Identifying and testing new targets for Parkinson Disease gene therapy

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不能再现在人类中看到的情况。虽然果蝇和酵母的模型是有用的，但它们没有控制PD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，我们相信这是传统小鼠转基因的一个有价值的辅助手段。利用这种方法，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性。在已经发表的研究中，我们展示了人类？-核蛋白(?大鼠黑质中的syn)会导致硝纹状体束的可重复性和进行性神经变性，多巴胺的丧失和PD典型的行为异常。这种方法的一个关键优点是，用于研究老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基因剂量可以操纵，基因表达是永久的。在本应用中，我们使用该技术来关注PD中的一个重要问题：它的功能是什么？-核蛋白吗?我们的初步数据显示了四件新奇的事情。首先，SNc中磷脂酶D2 （PLD2）的适度（2倍）过表达会导致急性和严重的神经病理。第二，那个？syn是一种PLD2病理抑制剂。第三，我们会看到相同类型的神经退化当我们击倒？syn与siRNA，我们看到PLD2过表达。第四，磷酸化？ser 129位点的syn在体内控制其抑制PLD2的能力，就像在体外一样。综上所述，这表明至少1 syn的一个重要功能是调节PLD2的活性。这已经被几个小组基于体外和细胞培养实验预测到，但之前没有在体内系统中显示出来。此外，我们的数据还首次显示？syn是一种必需的蛋白质，至少在SNc中是如此。这与之前在老鼠身上看到的情况背道而驰。并强调了这种替代遗传方法在研究帕金森病方面的潜在有用性。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PLD2和磷酸化1 syn的激酶。现在也有可能梳理出PLD2/?？Syn相互作用导致神经退行性变这将有希望确定额外的治疗靶点。该提案有三个具体目标：1)使用PLD2突变体来确定PLD2的哪个功能结构域负责PLD2毒性，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？3)验证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。

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.