Autophagic Clearance of Aberrant Tau: Biochemical and Therapeutic Implications

描述(由申请人提供)：细胞内错误折叠的蛋白质包涵体是许多神经退行性疾病的标志。泛素-蛋白酶体系统(UPS)和自噬-溶酶体系统(A-LS)这两条参与清除异常或陈旧细胞蛋白的蛋白分解途径的功能障碍可能是该病发生的基础。巨自噬(自噬)是溶酶体系统的一种主要降解途径，它在去除过大的细胞器和蛋白质聚集体方面发挥着重要作用，或者是那些不能被伴侣蛋白展开从而无法被UPS降解的细胞器和蛋白质聚集体。自噬小体的形成和溶酶体的清除之间存在着一个平衡，而不受影响的囊泡运输、异型细胞器融合和溶酶体功能是自噬小体降解的终末阶段的关键。A-LS已被证明在清除错误折叠的、容易聚集的蛋白质，如？-突触核蛋白和亨廷顿蛋白方面发挥着重要作用。一般来说，我们假设UPS在疾病早期被上调以清除错误折叠的tau物种，但随着更大的tau聚集，系统变得不堪重负。我们设想，随后将上调A-LS，以努力补偿丢失的UPS活动并清除聚集体，但最终两个系统都会失败，导致加速病理和衰退。Tau的积累、UPS和A-LS之间的相互作用以及相关的药物操作对与人类牵引症相关的结果测量的影响之间的关系将在三个具体目标中进行评估。目的1将研究4R转位症患者体内UPS、AL-S和tau蛋白在人体组织中的积聚之间的相互作用，并将其与两种4R转位症小鼠模型进行比较，这两种模型已经被修改为表达自噬标记。小鼠模型将允许我们操纵自噬途径的组件，以进一步研究与UPS的相互作用，并具体检查特定泛素化形式的蛋白质发生了什么，以确认自噬充足在体内自噬进展中的重要性。目的2将使用上述动物模型的原代神经元来测试特定途径的功能障碍(自噬空泡的异常运输导致自噬通量失败)的影响及其对肌病的影响，以及激活A-LS或降低过度磷酸化tau水平的化合物是否能改善病理表型。目的3将确定NCGC/NIH(国家化学和遗传中心)使用MLPCN(分子图书馆探测中心网络)鉴定的减少亨廷顿蛋白聚集和细胞死亡的化合物是否为可行的自噬增强剂，可用于治疗转位症。总而言之，这些研究将增加对清除途径之间的关系的洞察，这些途径是如何以及何时失败的，以及以自噬为靶点的药物作为对自噬的治疗干预的影响。 与公共卫生相关：几种神经退行性疾病统称为神经退行性疾病，作为病理的一部分，包括被称为神经原纤维缠结的细胞内包涵体。虽然我们不知道缠结是如何形成的，也不知道哪种类型的tau(错误折叠、过度磷酸化、寡聚或聚集tau)会导致细胞功能失调，但清除异常的、错误折叠的tau蛋白可能会减缓疾病的进展，并可能治愈或稳定疾病。建议的研究旨在更好地了解病理tau是如何通过两种清除途径中的任何一种从细胞中移除的-泛素-蛋白酶体清除途径或自噬清除途径。利用自噬作用的小鼠模型，我们将检查这些系统如何以及何时失效；它们之间的关系，细胞运输功能异常的后果；以及增强自噬清除的新候选药物在自噬作用的小鼠模型中是否有效。

DESCRIPTION (provided by applicant): Intracellular inclusions of misfolded proteins are the hallmark of many neurodegenerative diseases. Dysfunction of either of the two proteolytic pathways involved in clearing abnormal or obsolete cellular proteins, the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal system (A-LS) may underlie the development of the disease. Macroautophagy (autophagy), a major degradative pathway of the lysosomal system, plays a significant role in the removal of organelles and protein aggregates that are too large, or that cannot be unfolded by chaperone proteins and that are consequently unable to be degraded by the UPS. An equilibrium exists between autophagosome formation and clearance by lysosomes, and uncompromised vesicular trafficking, heterotypic organelle fusion and lysosomal function are critical for the terminal stages of autophagosomal degradation. The A-LS has been shown to play an important role in the clearance of misfolded, aggregate-prone proteins such as ?-synuclein and huntingtin. In general, we hypothesize that the UPS is upregulated to clear misfolded tau species early during the disease, but the system becomes overwhelmed as larger aggregates of tau accumulate. We envisage that the A-LS is then upregulated in an effort to compensate for the lost UPS activity and to clear the aggregates but ultimately both systems fail resulting in accelerated pathology and decline. The relationship between the accumulation of tau, the interplay between the UPS and A-LS, and the effect of relevant pharmacologic manipulations on outcome measures of relevance to human tauopathy will be assessed in three specific aims. Aim 1 will examine the interplay between the UPS, AL-S and tau accumulation in human tissue from patients with 4R tauopathies and will compare to two mouse models of 4R tauopathy that have been modified to express an autophagic marker. The mouse models will allow us to manipulate components of the autophagic pathways to further study the interplay with the UPS, with specific examination of what happens to specific ubiquitinated forms of proteins to confirm the significance of autophagic sufficiency in tauopathy progression in vivo. Aim 2 will use primary neurons from the aforementioned animal models to test the impact of dysfunction in a particular pathway (abnormal transport of autophagic vacuoles leading to failure of autophagic flux) and its impact on tauopathy, and whether compounds that activate A-LS or reduce the levels of hyperphosphorylated tau ameliorate the pathological phenotype. Aim 3 will identify if compounds identified by NCGC/NIH (National Chemical and Genetic Center) using the MLPCN (Molecular Library Probes Center Network) to reduce huntingtin aggregates and cell death are viable autophagic enhancers that can be used to treat tauopathy. Cumulatively, these studies will add insight into the relationship between clearance pathways, how and when they fail, and the impact of drugs that target autophagy as a therapeutic intervention for the tauopathies PUBLIC HEALTH RELEVANCE: Several neurodegenerative diseases known collectively as the tauopathies include, as part of the pathology, intracellular inclusions known as neurofibrillary tangles. Although we do not know how tangles form, or exactly what type of tau (misfolded, hyperphosphorylated, oligomeric or aggregated tau) causes the cell to become dysfunctional, but it is likely that clearing abnormal, misfolded tau proteins will attenuate disease progression and possibly cure, or stabilize the disease. Studies proposed aim to understand better how pathological tau is removed from the cell through either of the two clearance pathways - the ubiquitin-proteasome clearance pathway, or the autophagic clearance pathway. Using mouse models of tauopathy, we will examine how and when these systems fail; their relationship to each other, the consequence of abnormal cellular transport function; and whether novel drug candidates that enhance autophagic clearance are efficacious in a mouse model of tauopathy.