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: 8311777
• 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/8311777
• 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（由申请人提供）：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没有概括过人类所见。尽管果蝇和酵母模型很有用，但它们没有控制PD中症状的斑纹纹状体区的独特结构。我们的小组率先使用重组腺相关病毒（AAV）作为大脑的基因转移载体，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，包括胚胎致死性和发育适应。在已经出版的工作中，我们展示了人类的表达？大鼠黑质中的突触核蛋白（？syn）导致nitgrolstriatal束，多巴胺的丧失以及PD典型的行为异常的可重复性和进行性神经变性。这种方法的关键优势之一是，用于研究大鼠大脑中的遗传问题的相同结构也可以在灵长类动物中使用。这种遗传转移系统是独一无二的，因为可以操纵基因剂量，并且基因表达是永久性的。 在此应用程序中，我们使用该技术专注于PD中的一个重要问题：什么是功能？突触核素？我们的初步数据展示了四个新事物。首先，SNC中磷脂酶D2（PLD2）的适度（2倍）过表达会引起急性和严重的神经病理学。第二，那呢？ SYN是PLD2病理学的抑制剂。第三，当我们击倒时，我们会看到相同类型的神经变性？正如我们看到的，与PLD2的过表达一样。第四，那磷酸化？ SER 129位置的SYN可以控制其在体内抑制PLD2的能力，就像体外一样。综上所述，这表明1 SYN的至少一个重要功能是调节PLD2的活性。基于体外和细胞培养实验的几个组已经预期了这一点，但先前尚未在体内系统中显示。此外，我们的数据还首次显示？ SYN是必不可少的蛋白质，至少在SNC中。这与鼠标以前看到的东西相反吗？ SYN敲除并突出了这种替代遗传方法研究帕金森氏病的潜在有用性。 但是，这项工作的最重要方面是，我们有潜在地鉴定出几个用于治疗PD的新治疗靶标，包括PLD2和磷酸化1 Syn的激酶。现在也可以逗弄PLD2/??的机制。导致神经变性的SYN相互作用，这将有望确定其他治疗靶标。该提案具有三个特定的目的：1）使用PLD2突变体确定PLD2功能域的哪个负责PLD2 TOXCITY，其催化结构域或其蛋白质相互作用结构域，2）确定磷酸化的激酶？ SYN在体内，3）测试PLD2功能影响D2多巴胺受体活性的可能性，这是一种关键的多巴胺细胞调节剂。 公共卫生相关性：我们发现，尼格拉（Nigra）大鼠底层中PLD2的表达也会引起帕金森（Parkinson），例如神经变性，而这种共表达？突触核蛋白抑制PLD2毒性。这表明PLD2的正调可能是帕金森病的最初原因。我们的建议描述了旨在识别PLD2毒性的下游介质和体内磷酸化S129的激酶的实验，这将有望为帕金森氏病基因治疗提供新的靶标，我们将在大鼠模型中测试。