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（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无法重现人类的情况。尽管果蝇和酵母模型很有用，但它们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）作为大脑的基因转移载体，我们相信这是传统小鼠转基因的一个有价值的辅助手段。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。在已经发表的作品中，我们展示了人类的表达？大鼠黑质中的突触核蛋白 (? syn) 会导致黑质纹状体束的可重复性进行性神经变性、多巴胺丧失以及 PD 典型的行为异常。这种方法的主要优点之一是，用于研究大鼠大脑遗传问题的相同结构也可以用于灵长类动物。这种基因转移系统的独特之处在于可以操纵基因剂量并且基因表达是永久性的。 在这个应用中，我们利用这个技术来关注PD中的一个重要问题： 的作用是什么？突触核蛋白？我们的初步数据展示了四件新颖的事情。首先，SNc 中磷脂酶 D2 (PLD2) 的适度（2 倍）过度表达会导致急性和严重的神经病理学。第二，那个？ 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？正如我们在 PLD2 过表达中看到的那样，与 siRNA 一致。第四，磷酸化？ Ser 129 位点的 syn 控制其在体内抑制 PLD2 的能力，就像在体外一样。综上所述，这表明 1 syn 的至少一项重要功能是调节 PLD2 的活性。几个研究小组根据体外和细胞培养实验预测到了这一点，但之前并未在体内系统中得到证实。此外，我们的数据还首次表明？ syn 是一种必需蛋白，至少在 SNc 中是这样。这与之前用鼠标看到的情况背道而驰？ syn 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途。 然而，这项工作最重要的方面是我们可能确定了治疗 PD 的几个新治疗靶点，包括 PLD2 和磷酸化 1 syn 的激酶。现在还可以梳理出 PLD2/?? 的机制。 syn 相互作用会导致神经退行性变，这有望确定其他治疗靶点。该提案具有三个具体目标：1) 使用 PLD2 突变体来确定哪个 PLD2 功能域负责 PLD2 毒性、其催化结构域或其蛋白质相互作用结构域，2) 识别磷酸化的激酶？体内同步，3）测试PLD2功能影响D2多巴胺受体活性的可能性，D2多巴胺受体活性是关键的多巴胺细胞调节剂。 公共健康相关性：我们发现大鼠黑质中 PLD2 的表达也会导致帕金森样神经变性，并且 ? 的共表达突触核蛋白可抑制 PLD2 毒性。这表明 PLD2 的失调可能是帕金森病的最初原因。我们的提案描述了旨在识别 PLD2 毒性的下游介质和体内磷酸化 S129 的激酶的实验，这有望为帕金森病基因治疗提供新的靶点，我们将在大鼠模型中进行测试。