Drp1的调控对PINK1突变转基因果蝇的保护作用

帕金森病（PD）是以黑质致密部多巴胺神经元选择性减少和胞浆内路易小体的形成为特征的神经退行性疾病。研究发现,PTEN诱导激酶1(PINK1)基因突变导致家族性早发型帕金森病的发生。在转基因果蝇中,PINK1功能丢失导致间接飞行肌缺陷,线粒体结构、功能障碍,多巴胺神经元丢失。本研究在PINK1突变PD转基因果蝇中,进行发动蛋白相关蛋白1（Drp1）过表达和敲低,探索Drp1对PD转基因果蝇的保护作用及其可能机制。本研究选用MHC-Gal4/UAS系统的PD转基因果蝇模型,特异性启动PINK1B9基因于果蝇肌肉组织中表达;运用Drp1基因过表达和RNA干扰干预PINK1B9转基因果蝇,研究其对PD转基因果蝇的作用。结果显示,不论过表达Drp1还是Drp1敲低均可挽救PINK1突变转基因果蝇,降低翅膀异常率,改善飞行能力,恢复间接飞行肌排列,调节线粒体形态,提高ATP生成量,上调NDUFS3蛋白表达水平。本文结果提示,Drp1的调控挽救PINK1突变转基因果蝇与线粒体呼吸链有关。     

过表达 PINK1改善SCA3/MJD转基因果蝇模型的线粒体功能

脊髓小脑性共济失调3型（SCA3/MJD）,是一种因致病基因MJD1编码区内CAG异常重复扩增所致的常染色体显性遗传迟发性神经退行性疾病. 已知PINK1蛋白可通过抗氧化稳定线粒体,阻止帕金森疾病的发生,但其在SCA3/MJD中的作用尚不清楚. 本文旨在探索过表达PINK1对SCA3/MJD转基因果蝇模型的保护作用.本研究利用Mhc-Gal4启动子表达致病蛋白质片段（MJDtr-Q78）获得SCA3/MJD果蝇模型,分别运用过表达PINK1和RNA干扰PINK1研究其在SCA3/MJD果蝇模型中的功能.结果显示,疾病模型组翅膀异常率增高,线粒体呈过度融合状态,ATP值降低;PINK1 RNA干扰组翅膀异常率明显增高,线粒体呈显著过度融合状态,ATP值明显降低;PINK1过表达组翅膀异常率明显降低,线粒体清晰、完整,ATP值明显升高.本文的结果提示, 过表达PINK1对SCA3/MJD转基因果蝇模型起保护作用,而RNA干扰PINK1表达加重SCA3/MJD转基因果蝇模型病情.PINK1在SCA3/MJD果蝇模型中的功能可能通过改善细胞内线粒体功能实现.     

Parkin、PINK1、DJ-1和线粒体功能障碍与帕金森病

帕金森病(Parkinson’s disese,PD)是一种常见的神经退行性疾病,但到目前为止发病机制尚不明确,环境和遗传等因素与其发病有密切关系。研究表明,蛋白质异常积聚(泛素/蛋白酶体途径)和线粒体氧化损伤(线粒体途径),可能是导致PD患者发病的关键分子机制。Parkin、PINK1和DJ-1等基因突变与常染色体隐性的家族性PD有关,这些相关基因编码的蛋白对于维持线粒体形态和功能起着重要的作用。本文将主要从Parkin、PINK1、DJ-1和线粒体功能障碍与帕金森病的关系进行综述。     

以果蝇模型研究人类神经退行性疾病

人类神经退行性疾病是一类以神经元退行性病变导致个体行为异常乃至死亡为主要特征的疾病.果蝇以其独特的分子遗传学优势成为研究人类神经退行性疾病的理想模型.通过对果蝇模型的研究不仅可以揭示神经退行性疾病发生的细胞分子通路,还可以研究对疾病有调控作用的基因及其表达产物,寻找可能的药物作用靶点并进行药物筛选,为防治人类神经退行性疾病提供新的方法和有效的药物.     

DJ-1保护大鼠中脑黑质多巴胺能神经元抵抗鱼藤酮损伤的研究

目的:探讨过表达DJ-1蛋白能否保护大鼠中脑黑质多巴胺能神经元抵抗鱼藤酮所致的急性损伤.方法:构建表达DJ-1基因的腺相关病毒载体(rAAV-DJ-1)并转染HEK-293细胞,进行腺相关假病毒颗粒包装,所得假病毒颗粒注射到大鼠脑内感染黑质神经细胞;4周后,注射鱼藤酮于大鼠黑质相同脑区;通过免疫组织化学和免疫印迹试验鉴定TH蛋白表达情况,采用动物行为轨迹分析软件计算大鼠30 min内运动距离以及强迫游泳试验鉴定大鼠精神症状.结果:与对照组相比,过表达D J-1组大鼠运动损伤症状较轻,精神症状改善明显;损伤侧黑质TH阳性神经元数目和TH蛋白表达水平显著高于对照组.结论:过表达DJ-1蛋白能保护大鼠黑质多巴胺能神经元抵抗鱼藤酮所致的急性损伤,延缓多巴胺能神经元的退行性变,提示DJ-1可能对帕金森病的治疗有较好的应用前景.     

DJ-1基因与帕金森病

帕金森病是仅次于阿尔茨海默病的第二大神经退行性疾病。DJ-1基因的突变可以引起常染色体隐性遗传性帕金森病。该文将从DJ-1基因及DJ-1蛋白的结构,组织分布,及其在帕金森病发病机制中的功能等方面进行阐述,为研制开发帕金森病的治疗药物提供新的思路。     

帕金森病相关蛋白Parkin 与PINK1的相互作用研究

帕金森病(Parkinson's disease,PD)是常见的神经系统变性疾病．分子遗传学研究发现,突变的Parkin蛋白及PINK1蛋白均参与了帕金森病的致病过程,但二者之间是否存在相互作用以及是否能够相互调节仍不十分清楚．为明确生理状态下Parkin蛋白与PINK1蛋白之间的相互作用,首先运用蛋白体外结合实验(GST pull-down)技术及免疫共沉淀技术证实了Parkin与PINK1在体外及体内均可相互结合．进一步构建PINK1的不同截短型,运用GST pull-down技术验证了PINK1与Parkin相互结合的区段为PINK1的蛋白激酶结构域．免疫细胞化学实验也证实Parkin与PINK1蛋白在细胞中存在共定位．进一步运用免疫共沉淀技术证实Parkin可减少PINK1通过泛素蛋白酶体系统(ubiquitin proteasome system,UPS)的降解,从而稳定PINK1．PINK1可增加Parkin通过UPS的降解,从而减少Parkin的水平,降低其稳定性．这些结果提示,帕金森病相关蛋白Parkin与PINK1能够直接结合,二者通过泛素蛋白酶体降解系统相互调节,可能协同作用参与了帕金森病的致病过程．     

慢病毒介导的小鼠PINK1基因RNAi载体的构建及在NIH3T3细胞中的筛选

目的:构建筛选靶向特异PINK1-siRNA慢病毒表达载体,感染小鼠胚胎细胞(NIH3T3)验证该病毒载体的敲减效率,为研究帕金森病的发病机制奠定基础。方法:构建2对靶向小鼠PINK1-siRNA序列(KD1和KD2),将这2对序列连接在GV118上,将重组载体和病毒包装的辅助质粒共转染293 T细胞,获得慢病毒颗粒,再将该病毒颗粒转染入NIH3T3细胞,用qRT-PCR验证细胞内PINK1 mRNA表达水平以验证敲减效果。结果:筛选出可以用于后续实验的高效靶向KD2序列,并成功转染到小鼠NIH3T3细胞中,在感染复数(MOI)为50时,KD2的沉默效果最好,敲减率达到66.4%。结论:成功构建了高效靶向小鼠PINK1-siRNA慢病毒载体,其可稳定转染小鼠NIH3T3细胞,可高效抑制PINK1 mRNA的表达。为下一步利用其感染神经细胞或注入动物脑内,进一步研究PINK1基因在帕金森病发病过程中的作用环节和机制提供了分子生物学的技术基础。     

突触囊泡循环障碍与帕金森病

帕金森病是人类第二大神经退行性疾病,虽然通过对帕金森病遗传家族的研究已发现不少与之相关的致病基因,然而其病因尚不清楚。在已发现的与帕金森病相关基因中,α-synuclein、LRRK2、Parkin、PINK1以及DJ-1也在调节神经元突触前囊泡的神经递质释放以及突触前囊泡循环过程中发挥重要作用。最近的一些研究表明突触前囊泡循环障碍在帕金森病发病过程中扮演重要角色。本文对上述基因在突触囊泡循环以及其突变导致帕金森病病程中的作用作一概述,并推测囊泡循环障碍在帕金森病发病过程中的作用机理,最后指出目前在该研究领域需要解决的一些问题。     

盘基网柄菌DJ-1蛋白的亚细胞定位研究

DJ-1基因的突变或缺失导致帕金森病相关症状,但其在帕金森病中的作用以及其亚细胞位置尚存在争议。盘基网柄菌是研究神经退化性疾病的模式生物,通过绿色荧光蛋白(GFP)标记DJ-1蛋白,利用荧光蛋白技术及DJ-1与GFP共定位技术在正常和氧化情况下研究其在盘基网柄菌的亚细胞定位,可以为探索DJ-1蛋白亚细胞位置与致病机制之间的联系奠定基础。研究结果表明,正常情况下盘基网柄菌DJ-1蛋白位于细胞质内,一旦受到氧化应激, DJ-1蛋白则转移至线粒体,这个亚细胞位置转移与DJ-1蛋白C117位点的氧化相关。该研究为探索DJ-1蛋白如何在氧化应激条件下完成对细胞的保护提供了实验依据。     

Dopamine modulates metabolic rate and temperature sensitivity in Drosophila melanogaster

Homeothermal animals, such as mammals, maintain their body temperature by heat generation and heat dissipation, while poikilothermal animals, such as insects, accomplish it by relocating to an environment of their favored temperature. Catecholamines are known to regulate thermogenesis and metabolic rate in mammals, but their roles in other animals are poorly understood. The fruit fly, Drosophila melanogaster, has been used as a model system for the genetic studies of temperature preference behavior. Here, we demonstrate that metabolic rate and temperature sensitivity of some temperature sensitive behaviors are regulated by dopamine in Drosophila. Temperature-sensitive molecules like dTrpA1 and shi(ts) induce temperature-dependent behavioral changes, and the temperature at which the changes are induced were lowered in the dopamine transporter-defective mutant, fumin. The mutant also displays a preference for lower temperatures. This thermophobic phenotype was rescued by the genetic recovery of the dopamine transporter in dopamine neurons. Flies fed with a dopamine biosynthesis inhibitor (3-iodo-L-tyrosine), which diminishes dopamine signaling, exhibited preference for a higher temperature. Furthermore, we found that the metabolic rate is up-regulated in the fumin mutant. Taken together, dopamine has functions in the temperature sensitivity of behavioral changes and metabolic rate regulation in Drosophila, as well as its previously reported functions in arousal/sleep regulation.     

Drosophila melanogaster Parkin ubiquitinates peanut and septin1 as an E3 ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR-JP), a common familial form of Parkinson's disease, is caused by mutations of human Parkin. To deepen the understanding of Parkin biology in an in vivo model of Drosophila, we attempted to characterize the function of Drosophila melanogaster Parkin and found that D. melanogaster Parkin exhibited UbcH8-dependent E3 ubiquitin-protein ligase activity. Using E2 binding and in vitro ubiquitination assays, UbcH8 preferentially was found to bind to Parkin mutants harboring functional RING1 domains, but failed to bind to mutants harboring point mutants with complete loss of function. This inability of UbcH8 binding to such mutants was accompanied by abrogation of an E3 ligase activity, indicating that D. melanogaster Parkin as an E3 ligase interacts with UbcH8 through its RING1 domain. An in vivo ubiquitination assay revealed that D. melanogaster Parkin existed in ubiquitinated form in vivo. Moreover, peanut and septin1, D. melanogaster septin proteins, were also ubiquitinated by D. melanogaster Parkin. Co-immunoprecipitation with membrane protein Syntaxin indicated direct binding of septin proteins to syntaxin, implicating their relevance in the exocytosis of dopamine in cells. Western blot analysis and DNA fragmentation indicated that the rate and efficiency of p53-dependent apoptosis were significantly higher in the presence of dopamine than without the septin proteins. Therefore, our findings in the present study demonstrate that Parkin possibly influences septin protein effects on p53-mediated apoptosis, helping to extend the utility of Drosophila as a model system for the study of neurodegeneration.     

Suppression of Nrdp1 toxicity by Parkin in Drosophila models

Nrdp1 is a RING finger ubiquitin E3 ligase that interacts with Parkin, and promotes the degradation of Parkin, a causative protein for early onset Autosomal Recessive Juvenile Parkinsonism (AR-JP). To investigate if Nrdp1 plays a role in the pathogenesis of Parkinson's disease, we generated transgenic Drosophila that expressed Drosophila Nrdp1 (dNrdp1) and dNrdp1(D56V), an aspartic acid to valine mutant at residue 56 that disrupts its ring finger domain, resulting in impaired capacity to degrade its substrate ErbB3. Our data show that a pan-neuronal expression of transgenic dNrdp1 but not dNrdp1(D56V) mutant leads to the loss of dopaminergic neurons in brains, resulting in reduction of dopamine production. These flies also manifested decreased flight ability. Co-expression of human Parkin (hParkin) provides protection against toxicity induced by over-expression of dNrdp1, reversing the effects of dNrdp1 on death of dopaminergic neurons, reduction of dopamine production, and decreased flight ability. Taken together, we conclude that Nrdp1 plays a role in neurodegeneration and could be potentially targeted as a therapeutic strategy for Parkinson's disease.     

Experimental models of Parkinson's disease

Research into the pathogenesis of Parkinson's disease has been rapidly advanced by the development of animal models. Initial models were developed by using toxins that specifically targeted dopamine neurons, the most successful of which used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a toxin that causes parkinsonism in man. More recently, the identification of alpha-synuclein mutations as a rare cause of Parkinson's disease has led to the development of alpha-synuclein transgenic mice and Drosophila. Here, I discuss the merits and limitations of these different animal models in our attempts to understand the physiology of Parkinson's disease and to develop new therapies.     

Molecular and pharmacological characterization of two D(1)-like dopamine receptors in the Lyme disease vector, Ixodes scapularis

Advancements in tick neurobiology may impact the development of acaricides to control those species that transmit human and animal diseases. Here, we report the first cloning and pharmacological characterization of two neurotransmitter binding G protein-coupled receptors in the Lyme disease (blacklegged) tick, Ixodes scapularis. The genes IscaGPRdop1 and IscaGPRdop2 were identified in the I. scapularis genome assembly and predicted as orthologs of previously characterized D₁-like dopamine receptors in the fruit fly Drosophila melanogaster and honeybee Apis mellifera. Heterologous expression in HEK 293 cells demonstrated that each receptor functioned as a D₁-like dopamine receptor because significant increases in levels of intracellular cyclic adenosine monophosphate (cAMP) were detected following dopamine treatment. Importantly, the receptors were distinct in their pharmacological properties regarding concentration-dependent response to dopamine, constitutive activity, and response to other biogenic amines. Exposure to a variety of dopamine receptor agonists and antagonists further demonstrated a D₁-like pharmacology of these dopamine receptors and highlighted their differential activities in vitro.     

Minocycline,levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease

Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes.     

Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila

BACKGROUND: Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. RESULTS: We present evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. We used a startle-induced negative geotaxis assay and a locomotor tracking system to measure the effect of psychostimulants on fly behavior. Using these assays, we show that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, we found that dopamine is involved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. CONCLUSIONS: We show that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. We therefore suggest that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse.     

Paraquat-induced Oxidative Stress in Drosophila melanogaster: Effects of Melatonin, Glutathione, Serotonin, Minocycline, Lipoic Acid and Ascorbic Acid

The efficacy of melatonin, glutathione, serotonin, minocycline, lipoic acid and ascorbic acid in counteracting the toxicity of paraquat in Drosophila melanogaster was examined. Male Oregon wild strain flies were fed for 5 days with control food or food containing the test substance. They were transferred in groups of five to vials containing only filter paper soaked with 20 mM paraquat in 5% sucrose solution. Survival was determined 24 and 48 h later. All the substances assayed increased the survival of D. melanogaster. At equimolar concentrations (0.43 mM) melatonin was more effective than serotonin, lipoic acid and ascorbic acid. However, lower concentrations of glutathione (0.22 mM) and minocycline (0.05 mM) were as efficient as melatonin. The highest survival rate (38.6%) after 48 h of paraquat treatment was found with 2.15 mM of lipoic acid. No synergistic effect of melatonin with glutathione, serotonin, minocycline, lipoic acid and ascorbic acid was detected.