Identifying and testing new targets for Parkinson Disease gene therapy

识别和测试帕金森病基因治疗的新靶点

• 批准号: 8151115
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 31.41万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8151115
• 关键词: Acute; Affect; Architecture; Behavioral; Biochemical; Biological Assay; Brain; Bypass; Catalytic Domain; Cell Culture Techniques; Cell physiology; Cells; Cleaved cell; Contralateral; Data; Development; Dopamine; Dopamine D2 Receptor; Drosophila genus; Embryo; Event; Gene Dosage; Gene Expression; Gene Transfer; Genes; Genetic; Goals; Golgi Apparatus; Human; In Vitro; Intervention; Knock-out; Lecithin; Mediator of activation protein; Membrane; Membrane Fusion; Methods; Modeling; Mus; Mutation; Nerve Degeneration; Oxidative Stress; Parkinson Disease; Pathology; Pathway interactions; Phenotype; Phosphatidic Acid; Phosphorylation; Phosphotransferases; Physiological; Positioning Attribute; Primates; Proteins; Publishing; Rattus; Recombinant adeno-associated virus (rAAV); Regulation; Role; Rotation; Running; Signal Transduction; Signaling Molecule; Small Interfering RNA; Substantia nigra structure; Symptoms; System; Techniques; Technology; Testing; Time; Toxic effect; Transgenic Mice; Transgenic Model; Vesicle; Work; Yeast Model System; alpha synuclein; base; casein kinase I; design; gene therapy; in vivo; inhibitor/antagonist; knock-down; mutant; neuropathology; neurotoxicity; new therapeutic target; novel; overexpression; pars compacta; phospholipase D2; progressive neurodegeneration; public health relevance; relating to nervous system; research study; synuclein; therapeutic target; trafficking

DESCRIPTION (provided by applicant): One of the key problems in Parkinson Disease (PD) genetics is that mouse transgenic models have often not recapitulated what is seen in humans. Although Drosophila and yeast models have been useful, they do not have the unique architecture of the nigrostriatal tract that control the symptoms seen in PD. Our group has pioneered the use of recombinant Adeno-associated virus (AAV) as a gene transfer vehicle to the brain, which we believe is a valuable adjunct to conventional mouse transgenics. Using this method we can bypass many of the problems encountered in mouse transgenic systems, including embryonic lethality and developmental adaptation. In work already published, we showed that expression of human ? synuclein (? syn) in the rat substantia nigra causes reproducible and progressive neurodegeneration of the nitgrolstriatal tract, loss of dopamine, and behavioral abnormalities that are typical to PD. One of the key advantages of this approach is that the same construct used to study a genetic question in rat brains can be used in primates as well. This genetic transfer system is unique in that gene dosage can be manipulated and gene expression is permanent. In this application, we use this technology to focus on an important question in PD: What is the function of ? synuclein? Our preliminary data demonstrate four novel things. First, that modest (2 fold) overexpression of phospholipase D2 (PLD2) in the SNc causes acute and severe neuropathology. Second, that ? syn is an inhibitor of PLD2 pathology. Third, that we see the same type of neurodegeneration when we knock down ? syn with siRNA as we see with overexpression of PLD2. And fourth, that phosphorylation of ? syn at the ser 129 position controls its ability to inhibit PLD2 in vivo just as it does in vitro. Taken together, this demonstrates that at least one important function of 1 syn is to modulate the activity of PLD2. This had been anticipated by several groups based on in vitro and cell culture experiments, but had not been shown previously in an in vivo system. Moreover, our data also shows for the first time that ? syn is an essential protein, at least in the SNc. This runs counter to what had previously been seen with mouse ? syn knockouts and highlights the potential usefulness of this alternative genetic approach to studying Parkinson Disease. However, the most important aspect of this work is that we have potentially identified several new therapeutic targets for treating PD, including PLD2 and the kinase(s) that phosphorylate 1 syn. It is also now possible to tease out the mechanism of the PLD2/?? syn interaction that leads to neurodegeneration and this will hopefully identify additional therapeutic targets. The proposal has three specific aims: 1) To use PLD2 mutants to determine which of the PLD2 functional domains is responsible for PLD2 toxcity, its catalytic domain or its protein interaction domains, 2) To identify the kinase that phosphorylates ? syn in vivo, 3) To test the possibility that PLD2 function affects D2 dopamine receptor activity, which is a key dopamine cell regulator. PUBLIC HEALTH RELEVANCE: We have discovered that expression of PLD2 in the rat substantia nigra also causes a Parkinson like neurodegeneration, and that coexpression of ? synuclein suppresses PLD2 toxicity. This suggests that misregulation of PLD2 may be the initial cause of Parkinson Disease. Our proposal describes experiments designed to identify the downstream mediators of PLD2 toxicity and the kinase that phosphorylates S129 in vivo, and this will hopefully provide new targets for Parkinson Disease gene therapy which we will test in the rat model.

描述(申请人提供)：帕金森病(PD)遗传学的关键问题之一是小鼠转基因模型通常不能概括人类的情况。尽管果蝇和酵母模型很有用，但它们并不具有控制帕金森病症状的黑质纹状体束的独特结构。我们的团队率先使用重组腺相关病毒(AAV)作为基因转移到大脑的载体，我们认为这是传统小鼠转基因的有价值的辅助手段。使用这种方法，我们可以绕过小鼠转基因系统中遇到的许多问题，包括胚胎致死和发育适应。在已经发表作品中，我们展示了人类？突触核蛋白(？)在大鼠的黑质中，黑质可引起黑质纹状体束可复制的进行性神经退行性变、多巴胺的丢失以及帕金森病的典型行为异常。这种方法的主要优点之一是，用于研究老鼠大脑中的遗传问题的相同结构也可以用于灵长类动物。这种遗传转移系统的独特之处在于，基因剂量可以控制，基因表达是永久的。在这个应用中，我们使用这项技术来关注PD中的一个重要问题：什么是功能？突触核蛋白？我们的初步数据展示了四件新奇的事情。首先，在SNC中，磷脂酶D2(PLD2)适度(2倍)的过度表达会导致急性和严重的神经病理。第二，那个？SYN是PLD2的病理抑制因子。第三，当我们撞车时，我们会看到相同类型的神经退行性变吗？Syn与siRNA结合，我们可以看到PLD2的过表达。第四，磷酸化？位于Ser129位置的SYN在体内控制其抑制PLD2的能力，就像它在体外所做的那样。综上所述，这表明1syn的至少一个重要功能是调节PLD2的活性。根据体外和细胞培养实验，几个小组已经预料到了这一点，但之前在体内系统中没有显示出这一点。而且，我们的数据也是第一次显示？Syn是一种必需的蛋白质，至少在SNC中是如此。这与之前看到的鼠标背道而驰？SYN基因敲除，并强调了这种替代遗传方法在研究帕金森病方面的潜在用处。然而，这项工作最重要的方面是，我们可能已经确定了几个治疗帕金森病的新靶点，包括PLD2和使1syn磷酸化的激酶(S)。现在还可以梳理出PLD2/？？的机制。SYN相互作用导致神经变性，这将有望确定更多的治疗靶点。该提案有三个具体目标：1)使用PLD2突变体来确定PLD2的哪个功能结构域是导致PLD2毒性的，它的催化结构域还是它的蛋白质相互作用结构域，2)确定磷酸化的激酶？3)测试PLD2功能影响D2多巴胺受体活性的可能性，D2多巴胺受体是多巴胺细胞的关键调节因子。 公共卫生相关性：我们发现PLD2在大鼠黑质的表达也会导致帕金森样神经变性，并且？突触核蛋白抑制PLD2毒性。这表明PLD2的错误调控可能是帕金森病的最初原因。我们的建议描述了旨在确定PLD2毒性的下游介体和体内磷酸化S129的激酶的实验，这将有望为帕金森病的基因治疗提供新的靶点，我们将在大鼠模型中进行测试。