Determining the mechanism of alpha-synuclein dependent innate immune responses in the brain

项目摘要 神经系统传统上被认为独立于生物体的免疫反应而起作用， 一个“免疫特权网站”越来越多的科学证据表明，神经系统也不能免疫。 特权，而是具有独特的免疫反应，这对维持体内平衡至关重要，对 中枢神经系统（CNS）功能。神经炎症领域的许多工作都集中在 小胶质细胞的功能，但鲜为人知的是，神经元在调节神经炎症反应的作用 在CNS。五年前，我们发现神经元蛋白，α-突触核蛋白（asyn）， 保护神经元免受病毒感染我们已经扩展了这些数据，以表明ayn调制1型 干扰素（T1 IFN）信号传导。Asyn被认为是帕金森病（PD）的一个原因， 在神经退行性疾病、创伤性脑损伤和其他不同的CNS疾病中，神经胶质瘤的表达失调。尽管 尽管asyn在CNS疾病状态中的重要性，但是asyn表达的功能作用还没有很好地理解。 我们已经发现asyn表达对于支持特异性干扰素刺激的表达是必需的。 基因（ISG）在脑中的T1 IFN信号，独立于小胶质细胞激活。使用诱导 多能干细胞（iPSC）和CRISPR介导的SNCA缺失以产生asyn KO人多巴胺能神经元受体。 神经元，我们发现，在asyn表达的存在下，神经元中的病毒生长受到抑制， 诱导的ISG如IFIT 1、OAS 1和TRIM 25表达降低了asyn KO神经元中的表达。我们接下来 发现asyn KO神经元在用poly I：C或1型处理后表现出广泛的ISG表达丧失 干扰素（IFN-2）治疗由于IFN-2依赖性STAT 2活化和asyn核定位的丧失。采取 总之，我们的数据首次表明asyn的功能是支持神经元中的干扰素反应。的 这项建议的目的是确定在免疫系统中依赖于免疫球蛋白的先天免疫应答的具体机制。 神经元我们假设asyn是一种新的中枢神经系统先天免疫反应的神经元内在调节因子。 我们将从三个方面来检验我们的假设。目的1将使用asyn KO和WT人类神经元来定义特定的 在神经元中asyn、干扰素信号传导和囊泡运输之间的相互作用。目标2将确定以下方面的作用： 使用可诱导的nestin-Cre-lox对脑中先天性T细胞应答的神经元-内源性asyn产生 敲除小鼠中的asyn基因（Snca）。目标3将评价PD特异性和种属特异性变化， asyn可能影响其在神经元中的天然功能。总体而言，拟议的研究将大大 推进我们对中枢神经系统先天免疫反应的神经元内在控制的理解，并提供 新的洞察力的潜在免疫发病机制，有助于不同的人类疾病的中枢神经系统。

Project Summary The nervous system was traditionally thought to act independently of an organism’s immune response and be an “immune privileged site”. Increasing scientific evidence has shown that the nervous system is not immune privileged but instead has a unique immune response that is critical for maintaining homeostasis and critical for central nervous system (CNS) function. Much of the work in the field of neuroinflammation has focused on the function of microglia, but little is known about the role of neurons in modulating neuroinflammatory responses in the CNS. Five years ago, we discovered that the neuronal protein, alpha-synuclein(asyn), was critical in protecting neurons from viral infection. We have extended these data to show that ayn modulates type 1 interferon (T1IFN) signaling. Asyn is known as a cause of Parkinson’s disease (PD) and is known to be dysregulated in neurodegenerative diseases, traumatic brain injury, and other diverse CNS diseases. Despite the importance of asyn in CNS disease states, the functional role of asyn expression is not well understood. We have discovered that asyn expression is necessary to support expression of specific interferon stimulated genes (ISGs) in the brain during T1IFN signaling, independent of microglia activation. Using induced pluripotent stem cells (iPSC) and CRISPR-mediated SNCA deletion to create asyn KO human dopaminergic neurons, we found that viral growth in neurons is inhibited in the presence of asyn expression and that viral- induced ISGs such as IFIT1, OAS1, and TRIM25 exbibit decreased expression in asyn KO neurons. We next found that asyn KO neurons exhibit a broad loss of ISG expression following treatment with poly I:C or type 1 interferon (2) treatment due to loss of asyn-dependent STAT2 activation and asyn nuclear localization. Taken together, our data show for the first time that asyn functions to support interferon responses in neurons. The goal of this proposal is to determine the specific mechanism of asyn-dependent innate immune responses in neurons. We hypothesize that asyn is a novel neuron-intrinsic regulator of the CNS innate immune response. We will test our hypothesis in three aims. Aim 1 will use asyn KO and WT human neurons to define the specific interactions between asyn, interferon signaling, and vesicle transport in neurons. Aim 2 will define the role of neuron-intrinsic asyn production on the innate T-cell response in the brain using an inducible, nestin-Cre-lox knockout of the asyn gene (Snca) in mice. Aim 3 will evaluate PD-specific and species specific changes in asyn that may influence its native function in neurons. Taken together, the proposed studies will significantly advance our understanding of neuron-intrinsic control of the innate immune response in the CNS and provide novel insight into the underlying immunopathogenesis that contributes to diverse human diseases of the CNS.