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）遗传学中的关键问题之一是小鼠转基因模型通常不能重现在人类中观察到的情况。虽然果蝇和酵母模型是有用的，但它们不具有控制PD中所见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，包括胚胎致死和发育适应。在已经发表的工作中，我们展示了人类的表达？突触核蛋白（？syn）引起可再现的和进行性的硝基-纹状体束神经变性、多巴胺损失和PD典型的行为异常。这种方法的一个关键优势是，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。这种遗传转移系统的独特之处在于基因剂量可以被操纵，并且基因表达是永久性的。 在这个应用中，我们使用这种技术来关注PD中的一个重要问题：什么是功能？突触核蛋白？我们的初步数据证明了四个新的东西。首先，SNc中磷脂酶D2（PLD 2）的适度（2倍）过表达引起急性和严重的神经病理学。其次，这？syn是PLD 2病理学的抑制剂。第三，当我们击倒对手时，我们看到的是同一类型的神经退化？与我们在PLD 2的过表达中看到的一样。第四，磷酸化？在ser 129位置的syn控制其在体内抑制PLD 2的能力，就像它在体外一样。综上所述，这表明1 syn的至少一个重要功能是调节PLD 2的活性。基于体外和细胞培养实验，几个小组已经预期到了这一点，但以前在体内系统中没有显示出这一点。此外，我们的数据还首次显示，？syn是一种必需蛋白质，至少在SNc中是如此。这与以前在鼠标上看到的情况背道而驰？syn基因敲除，并强调了这种替代遗传方法对研究帕金森病的潜在有用性。 然而，这项工作最重要的方面是，我们已经潜在地确定了治疗PD的几个新的治疗靶点，包括PLD 2和磷酸化1 syn的激酶。现在也有可能梳理出PLD 2/？？突触相互作用导致神经退行性变，这将有望找到更多的治疗靶点。该提案有三个具体目标：1）使用PLD 2突变体来确定PLD 2功能结构域中的哪一个负责PLD 2毒性，其催化结构域或其蛋白质相互作用结构域，2）鉴定磷酸化的激酶？3）测试PLD 2功能影响D2多巴胺受体活性的可能性，D2多巴胺受体是关键的多巴胺细胞调节剂。 公共卫生相关性：我们已经发现，表达PLD 2在大鼠黑质也会导致帕金森样神经变性，和共表达？突触核蛋白抑制PLD 2毒性。这表明PLD 2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鉴定PLD 2毒性的下游介质和体内磷酸化S129的激酶的实验，这将有望为帕金森病基因治疗提供新的靶点，我们将在大鼠模型中进行测试。