Parkin Disrupts the α-Synuclein/Dopamine Transporter Interaction: Consequences Toward Dopamine-induced Toxicity

Parkinson’s disease is characterized by progressive neuronal degeneration of dopaminergic neurons in the substantia nigra. Many factors are thought to contribute to the neuronal cell death that occurs in Parkinson’s disease, including α-synuclein-mediated toxicity. Previously, we have reported that α-synuclein directly couples to the carboxyl tail of the dopamine transporter (DAT) and that the α-synuclein/DAT protein complex formation accelerates DAT-mediated cellular dopamine (DA) uptake and DA-induced cellular apoptosis. In the present study, we report that parkin, an E2-dependent E3 protein ubiquitin ligase associated with recessive early onset Parkinson’s disease, exerts a protective effect against DA-induced α-synuclein-dependent cell toxicity. Parkin impairs the α-synuclein/DAT coupling by interacting with the carboxyl-terminus of the DAT and blocks the α-synuclein-induced enhancement in both DAT cell surface expression and DAT-mediated DA uptake. Moreover, we have found that parkin protects against DA-induced cell toxicity in dopaminergic SK-N-SH cells. These findings will help identify the role of these proteins in the etiology and/or maintenance of Parkinson’s disease.     

线粒体自噬最新研究进展

活性氧由线粒体产生且作用于线粒体.在压力环境下,选择性降解损伤的线粒体,并维持正常的线粒体数量对维持细胞的正常结构、生长起重要作用.线粒体通透性改变、过氧化氢酶失活等诸多影响因素都会促进线粒体自噬的发生.最近研究发现PINK1-Parkin信号通路,Mfn1、Mfn2和OPA1等蛋白与线粒体关系紧密,调控线粒体的融合、分裂、自噬.线粒体自噬可能导致神经退行性疾病,主要的神经退行性疾病包括帕金森病(Parkinson's disease,PD)、肌萎缩侧索硬化症(Alzheimer's disease,AD)、亨廷顿舞蹈病等(Huntington's disease,HD),这些与线粒体的动态改变有关.     

A meta‐analysis of the relationship of the Parkin p.Val380Leu polymorphism to Parkinson's disease

Parkinson's disease (PD) is one of the most common movement disorders. Parkin p.Val380Leu polymorphism (c.1239G > C) has been investigated as a potential genetic hallmark of PD, but studies examining the association between the polymorphism and PD have reported conflicting results. Therefore, we conducted a meta‐analysis to assess the influence of Parkin p.Val380Leu polymorphism on the susceptibility of PD. Computer and hand searches of the literature were conducted using the MEDLINE, EMBASE, Cochrane Library, and China Academic Journals databases to identify studies addressing the association between the Parkin p.Val380Leu polymorphism and PD risk. We performed analyses of study characteristics, heterogeneity, and funnel plot asymmetry in analyses analogous to additive, dominant, recessive, homozygous, and heterozygous genetic models with the odds ratio (OR) as the measure of association. A total of 11 case–control studies involving 2,073 PD cases and 2,131 controls were included. When all 11 studies were pooled into the analysis, the presence of the Leu allele at the Parkin p.Val389Leu polymorphism was associated with decreased risk for PD in three genetic comparison models: OR in additive model: 0.79, 95% confidence interval (CI) = 0.64–0.98, P = 0.029; OR in recessive model: 0.55, 95% CI = 0.35–0.89, P = 0.014; OR in homozygous model: 0.51, 95% CI = 0.32–0.82, P = 0.005. Begg's funnel plot and Egger's test provided visual and statistical evidences for funnel plot symmetry, without evidence presence of publication bias. We conclude that the presence of the Leu allele at the Parkin p.Val380Leu polymorphism is associated decreased risk for PD. © 2013 Wiley Periodicals, Inc.     

In Brief: Mitophagy: mechanisms and role in human disease

Mitophagy is a selective form of macro‐autophagy in which mitochondria are specifically targeted for autophagic degradation. Mitophagy plays an important role in cellular homeostasis by eliminating dysfunctional mitochondria and reducing mitochondrial mass as an adaptive response to stress. Cells execute mitophagy through several non‐redundant mechanisms, including the PINK1/Parkin partnership, which modulates turnover of depolarized mitochondria, and stress‐induced BNIP3, NIX, and FUNDC1 molecular adaptors, which interact directly with LC3 to promote mitophagy. These pathways are deregulated in human diseases, including cancer, neurodegeneration, metabolic disorders, muscle atrophy, ageing, and inflammation, reflecting the importance of mitophagy as a cellular housekeeping function. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

锰接触工人神经行为改变与Parkin基因7号外显子多态性的关系

[目的]探讨锰接触工人神经行为改变与Parkin基因7号外显子Gly284Arg多态性的相关性. [方法]选工龄在半年以上的职业性锰暴露工人200名和劳动强度相似,但工作环境不接触锰工人94名,按猛累积暴露指数将其分为高、低猛暴露组和对照组.利用沟槽稳定试验和拼板试验进行神经行为改变检测;利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法对Parkin基因7号外显子多态性位点进行基因分型. [结果]高暴露组中,Parkin基因7号外显子CC型工人的划痕长度、拼板试验得分均低于GG型和GC型,而碰撞次数高于GG型和GC型,其差别有统计学意义(P＜0.05);低暴露组中,Parkin基因7号外显子CC型工人的划痕长度、拼板试验得分均低于GG型和GC型,其差别有统计学意义(P＜0.05),3个基因型工人碰撞次数间差别无统计学意义(P＞0.05);对照组中,Parkin基因7号外显子3个基因型划痕长度、碰撞次数、拼板试验得分之间差异无统计学意义(P＞0.05). [结论]环境因素和遗传因素共同作用更易导致锰引起神经行为改变;携带Parkin基因7号外显子CC基因型的工人是锰损伤的易感者;Parkin基因7号外显子G951C突变导致Gly284Arg多态性有可能作为检测锰致神经行为改变以及锰中毒风险评估的指标.     

基于PINK1/Parkin通路研究槲皮素减轻血管性痴呆大鼠海马神经元损伤的作用机制

目的 基于PTEN诱导激酶1（PTEN-induced putative kinase 1,PINK1）/帕金蛋白（Parkin）通路研究槲皮素减轻血管性痴呆（vascular dementia,VD）大鼠海马神经元损伤的作用机制。方法 通过双侧颈总动脉永久性结扎法建立VD大鼠模型,分为模型组及槲皮素低、中、高（25、50、100 mg/kg）组和欧来宁（14.8 g/kg）组,另取12只大鼠作为假手术组,给予药物干预21 d后,采用穿梭箱实验评价大鼠学习记忆能力,比较各组大鼠步入潜伏期（step-through latency,STL）及在暗室中花费的总时间（total time spent in the dark chamber,TDC）;采用Nissl染色检测大鼠海马组织神经元损伤情况;采用透射电镜观察大鼠海马区神经元超微结构;采用ELISA法检测大鼠脑组织中乙酰胆碱（acetylcholine,Ach）、5-羟色胺（5-hydroxytryptamine,5-HT）水平和血清中白细胞介素-6（interleukin-6,IL-6）、IL-17、IL-1β水平;采用Western blotting检测大鼠脑组织中自噬相关蛋白与PINK1/Parkin信号通路相关蛋白表达。结果 与假手术组比较,模型组大鼠海马组织神经元损伤及超微结构受损严重,神经元内出现少量自噬体;STL显著降低（P＜0.05）,TDC显著升高（P＜0.05）;脑组织中Ach和5-HT水平显著降低（P＜0.05）,血清中IL-6、IL-17和IL-1β水平显著升高（P＜0.05）;脑组织中微管相关蛋白1轻链3 II（microtubule-associated protein 1 light 3 II,LC3II）/LC3I、Beclin1、PINK1和Parkin蛋白表达水平均显著升高（P＜0.05）。与模型组比较,槲皮素组大鼠海马组织神经元损伤及超微结构受损减轻,且神经元自噬现象增强;STL显著升高（P＜0.05）,TDC显著降低（P＜0.05）;脑组织中Ach及5-HT水平显著升高（P＜0.05）,血清中IL-6、IL-17和IL-1β水平均显著降低（P＜0.05）;脑组织中LC3II/LC3I、Beclin1、PINK1和Parkin蛋白表达水平均显著升高（P＜0.05）。结论 槲皮素可激活PINK1/Parkin信号通路,降低VD大鼠炎性细胞因子水平,减轻海马神经元炎症损伤,提高大鼠学习记忆能力,改善其痴呆症状。     

饥饿条件下活性氧通过PINK1/Parkin通路调控人牙周膜细胞的线粒体自噬

目的 探究人牙周膜细胞（hPDLC）在饥饿条件下活性氧（ROS）与PINK1/Parkin通路介导hPDLC线粒体自噬的具体机制。 方法 分离培养正常牙周组织的hPDLSC,利用Earle’s平衡盐溶液（EBSS）模拟饥饿环境诱导hPDLC线粒体自噬,利用N-乙酰-L-半胱氨酸（NAC）抑制ROS生成以探讨ROS在hPDLC线粒体自噬的作用,利用环孢素A（CsA）抑制PINK1/Parkin通路以研究ROS与PINK1/Parkin通路在饥饿条件下激活hPDLC中的作用。采用流式细胞术及JC-1线粒体膜电位检测试剂盒,检测线粒体膜电位;采用透射电镜观察线粒体自噬体的生成及线粒体形态变化;采用线粒体红色荧光探针定位线粒体,溶酶体绿色荧光探针定位溶酶体;采用DCFH-DA ROS荧光探针检测ROS生成强度;采用实时荧光定量聚合酶链反应（RT-qPCR）检测细胞中线粒体自噬基因（Tomm20、Timm23）及PINK1/Parkin通路的表达水平,采用蛋白质印迹 （Western blot）检测细胞中线粒体自噬蛋白（Tomm20、Timm23）及PINK1/Parkin通路蛋白表达水平。 结果 EBSS饥饿作用30 min后,诱导激活hPDLC线粒体自噬的作用最强,ROS表达增加,且线粒体自噬相关基因（Tomm20、Timm23）下调（P<0.001）,PINK1/Parkin通路表达上调（P<0.001）。NAC抑制ROS的产生后,自噬被抑制,同时Tomm20、Timm23表达上调（P<0.001,P<0.05）,PINK1/Parkin通路表达下调（P<0.001,P<0.05）。而当CsA抑制PINK1/Parkin通路表达时（P<0.05,P<0.05）,自噬被逆转,同时Tomm20、Timm23表达上调（P<0.001,P<0.01）。 结论 ROS在饥饿条件下主要通过PINK1/Parkin通路增强hPDLC线粒体自噬。     

早发性帕金森病患者中Parkin, LRRK2基因序列变异研究

目的观察散发性早发帕金森病(Parkinsons disease,PD)患者遗传易感基因突变的形式和分布,探讨易感基因突变在PD发病中的可能作用。方法病例组由13例散发性早发帕金森患者组成。以基因组DNA为模板,扩增Parkin基因的第1、4、6号外显子和LRRK2基因的第31号外显子。观察PCR产物测序后的突变情况。结果发现样本中存在突变,在正常人中存在单核苷酸多态性(single nucleotide polymorphism,SNP)。结论Parkin基因外显子的突变是我国散发性早发PD患者的致病原因之一。     

Premutations in the FMR1 gene as a modifying factor in Parkin‐associated Parkinson's disease?

Premutations in the FMR1 gene may be associated with some cases of parkinsonism. To test this hypothesis, we determined the CGG repeat number in FMR1 in 673 individuals with and without parkinsonism and detected 3 premutation carriers (2 patients, 1 control). Of note, 1 of the affected premutation carriers had a heterozygous Parkin mutation.     

帕金森病患者parkin基因多态性位点的研究

目的 观察汉族帕金森病(PD)患者中parkin基因多态性位点等位基因频率，探讨parkin基因多态性位点与PD发病的关系。方法 PD患者组及对照组各70例。以提取基因组DNA为模板，扩增parkin基因的外显子4、外显子10，然后行酶切与聚丙烯酰胺电泳，观察S／N167、R／W366、V/L380多态性位点等位基因频率。结果 PD患者组与对照组间三个多态性位点等位基因频率均无明显差异。结论 parkin基因S／N167、R／W366、V/L380多态性位点与汉族PD患者的发病无明显相关性。