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

描述(由申请人提供)： 帕金森氏病(PD)是导致退伍军人发病的重要原因。目前的治疗方法并不能防止帕金森病进展导致的残疾。因此，了解常见的散发性帕金森病的发病机制是一个紧迫的目标，以便开发适当的治疗干预措施。多条趋同的证据表明，线粒体呼吸链功能受损和1-突触核蛋白代谢异常可能是散发性帕金森病的中心。尽管体外证据表明1-突触核蛋白和线粒体之间存在双向相互作用，但尚不清楚这些相互作用是否导致帕金森病患者的多巴胺能神经元变性。在这个方案中，我们将利用两个最新的技术进展来询问1-突触核蛋白是否是体内发生的多巴胺能神经元变性的一个重要因素，这种变性是由于部分系统性线粒体损伤，在散发性帕金森病中发现的类型。首先，我们开发了新型的腺相关病毒载体，可以表达针对内源性大鼠1-突触核蛋白转录本的短发夹状RNA(ShRNA)，并优化了AAV载体在体内向大鼠黑质的递送，允许在体内调节大鼠黑质-纹状体投射中1-突触核蛋白的表达。第二，我们开发了一种新的散发性帕金森病模型，在该模型中，每天经腹注射鱼藤酮导致部分系统性线粒体复合体I损伤，导致黑质高度可复制的损害，包括形成类似路易体的突出的1-突触核蛋白免疫反应包涵体和多巴胺能神经元的变性。由于它可重现散发性帕金森病的多种相关异常特征，因此，腹膜内鱼藤酮模型将是唯一适合于确定1-突触核蛋白是否代表疾病发病机制中的关键介质的模型。在拟议的实验中，我们将研究1-突触核蛋白敲除对成年动物的行为、神经化学和组织学影响(目标1)。我们从1-突触核蛋白基因敲除小鼠的数据中预计，多巴胺能神经传递将出现微妙的减少。然而，在成人的这些实验中，可能出现在种系零突变中的发育补偿/适应性变化将不存在；因此，结果可能与在小鼠基因敲除中报道的结果不同。这些数据将成为解释后续实验的基础，并将告知潜在的安全问题的可能性，这些潜在的安全问题将使针对成人大脑中1-突触核蛋白表达的神经保护策略复杂化。接下来，我们将询问1-突触核蛋白敲除是否对慢性鱼藤酮暴露后1-突触核蛋白包涵体的形成、神经行为缺陷和黑质细胞丢失具有保护作用(目标2)。我们假设黑质病变的发病机制依赖于线粒体和1-突触核蛋白之间的关键相互作用，因此，shRNA载体转导后内源性1-突触核蛋白表达的减少将改善病理，导致显著保留多巴胺能功能。本实验将揭示降低细胞内1-突触核蛋白水平或靶向1-突触核蛋白功能异常在预防PD进展中的可能效果。总之，这些研究将确定帕金森病两个主要特征异常在神经变性发病机制中体内相互作用的重要性，并将确定1-突触核蛋白是否代表常见散发性帕金森病神经保护的有效治疗靶点。 公共卫生相关性： 帕金森氏病(PD)在老年退伍军人中非常普遍。这种疾病对退伍军人的后果是严重的。进行性帕金森病会导致运动、记忆、情绪和睡眠问题、膀胱和肠道症状以及跌倒，导致残疾，并对与身心健康相关的生活质量指标产生强烈的负面影响。我们想了解帕金森氏症的病因，以便我们能够设计出预防退伍军人帕金森氏症相关残疾的治疗方法。一些证据表明，关闭一种名为“1-突触核蛋白”的关键大脑蛋白质可能会阻止帕金森氏症的疾病进展。在目前的研究中，我们将通过使用基因工程病毒来确定关闭1-突触核蛋白是否可以预防一种新的大鼠模型中的帕金森氏病样症状。如果这项概念验证研究是积极的，那么可以在未来的研究中设计和开发针对1-突触核蛋白的药物，以防止帕金森病的进展。

DESCRIPTION (provided by applicant): 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 1-synuclein metabolism may be central to sporadic PD. Although evidence in vitro suggests the presence of bidirectional interactions between 1-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 1-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 1-synuclein transcript, and have optimized in vivo delivery of AAV vectors to the rat substantia nigra, allowing modulation of 1- 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 1-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 1-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 1-synuclein knockdown in an adult animal (objective 1). We anticipate from data in 1-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 1-synuclein expression in the adult brain. We will next ask whether 1-synuclein knockdown is protective against the formation of 1-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 1-synuclein, and consequently that reduction of endogenous 1-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 1- synuclein levels or targeting abnormal functions of 1-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 1-synuclein represents a valid therapeutic target for neuroprotection in the common sporadic form of Parkinson's disease. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is highly prevalent amongst the aging veteran population. The consequences of the disease for veterans are serious. Progressive PD causes movement, memory, mood and sleep problems, bladder and bowel symptoms, and falls, resulting in disability and strongly negative impacts on physical and mental health-related quality of life measures. We would like to understand what causes Parkinson's disease, in order that we can design treatments to prevent PD-related disability in veterans. Several lines of evidence suggest that switching off a key brain protein called "1-synuclein" might prevent disease progression in PD. In the present study, we will establish whether turning off 1- synuclein, by using a genetically-engineered virus, prevents Parkinson's disease-like symptoms in a novel rat model. If this proof-of-concept study is positive, drugs that target 1-synuclein could be designed and developed in future studies, in order to prevent the progression of PD.