Pathophysiologic roles of alpha-synuclein at the synapse

该提案的总体目标是阐明路易体（LB）痴呆的机制病理生物学事件-一种影响超过一百万美国人的认知障碍的痴呆疾病。LB痴呆的一个确定的分子参与者是小的突触前蛋白α-突触核蛋白。在众多有罪证据中，α-突触核蛋白的基因组扩增和突变在患有这些疾病的家庭中可见；人们早就认识到，了解导致α-突触核蛋白介导的LB痴呆毒性的机制事件至关重要。十多年来，该领域的主要焦点一直是破译α-突触核蛋白的正常功能，最终目的是了解向病理状态的转变。然而，尽管付出了巨大的努力，α-突触核蛋白正常功能的确切机制，以及导致病理聚集的早期触发因素仍然难以捉摸。我们的建议的基础是一系列的先导实验，我们发现α-突触核蛋白的两个功能伙伴的新作用，我们假设这些关联的异常是LB痴呆的初始病理触发因素。我们和其他人之前的工作帮助形成了一个共识，即α-突触核蛋白是神经递质释放的生理衰减剂，尽管潜在的机制事件尚不清楚。在这些先前的研究中，我们提出了一个模型，其中α-syn组织成高阶多聚体，在生理上束缚突触囊泡（SVs），导致SVs动员，SVs回收减少，从而减少神经递质释放。在新的先导实验中，我们发现了另外两种突触前蛋白VAMP2和synapsin在帮助α-突触核蛋白减弱神经传递中的新作用。最终，我们的数据使我们建立了一个工作模型，其中突触蛋白和VAMP2在执行α-突触核蛋白功能中依次发挥作用。该模型的原理将在Aims 1/2中进行测试。此外，该领域的一个新兴观点是，生理关联的破坏可能允许游离α-突触核蛋白单体聚集——引发病理——这可能是疾病中最早的病理事件之一；然而，缺乏体内证据。利用我们对功能性α-突触核蛋白伴侣的发现，Aims 2/3将询问这些关联的破坏是否也可能加速LB痴呆细胞和动物模型的病理。我们的目标是：目的1：确定VAMP2在α-突触核蛋白介导的突触衰减中的作用。目的2：确定突触素在α-突触核蛋白介导的突触衰减和病理中的作用。目的3：验证破坏生理关联可以在体内引发α-突触核蛋白病理的假设。一旦完成，我们的研究将揭示α-突触核蛋白正常功能的重要线索，以及在这些毁灭性疾病中引发痴呆和认知障碍的事件。

The overall goal of this proposal is to clarify mechanistic pathobiological events underlying Lewy body (LB) dementias – a dementing illness with cognitive impairment that affects more than a million Americans. An established molecular player in LB dementia is the small presynaptic protein α-synuclein. Amongst a plethora of incriminating evidence, genomic multiplications and mutations of α-synuclein are seen in families harboring these diseases; and it has been long recognized that understanding the mechanistic events that lead to α-synuclein-mediated toxicity in LB dementia is of utmost importance. For over a decade, a primary focus in the field has been to decipher the normal function of α-synuclein, with the ultimate goal of understanding transition to pathologic states. However, despite considerable effort, the precise mechanisms underlying the normal function of α-synuclein, and early triggers leading to pathologic aggregation remain elusive. The basis of our proposal is a series of pilot experiments, where we uncovered novel roles for two functional partners of α-synuclein, and we hypothesize that abnormalities in these associations are the initial pathologic triggers for LB dementias. Previous work from us and others has helped shape a consensus that α-synuclein is a physiologic attenuator of neurotransmitter release, though underlying mechanistic events are unclear. In these previous studies, we proposed a model where α-syn organizes into higher-order multimers that physiologically tether synaptic vesicles (SVs) – leading to a diminution in SV-mobilization, SV-recycling, and consequently, neurotransmitter release. In new pilot experiments, we discovered novel roles for two other presynaptic proteins – VAMP2 and synapsin – in helping α-synuclein attenuate neurotransmission. Eventually, our data led us to a working model where synapsin and VAMP2 play sequential roles in executing α-synuclein function. Tenets of this model will be tested in Aims 1/2. Additionally, an emerging idea in the field is that disruption of physiologic associations might allow free α-synuclein monomers to aggregate – triggering pathology – and that this might be one of the earliest pathologic events in disease; however, in vivo evidence is lacking. Leveraging our discoveries on functional α-synuclein partners, Aims 2/3 will ask if a disruption of these associations might also accelerate pathology in cellular and animal models of LB dementias. Our aims are: Aim #1: Identify the role of VAMP2 in α-synuclein mediated synaptic attenuation. Aim #2: Identify the role of synapsin in α-synuclein mediated synaptic attenuation and pathology. Aim #3: Test the hypothesis that disrupting physiologic associations can trigger α-synuclein pathology in vivo. Upon completion, our studies should reveal vital clues into the normal function of α-synuclein, as well as events that trigger dementia and cognitive impairment in these devastating illnesses.