Alpha-synuclein RNAi in a model of sporadic Parkinson's disease

摘要 帕金森病（PD）是退伍军人群体发病的重要原因。 电流 治疗不能预防PD进展导致的残疾。 了解 因此，研究常见的散发性PD的发病机制是一个迫切的目标， 可以制定适当的治疗干预措施。 多重证据表明 线粒体呼吸链功能受损和突触核蛋白异常 代谢可能是散发性PD的核心。 尽管体外实验的证据表明 突触核蛋白和突触子之间的双向相互作用，目前还不清楚这些 是帕金森病中多巴胺能神经元变性的基础。 在本建议中，我们将利用两个最近的 技术进步，以询问是否<$-突触核蛋白是多巴胺能神经递质的一个重要因素。 体内发生的神经元变性，响应于部分系统性线粒体损伤， 是偶发性帕金森病的类型 首先，我们开发了新型腺相关病毒（AAV）载体 表达靶向内源性大鼠突触核蛋白转录物的短发夹RNA（shRNA）， 优化了AAV载体向大鼠黑质的体内递送，允许调节 突触核蛋白在体内大鼠黑质-纹状体投射中的表达。 第二，我们开发了一种新的 散发性PD模型，其中部分系统性线粒体复合物I损伤，每天通过 腹膜内鱼藤酮递送，引起黑质的高度可再现的损伤，包括 形成类似路易体的突出的突触核蛋白免疫反应性包涵体， 多巴胺能神经元变性。 因为它可重复地概括了多个相关的 异常特征的散发性PD，腹膜内鱼藤酮模型将是唯一的 适用于确定突触核蛋白是否代表在发病机制中的关键介质， 这种疾病 在拟议的实验中，我们将检查行为，神经化学和 在成年动物中敲低<$-突触核蛋白的组织学效应（目的1）。 我们根据数据预测 在突触核蛋白基因敲除小鼠中，多巴胺能神经传递会有细微的减少。 然而，发育补偿/适应性变化，可能存在于生殖细胞空 突变体将缺席这些实验中的成年人;因此，有可能发现将 与小鼠基因敲除中报道的那些不同。 这些数据将构成解释 随后的实验，并将告知潜在的安全问题的可能性， 神经保护策略针对成年大脑中突触核蛋白表达的调节。 我们将 接下来要问的是，突触核蛋白的敲低是否能防止突触核蛋白内含物的形成， 慢性鱼藤酮暴露后神经行为缺陷和黑质细胞丢失（目的2）。 我们 假设黑质病变发病机制取决于 线粒体和<$-突触核蛋白，并因此减少内源性<$-突触核蛋白表达 随后的shRNA载体转导将改善病理学，导致显著的保护， 多巴胺能功能 这项实验将告知减少细胞毒性的可能功效。 突触核蛋白水平或靶向突触核蛋白的异常功能以预防PD进展。 总之，这些研究将确定两种主要的免疫抑制剂之间的体内相互作用的重要性。 PD在神经变性发病机制中的特征性异常，并将确定 突触核蛋白是否是一个有效的治疗靶点，用于在常见的散发性 帕金森病的症状

Abstract Parkinson's disease (PD) is an important cause of morbidity in the Veteran population. Current treatments do not prevent disability resulting from progression of PD. Understanding the pathogenesis of the common sporadic form of PD is therefore an urgent objective, in order that appropriate therapeutic interventions can be developed. Multiple convergent lines of evidence suggest that impairment of mitochondrial respiratory chain function and abnormalities of ¿-synuclein metabolism may be central to sporadic PD. Although evidence in vitro suggests the presence of bidirectional interactions between ¿-synuclein and the mitochondrion, it is unclear whether these underlie dopaminergic neuronal degeneration in PD. In this proposal, we will exploit two recent technical advances in order to ask whether ¿-synuclein is an important factor in the dopaminergic neuronal degeneration that occurs in vivo in response to partial systemic mitochondrial impairment, of the type found in sporadic PD. First, we have developed novel adeno-associated virus (AAV) vectors that express short hairpin RNA (shRNA) targeting the endogenous rat ¿-synuclein transcript, and have optimized in vivo delivery of AAV vectors to the rat substantia nigra, allowing modulation of ¿- synuclein expression in the rat nigro-striatal projection in vivo. Second, we have developed a novel model of sporadic PD, in which partial systemic mitochondrial complex I impairment, induced by daily intraperitoneal rotenone delivery, causes a highly reproducible lesion of the substantia nigra, including formation of prominent ¿-synuclein immunoreactive inclusions resembling Lewy bodies and degeneration of dopaminergic neurons. Since it reproducibly recapitulates multiple relevant abnormalities characteristic of sporadic PD, the intraperitoneal rotenone model will be uniquely suitable for determination of whether ¿-synuclein represents a critical mediator in the pathogenesis of the disease. In the proposed experiments, we will examine the behavioral, neurochemical and histological effects of ¿-synuclein knockdown in an adult animal (objective 1). We anticipate from data in ¿-synuclein knockout mice that there will be a subtle reduction in dopaminergic neurotransmission. However, developmental compensatory/adaptive changes that may be present in germline null mutants will be absent from these experiments in adults; consequently, it is possible that findings will be different to those reported in mouse knockouts. These data will form a basis for interpretation of subsequent experiments, and will inform on the likelihood of potential safety issues complicating neuroprotective strategies directed at modulation of ¿-synuclein expression in the adult brain. We will next ask whether ¿-synuclein knockdown is protective against the formation of ¿-synuclein inclusions, neurobehavioral deficits and nigral cell loss after chronic rotenone exposure (objective 2). We hypothesize that pathogenesis of the nigral lesion is dependent on a key interaction between mitochondria and ¿-synuclein, and consequently that reduction of endogenous ¿-synuclein expression following shRNA vector transduction will ameliorate pathology, resulting in significant preservation of dopaminergic function. This experiment will inform on the possible efficacy of reducing cellular ¿- synuclein levels or targeting abnormal functions of ¿-synuclein in preventing PD progression. Together, these studies will determine the importance of an interaction in vivo between two major characteristic abnormalities of PD in the pathogenesis of neurodegeneration, and will determine whether ¿-synuclein represents a valid therapeutic target for neuroprotection in the common sporadic form of Parkinson's disease.