Elucidating the contribution of amyloidogenic APP processing to AD-relevant impaired synaptic protein turnover

项目摘要 阿尔茨海默病（AD）是一种使人衰弱的神经退行性疾病， 老年痴呆症AD的病理特征是两种错误折叠和聚集的蛋白质： 淀粉样β肽（Aβ42）和过度磷酸化的tau蛋白。虽然Aβ42蓄积， 产生淀粉样前体蛋白（APP）的淀粉样蛋白形成过程，是最早的 病理事件，蛋白质稳态失衡的初始触发因素仍然未知。探讨 为了研究AD中的蛋白质稳态障碍，我们的研究利用代谢脉冲追踪（pc）标记， 稳定同位素结合基于定量质谱（MS）的蛋白质组学 分析.使用这种策略与最近开发的APP敲入（App KI）小鼠模型， 在淀粉样蛋白病理学中，我们发现轴突终末是受损蛋白的选择性位点， 降解，特别是突触囊泡（SV）和SV相关蛋白。这种改变 发生在斑块病理学或Aβ42水平升高之前。这一点很重要，因为它表明， 已经确定了在淀粉样蛋白之前发生的蛋白质周转的最早突触损伤 病理此外，我最近发现，用小分子抗癫痫药靶向SV， 药物左乙拉西坦在App KI小鼠中通过降低Aβ42蓄积减轻AD病理学， 改变淀粉样蛋白的加工。我提出的项目的目标是揭示 在临床前淀粉样蛋白病理模型中， 是AD中所见的病理级联中的初始触发因素的基础。一种周转机制 在突触前被认为依赖于泛素-蛋白酶体系统（UPS）标记蛋白 用于从轴突末端运输到索马进行降解。我的核心假设是 一种建议是APP的淀粉样蛋白形成过程导致这一关键过程的缺陷， 轴突末端蛋白质周转受损。为了解决中断这一过程是否会损害 轴突终末蛋白质稳态，我提出以下目标。首先，我将在体内研究UPS是否 在App KI大脑中使用先前的PC组织和先进的MS技术进行破坏， 泛素化蛋白的分离和定量。其次，我将确定SV的中断是否 转运由APP的淀粉样蛋白加工引起，如果这导致APP的错误定位， 并最终将在来自AD患者的人类神经元中证实这些发现。采取 总的来说，这个拟议的项目将确定AD相关蛋白的初始机制， 降解损伤，对确定AD中蛋白质积累的原因至关重要， 目前仍然未知。

Project Summary Alzheimer’s disease (AD) is a debilitating neurodegenerative disease and the most prevalent form of dementia. AD is pathologically characterized by two misfolded and aggregated proteins: amyloid-beta peptides (Aβ42) and hyperphosphorylated tau. Although Aβ42 accumulation, produced amyloidogenic processing of the amyloid precursor protein (APP), is one of the earliest pathological events, the initial trigger in proteostasis imbalance remains unknown. To investigate proteostasis impairments in AD, our research utilizes metabolic pulse-chase (pc) labeling with stable isotopes in combination with quantitative mass spectrometry (MS) based proteomic analysis. Using this strategy with the recently developed APP knock-in (App KI) mouse models of amyloid pathology, we discovered that axon terminals are selective sites of impaired protein degradation, specifically synaptic vesicle (SV) and SV-associated proteins. This alteration occurred before plaque pathology or elevated Aβ42 levels. This is important as it suggests we have identified the earliest synaptic impairment in protein turnover that occurs before amyloid pathology. Additionally, I recently discovered that targeting SVs with small molecule antiepileptic drug levetiracetam in App KI mice mitigated AD pathology by decreasing Aβ42 accumulation via alteration of amyloidogenic processing of APP. The goal of my proposed project is to uncover the mechanism for impaired synaptic proteostasis in models of preclinical amyloid pathology that may underlie the initial trigger in the cascade of pathologies seen in AD. One mechanism for turnover at the presynapse is thought to rely on the ubiquitin-proteasome system (UPS) marking proteins for transport out of the axon terminal to the soma for degradation. The central hypothesis of my proposal is that amyloidogenic processing of APP leads to a deficit to this key process resulting in an impairment in axon terminal protein turnover. To address if disrupting this process impairs axon terminal proteostasis, I propose the following aims. First, I will investigate in vivo if the UPS is disrupted in App KI brains using previously pc-ed tissue and advanced MS techniques for isolation and quantification of ubiquitinated proteins. Second, I will determine if disruptions in SV transport result from amyloidogenic processing of APP and if this leads to mislocalization of APP in vitro and finally will confirm these findings in human neurons derived from AD patients. Taken all together, this proposed project will determine the initial mechanisms of AD-relevant protein degradation impairments, crucial to determining the cause of protein accumulation in AD which currently remains unknown.