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)是一种衰弱的神经退行性疾病，也是最常见的形式 痴呆症。AD的病理特征是两个错误折叠和聚集的蛋白质： 淀粉样β多肽(Aβ42)和过度磷酸化的tau。虽然Aβ42积累， 产生的淀粉样前体蛋白(APP)，是最早产生的淀粉样前体蛋白之一 病理事件中，蛋白平衡失衡的初始触发机制尚不清楚。去调查 AD的蛋白稳定性损害，我们的研究利用代谢脉冲追逐(PC)标记 稳定同位素与定量质谱学结合的蛋白质组学研究 分析。将这一策略与最近开发的App-In(App Ki)鼠标模型一起使用 淀粉样变病理，我们发现轴突终末是受损蛋白的选择性部位 降解，特别是突触小泡(SV)和SV相关蛋白。这一改变 发生在斑块病理之前或Aβ42水平升高。这一点很重要，因为它表明我们 已经确定了蛋白质周转中最早的突触损伤发生在淀粉样蛋白之前 病理学。此外，我最近发现，靶向SVS的小分子抗癫痫药物 左乙拉西坦通过减少A-β-42蓄积减轻AD病理 APP淀粉样蛋白形成过程的改变。我提出的项目的目标是揭示 临床前淀粉样蛋白病理模型中突触蛋白平衡受损的机制可能 在AD所见的一连串病理中，最初的触发因素是基础。一种人员流动的机制 在突触前，被认为依赖于泛素-蛋白酶体系统(UPS)标记蛋白 从轴突末端运输到胞体进行降解。我的核心假设是 有观点认为，APP的淀粉样变性加工会导致这一关键过程的缺陷 轴突终末蛋白周转受损。以解决如果中断这一进程会损害 轴突终末蛋白稳定期，我提出了以下目标。首先，我会在体内调查UPS是否 在App Ki大脑中被破坏，使用以前的PC组织和高级MS技术 泛素化蛋白质的分离和定量。其次，我将确定SV的中断是否 APP的淀粉样变性加工造成的运输，以及这是否导致APP的错误定位 在体外，最终将在AD患者的人类神经元中证实这些发现。已被占用 总之，这个拟议的项目将确定AD相关蛋白质的初步机制 降解损伤，对确定AD中蛋白质堆积的原因至关重要 目前仍不得而知。

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.