How Serine-129 Phosphorylation Status Affects the Spreading of α-Synuclein Pathology in Vivo: a Study in Knock-in Animals

总结/摘要 帕金森氏病（PD）、路易体痴呆（DLB）、多系统萎缩（MSA）和某些 阿尔茨海默氏病（AD）的形式是“突触核蛋白病”--以病变为特征的脑疾病（Lewybod.com）。 Lewy神经突）富含α-突触核蛋白（αS）。没有改善疾病的治疗方法，部分原因是缺乏 了解原生αS动力学如何变得异常。 我们的长期目标是详细了解 正常的αS生物学发生了差错，并制定了保持/重建正常生理状态的策略 和αS的函数。路易体/神经突中的αS通常在丝氨酸-129（pS129）上磷酸化， 已被认为是潜在的药物靶点。然而，我们最近的研究也表明， pS129在调节突触活动中的生理作用（pS129由神经活动可逆地诱导）。我们 本申请的总体目的是（i）鉴定pS129对接种的神经元内聚集的作用 和（ii）确定pS129与αS病理在动物脑中传播的相关性。我们的中央 一种假设是，pS129的缺乏和过量都可能首先触发αS聚集（因为正常的 αS动力学在缺少“正确”水平的pS129的情况下受到扰动。对扩散的后续影响是 试探性的该项目的基本原理是，了解正常和种子αS磷酸化是 可能为保护αS稳态，纠正αS失衡的策略的发展提供新的见解 并量化αS病变的特征利用我们的新型S129磷酸缺陷（S129 A）和- 模拟（S129 D）基因敲入（KI）小鼠模型，我们提出以下具体目标：1）PFF的作用， 患者源性种子对培养的WT、S129 AKI和S129 DKI神经元中αS聚集的影响。2)PFF的影响和 患者源性种子对WT、S129 AKI和S129 DKI小鼠中αS聚集和病原性传播的影响。目标之下 1，原代啮齿动物皮层神经元培养物（WT、S129 AKI和S129 DKI）将用于确定接种 PFF和源自患者的脑提取物的作用±突触活性。将研究突触毒性机制 所有基因型的海马切片中。在目标2中，我们将确定pS129对αS致病性传播的影响 in our animal动物models模型in vivo体内.作为一个结果，我们希望有一个全面的看法，对启动与蔓延 pS129的病理学功能。拟议的研究是创新的，因为它采用了新的啮齿动物KI pS129缺失和pS129过量的模型。据我们所知，这类模型从未用αS处理过 “种子”之前。目前还没有系统分析pS129对异常αS生物学的启动和传播的影响， 完了我们的细胞、脑切片和动物模型的组合也是创新的。我们的贡献将是 意义重大，因为它有望提供新的，范式转变的洞察正常αS生物学是如何进行的， 以及这种扰动如何在大脑中传播。证实了pS129在 在疾病的开始和传播，并了解如何和为什么，是一个重要的一步，以消除母鸡- αS在健康和疾病中的作用，对药物和生物标志物的开发具有重要意义。

SUMMARY/ABSTRACT Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple-system atrophy (MSA), and certain forms of Alzheimer’s disease (AD) are ‘synucleinopathies’ - brain diseases characterized by lesions (Lewy bod- ies/Lewy neurites) rich in α-synuclein (αS). Disease-modifying treatments are not available, in part due to a lack of insight into how native αS dynamics becomes aberrant. Our long-term goal is to understand in detail how normal αS biology goes awry and to develop strategies to preserve/reestablish the normal physiological state and function of αS. αS in Lewy bodies/neurites is often phosphorylated on serine-129 (pS129), and the kinase(s) involved have been discussed as potential drug targets. However, our recent research also suggests a normal physiological role of pS129 in regulating synaptic activity (pS129 is reversibly induced by neural activity). Our overall objectives in this application are to (i) identify the effect of pS129 on the seeded intraneuronal aggregation of αS, and (ii) to determine the relevance of pS129 for the spreading of αS pathology in animal brains. Our central hypothesis is that both lack and excess of pS129 may trigger αS aggregation in the first place (because normal αS dynamics are perturbed in the absence of the “right” level of pS129). The subsequent effect on spreading is exploratory. The rationale for this project is that understanding both normal and seeded αS phosphorylation is likely to offer new insight for the development of strategies to preserve αS homeostasis, correct αS imbalance and quantify signatures of αS pathology. Taking advantage of our novel S129 phospho-deficient (S129A) and - mimicking (S129D) knock-in (KI) mouse models, we propose the following specific aims: 1) Effects of PFFs and patient-derived seed on αS aggregation in cultured WT, S129AKI, and S129DKI neurons. 2) Effects of PFFs and patient-derived seed on αS aggregation and pathogenic spread in WT, S129AKI, and S129DKI mice. Under Aim 1, primary rodent cortical neuron cultures (WT, S129AKI, and S129DKI) will be used to determine the seeding effects of PFFs and patient-derived brain extracts± synaptic activity. Synaptotoxicity mechanisms will be studied in hippocampal slices of all genotypes. In Aim 2, we will determine the effects of pS129 on αS pathogenic spread in our animal models in vivo. As one outcome, we hope to have a comprehensive view on initiation vs. spreading of pathology as a function of pS129. The proposed research is innovative because it employs novel rodent KI models of pS129 loss and pS129 excess. To our knowledge, such models have never been treated with αS “seed” before. A systemic analysis of pS129 on initiation and spreading of abnormal αS biology has not been done. Our combination of cellular, brain slice, and animal models is also innovative. Our contribution will be significant because it is expected to provide novel, paradigm-shifting insight into how normal αS biology is per- turbed in disease, and how the perturbation spreads within the brain. Corroborating that pS129 plays a key role in disease initiation and spreading, and understanding how and why, is an important step towards a comprehen- sive view of αS in health and disease with major implications for drug and biomarker development.