylnorharman with a phage display cDNA library that expressed a library of human brain proteins on the surface of bacteriophage T7. We washed off unbound phage, amplified the phage that were bound to 2-methylnorharman, and enriched for toxin-interacting phage by repeating the biopanning and amplification steps. The cDNA sequences from the toxin-interacting phage were used to derive the amino acid sequences of the phage-displayed proteins. Five of the six 2-methylnorharman-interacting proteins may have relevance to Parkinson's disease: alpha-tubulin, paraoxonase, dorfin, fatty acid binding protein, and platelet-activating factor acetylhydrolase. Dorfin has sequence homology with parkin, which is interesting because mutations in the parkin gene associate with early-onset Parkinson's disease. Our findings are the basis for future studies aimed at determining whether 2-methylnorharman affects the function of these specific proteins in vitro and in vivo.     

A novel role for parkin in trauma-induced central nervous system secondary injury

Recently, loss-of-function mutations of parkin have been identified as being causally related to autosomal recessive juvenile parkinsonism, the most common form of familial Parkinson's disease. In addition to functioning as an E3 ubiquitin ligase that facilitates the proteasomal degradation of proteins with abnormal conformations, parkin protects dopaminergic neurons from oxidative stress-mediated death by regulating mitochondrial function. Parkin is expressed throughout the brain in a variety of functional and neurochemical systems. We propose that parkin's role in protecting neurons from oxidative stress may extend beyond the nigrostriatal system to include neurons in other regions of the central nervous system. This is relevant for therapeutic strategies for brain and spinal cord injury because oxidative stress leading to lipid peroxidation and protein and nucleic acid oxidation is a significant cause of secondary injury and thus neuronal death following traumatic injuries to the central nervous system. A novel model system to verify the process of oxidative stress as a causative factor in trauma-induced secondary injury mechanisms would be to induce traumatic brain and spinal cord injury in parkin-null mice. This is expected to provide the proof-of-principle that a cascade of oxidative stress is a causal event leading to secondary neuronal injury, that parkin functions outside of the dopaminergic system to protect other neurons from oxidative stress, and that antioxidant pharmacotherapy is a rational therapeutic approach to decrease trauma-induced neuronal injury.     

Somatotopic mapping of the human primary sensorimotor cortex during motor imagery and motor execution by functional magnetic resonance imaging

Autosomal recessive parkinsonism associated with mutations in the parkin gene represents a monogenic form of hereditary parkinsonism. We performed [(18)F]6-fluorodopa (FDOPA) positron emission tomography as a measurement of the nigrostriatal dopaminergic system as well as extensive haplotype analysis of the PARK 2 gene locus in 14 subjects with parkin mutations. In parkin subjects, the reduction of striatal FDOPA uptake increased with the number of mutated alleles and was also slightly obvious in asymptomatic parkin gene carriers in the heterozygous state. The abnormal FDOPA uptake pattern in parkin patients did not significantly differ from that of sporadic Parkinson's disease. Our data are in agreement with an enzymatic dysfunction of the gene's translational product, which has been shown to promote protein degradation as an ubiquitin-protein ligase. Thus, parkinsonism in parkin gene carriers may be related to abnormal nigral protein accumulation in the presence of a suprathreshold enzyme dysfunction.     

parkin基因的一个新的点突变

目的：研究parkin基因外显子2-10点突变与散发性早发帕金森病发病的关系。方法：应用聚合酶链反应（polymerase chain reaction ，PCR）、琼脂糖电泳、单链构象多态性（single strand conformation polymorphism，SSCP）、DNA测序及限制性核酸内切酶酶切方法，检测了60例散发性早发帕金森病患者以及120名正常人外周血白细胞DNA的parkin基因外显子2-10点突变。结果：发现1例患者的parkin基因外显子2存在纯合突变（G237→C），限制性内切酶酶切证实，其它外显子未见突变，120名正常对照也未见突变。结论：parkin基因外显子存在点突变，可能与部分散发性早发帕金森病发病有关。     

早发性帕金森病parkin基因的一个新的点突变

目的：观察不同亚型帕金森病（Parkinson's disease,PD）患者中是否存在parkin基因新的突变以及突变的分布，探讨parkin基因在PD发病机理中的可能作用。方法：70例患者被分为早发性PD和晚发性PD，70名正常体检者为对照组。以基因组DNA为模板，扩增parkin基因的全部12个外显子，然后行单链构象多态性（single-strand conformation polymorphism,SSCP）电泳观察，对泳动异常者进行DNA序列测定，以确定外显子中存在的突变及其分布。结果：70例患者中有4例SSCP泳动异常，测序证实1例早发性PD患者的外显子7存在1个未曾报道过的新的点突变位点Gly284Arg。结论：parkin基因点突变也是我国早发性PD患者的致病原因之一。     

The Machado-Joseph disease-associated mutant form of ataxin-3 regulates parkin ubiquitination and stability

Machado-Joseph disease (MJD), the most common dominantly inherited ataxia worldwide, is caused by a polyglutamine (polyQ) expansion in the deubiquitinating (DUB) enzyme ataxin-3. Interestingly, MJD can present clinically with features of Parkinsonism. In this study, we identify parkin, an E3 ubiquitin-ligase responsible for a common familial form of Parkinson's disease, as a novel ataxin-3 binding partner. The interaction between ataxin-3 and parkin is direct, involves multiple domains and is greatly enhanced by parkin self-ubiquitination. Moreover, ataxin-3 deubiquitinates parkin directly in vitro and in cells. Compared with wild-type ataxin-3, MJD-linked polyQ-expanded mutant ataxin-3 is more active, possibly owing to its greater efficiency at DUB K27- and K29-linked Ub conjugates on parkin. Remarkably, mutant but not wild-type ataxin-3 promotes the clearance of parkin via the autophagy pathway. The finding is consistent with the reduction in parkin levels observed in the brains of transgenic mice over-expressing polyQ-expanded but not wild-type ataxin-3, raising the intriguing possibility that increased turnover of parkin may contribute to the pathogenesis of MJD and help explain some of its parkinsonian features.     

Stress-induced alterations in parkin solubility promote parkin aggregation and compromise parkin's protective function

Mutations in parkin are currently recognized as the most common cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for the development of the more common, sporadic form of Parkinson's disease (PD). Supporting this, we have recently demonstrated that parkin solubility in the human brain becomes altered with age. As parkin apparently functions as a broad-spectrum neuroprotectant, the resulting decrease in the availability of soluble parkin with age may underlie the progressive susceptibility of the brain to stress. Interestingly, we also observed that many familial-PD mutations of parkin alter its solubility in a manner that is highly reminiscent of our observations with the aged brain. The converging effects on parkin brought about by aging and PD-causing mutations are probably not trivial and suggest that environmental modulators affecting parkin solubility would increase an individual's risk of developing PD. Using both cell culture and in vivo models, we demonstrate here that several PD-linked stressors, including neurotoxins (MPP+, rotenone, 6-hydroxydopamine), paraquat, NO, dopamine and iron, induce alterations in parkin solubility and result in its intracellular aggregation. Furthermore, the depletion of soluble, functional forms of parkin is associated with reduced proteasomal activities and increased cell death. Our results suggest that exogenously introduced stress as well as endogenous dopamine could affect the native structure of parkin, promote its misfolding, and concomitantly compromise its protective functions. Mechanistically, our results provide a link between the influence of environmental and intrinsic factors and genetic susceptibilities in PD pathogenesis.     

Association between early-onset Parkinson's disease and mutations in the parkin gene

BACKGROUND: Mutations in the parkin gene have recently been identified in patients with early-onset Parkinson's disease, but the frequency of the mutations and the associated phenotype have not been assessed in a large series of patients. METHODS: We studied 73 families in which at least one of the affected family members was affected at or before the age of 45 years and had parents who were not affected, as well as 100 patients with isolated Parkinson's disease that began at or before the age of 45 years. All subjects were screened for mutations in the parkin gene with use of a semiquantitative polymerase-chain-reaction assay that simultaneously amplified several exons. We sequenced the coding exons in a subgroup of patients. We also compared the clinical features of patients with parkin mutations and those without mutations. RESULTS: Among the families with early-onset Parkinson's disease, 36 (49 percent) had parkin mutations. The age at onset ranged from 7 to 58 years. Among the patients with isolated Parkinson's disease, mutations were detected in 10 of 13 patients (77 percent) with an age at onset of 20 years or younger, but in only 2 of 64 patients (3 percent) with an age at onset of more than 30 years. The mean (+/-SD) age at onset in the patients with parkin mutations was younger than that in those without mutations (32+/-11 vs. 42+/-11 years, P<0.001), and they were more likely to have symmetric involvement and dystonia at onset, to have hyperreflexia at onset or later, to have a good response to levodopa therapy, and to have levodopa-induced dyskinesias during treatment. Nineteen different rearrangements of exons (deletions and multiplications) and 16 different point mutations were detected. CONCLUSIONS: Mutations in the parkin gene are a major cause of early-onset autosomal recessive familial Parkinson's disease and isolated juvenile-onset Parkinson's disease (at or before the age of 20 years). Accurate diagnosis of these cases cannot be based only on the clinical manifestations of the disease.     

Parkin protects human dopaminergic neuroblastoma cells against dopamine-induced apoptosis

Parkinson's disease (PD) is characterized by the selective degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc). A combination of genetic and environmental factors contributes to such a specific loss. Among the five PD-linked genes identified so far, parkin, a protein-ubiquitin E3 ligase, appears to be the most prevalent genetic factor in PD. Although a variety of substrates have been identified for parkin, none of them is selectively expressed in nigral DA neurons. It remains unclear how accumulation of these substrates in the absence of functional parkin may cause the selective death of DA neurons in SNpc. Here, we show that overexpression of parkin protected human DA neuroblastoma cell line (SH-SY5Y) against apoptosis induced by DA or 6-OHDA, but not by H(2)O(2) or rotenone. Parkin significantly attenuated dopamine-induced activation of c-Jun N-terminal kinase (JNK) and caspase-3. It also decreased the level of reactive oxygen species (ROS) and protein carbonyls in the cell. Inhibiting DA uptake through dopamine transporter or treating the cell with antioxidants significantly reduced oxidative stress and dopamine toxicity. Furthermore, PD-linked mutations of parkin significantly abrogated the protective effect of wild-type parkin, as well as its ability to suppress ROS and protein carbonylation. These results suggest that parkin protects against dopamine toxicity by decreasing oxidative stress and ensuing activation of apoptotic programs such as the JNK/caspase pathway. This protective function of parkin, which is greatly attenuated by its PD-linked mutations, may be uniquely important for the survival of DA neurons, as they are constantly threatened by oxyradicals produced during dopamine oxidation.     

Random forest fishing: a novel approach to identifying organic group of risk factors in genome-wide association studies

Genome-wide association studies (GWAS) has brought methodological challenges in handling massive high-dimensional data and also real opportunities for studying the joint effect of many risk factors acting in concert as an organic group. The random forest (RF) methodology is recognized by many for its potential in examining interaction effects in large data sets. However, RF is not designed to directly handle GWAS data, which typically have hundreds of thousands of single-nucleotide polymorphisms as predictor variables. We propose and evaluate a novel extension of RF, called random forest fishing (RFF), for GWAS analysis. RFF repeatedly updates a relatively small set of predictors obtained by RF tests to find globally important groups predictive of the disease phenotype, using a novel search algorithm based on genetic programming and simulated annealing. A key improvement of RFF results from the use of guidance incorporating empirical test results of genome-wide pairwise interactions. Evaluated using simulated and real GWAS data sets, RFF is shown to be effective in identifying important predictors, particularly when both marginal effects and interactions exist, and is applicable to very large GWAS data sets.     

Pael receptor induces death of dopaminergic neurons in the substantia nigra via endoplasmic reticulum stress and dopamine toxicity, which is enhanced under condition of parkin inactivation

Selective loss of dopaminergic neurons is the final common pathway in Parkinson's disease. Expression of Parkin associated endothelin-receptor like receptor (Pael-R) in mouse brain was achieved by injecting adenoviral vectors carrying a modified neuron-specific promoter and Cre recombinase into the striatum. Upregulation of Pael-R in the substantia nigra pars compacta of mice by retrograde infection induced endoplasmic reticulum (ER) stress leads to death of dopaminergic neurons. The role of ER stress in dopaminergic neuronal vulnerability was highlighted by their decreased survival in mice deficient in the ubiquitin-protein ligase Parkin and the ER chaperone ORP150 (150 kDa oxygen-regulated protein). Dopamine-related toxicity was also a key factor, as a dopamine synthesis inhibitor blocked neuronal death in parkin null mice. These data suggest a model in which ER- and dopamine-related stress are major contributors to decreased viability of dopaminergic neurons in a setting relevant to Parkinson's disease.     

Integrating genetic and gene expression evidence into genome-wide association analysis of gene sets

Single variant or single gene analyses generally account for only a small proportion of the phenotypic variation in complex traits. Alternatively, gene set or pathway association analyses are playing an increasingly important role in uncovering genetic architectures of complex traits through the identification of systematic genetic interactions. Two dominant paradigms for gene set analyses are association analyses based on SNP genotypes and those based on gene expression profiles. However, gene-disease association can manifest in many ways, such as alterations of gene expression, genotype, and copy number; thus, an integrative approach combining multiple forms of evidence can more accurately and comprehensively capture pathway associations. We have developed a single statistical framework, Gene Set Association Analysis (GSAA), that simultaneously measures genome-wide patterns of genetic variation and gene expression variation to identify sets of genes enriched for differential expression and/or trait-associated genetic markers. Simulation studies illustrate that joint analyses of genomic data increase the power to detect real associations when compared with gene set methods that use only one genomic data type. The analysis of two human diseases, glioblastoma and Crohn's disease, detected abnormalities in previously identified disease-associated pathways, such as pathways related to PI3K signaling, DNA damage response, and the activation of NFKB. In addition, GSAA predicted novel pathway associations, for example, differential genetic and expression characteristics in genes from the ABC transporter family in glioblastoma and from the HLA system in Crohn's disease. These demonstrate that GSAA can help uncover biological pathways underlying human diseases and complex traits.     

parkin mutation analysis in clinic patients with early-onset Parkinson [corrected] disease

parkin Mutations are the most common identified cause of Parkinson's disease (PD). It has been suggested that patients with young-onset PD be screened for parkin mutations as a part of their clinical work-up. The aim of this study was to assess parkin mutation frequency in a clinical setting, correlate genotype with phenotype, and evaluate the current justification for clinical parkin testing. Patients were selected from a movement disorder clinic based on diagnosis of PD and onset age 相似文献   

早发性帕金森病parkin基因第1～6外显子缺失突变的初步研究

目的 探讨中国人早发性帕金森病（ｐｒａｅｃｏｘ Ｐａｒｋｉｎｓｏｎ ｄｉｓｅａｓｅ,ＰＰＤ）中ｐａｒｋｉｎ基因第１～６外显子是否存在突变,及其与该病临床特点的关系。方法 用ＰＰＤ患者外周血液提取ＤＮＡ,通过ＰＣＲ扩增,琼脂糖凝胶电泳鉴定ｐａｒｋｉｎ基因外显子缺失突变,并结合临床资料分析。结果 ２１中层得中发现有２例第１外显子缺失,２例第４外显子缺失,１例第６外显子缺失;发生基因缺失突变的病例年龄为     

Prestimulus modification of the startle reflex: relationship to personality and physiological markers of dopamine function

Prepulse inhibition (PPI), a measure of sensorimotor gating, is regulated by dopamine (DA) in rodents. We examined the relationship of PPI in humans to putative markers of brain DA function: (1) novelty seeking (NS; Cloninger's Tridimensional Personality Questionnaire (TPQ)), which is associated with specific DA receptor subtypes, and is reduced in Parkinson's Disease; (2) blink rate, which is increased in primates by DA agonists, and is reduced in Parkinson's Disease. PPI, TPQ and blink rate were measured in 79 normal adult males. A significant negative correlation was observed between resting blink rate and mean PPI, but not between NS and PPI. Blink rate correlated positively with resting EMG level, but this did not account for the relationship between blink rate and PPI. In normal male humans, PPI is inversely related to a physiological marker of resting DA tone (blink rate), but not to a putatively DA-linked personality trait (high NS).     

A cell-based luciferase assay amenable to high-throughput screening of inhibitors of arenavirus budding

Several arenaviruses cause hemorrhagic fever (HF) disease in humans for which there are no licensed vaccines, and current therapy is limited to the use of ribavirin (Rib) that is only partially effective and associated with significant side effects. In addition, compelling evidence indicates that the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Therefore, it is important to develop novel and effective anti-arenaviral drugs. The arenavirus Z protein is the driving force of arenavirus budding, and PPPY and PTAP late (L) domain motifs within Z are critical for Z-mediated budding, which involves the interaction of Z with a variety of host cellular factors. Compounds capable of inhibiting these virus-host cell interactions represent candidate anti-arenaviral drugs. The identification of these candidate compounds would be facilitated by the availability of a Z budding assay amenable to high-throughput screens (HTS). To this end, we have developed a novel assay that allows for rapid and quantitative assessment of Z-mediated budding. We provide evidence that this novel assay is amenable to HTS to identify small molecule inhibitors of Z-mediated budding, as well as to uncover cellular genes contributing to arenavirus budding.