Defective lysosome acidification in dystrophic neurites in Alzheimer's disease contributes to failure of autophagy

阿尔茨海默病是一个国家危机，这种可怕的疾病的负担正在上升。后 几十年来一直未能产生一种改善阿尔茨海默病的疾病治疗方法， 我们确定药物靶点，这些靶点与各种疾病背后的因果机制有关， 阿尔茨海默病的神经病理学基础。我们的研究重点是 轴突结构中的溶酶体称为营养不良性神经突。营养不良的神经突形成时， 轴突节段通过β-淀粉样蛋白斑块附近，并变得膨胀和堵塞， 溶酶体和相关的细胞器。Tau聚集体也存在于一些营养不良的细胞中。 所以这种病理学似乎与阿尔茨海默氏症的主要病理学特征有关 疾病由于溶酶体对蛋白质稳态至关重要，我们假设， 这些溶酶体的功能是形成毒性蛋白质聚集体的关键步骤。我们 证明了这种溶酶体池功能的变化模式， 解释为酸化失败，因此在本提案中，我们将评估1）溶酶体是否 营养不良的神经突能够酸化，以及失败的分子机制可能是什么 2）溶酶体酸化的丧失是否导致缺陷性自噬， 病理性自噬中间体在营养不良的神经突内的积累，以及3） 一种新的阿尔茨海默病遗传风险因子RBFOX 1在多大程度上改变了 神经元溶酶体中的自噬和pH调节。对于每一个目标，我们都利用 先进的体外方法，包括来自成年模型动物的器官型脑切片培养物 和源自诱导多能干细胞的三维人神经元培养物 在定制的水凝胶基质中生长。使用实时成像，我们将定量测量 营养不良神经突中溶酶体的pH值。我们会进行详细的神经病理学研究 同时在人和小鼠模型系统中识别液泡分布的改变 ATP酶亚基，ARL 1和Golgin A4，它们是自噬的关键调节因子，以及RBFOX 1。 此外，我们还想了解每种疾病的认知和神经病理学联系， 导致酸化失败、自噬缺陷的分子途径的组成部分 和tau聚集，因此我们将利用大型临床研究的RNAseq和蛋白质组学数据， 特别是宗教秩序研究/记忆和阿尔茨海默病项目，以确保这些 路径在功能上是重要的。这些协同的数据流将有力地汇聚到 建立了营养不良大鼠溶酶体酸化丧失的机制和后果， 并为未来的药物发现工作确定分子靶点。

Alzheimer’s disease is a national crisis and the burden of this terrible disease is rising. After decades of failure to produce a disease modifying treatment for Alzheimer’s disease, it is critical that we identify drug targets that are linked to causal mechanisms underlying the diverse neuropathological substrates of Alzheimer’s disease. Our research focuses on the function of lysosomes within an axonal structure called a dystrophic neurite. Dystrophic neurites form when axon segments pass near a β-amyloid plaque and become distended and clogged with lysosomes and related organelles. Tau aggregates are also present in some dystrophic neurites, so this pathology seems to link the major pathological hallmarks of Alzheimer’s disease. Because lysosomes are critical for protein homeostasis, we hypothesize that loss of function of these lysosomes is a key step in the formation of toxic protein aggregates. We demonstrated a pattern of changes in the function this pool of lysosomes which could be explained by failure of acidification, so in this proposal we will evaluate 1) whether lysosomes in dystrophic neurites are able to acidify and what molecular mechanisms may underlie failed acidification, 2) whether loss of lysosome acidification results in defective autophagy and accumulation of pathologic autophagic intermediates within dystrophic neurites and 3) the degree to which a novel Alzheimer’s disease genetic risk factor, RBFOX1, contributes to altered autophagy and pH regulation in neuronal lysosomes. For each of these aims, we leverage advanced in vitro methods including organotypic brain slice cultures from adult model animals and three-dimensional human neuronal cultures derived from induced pluripotent stem cells grown in a custom designed hydrogel matrix. Using live-imaging, we will quantitatively measure the pH in lysosomes in dystrophic neurites. We will conduct detailed neuropathological studies simultaneously in human and mouse model systems to identify altered distribution of vacuolar ATPase subunits, ARL1 and Golgin A4 which are key regulators of autophagy, and RBFOX1. Additionally, we want to understand the cognitive and neuropathological associations of each component of the molecular pathways that contribute to failed acidification, defective autophagy and tau aggregation, so we will leverage RNAseq and proteomic data from large clinical studies, in particularly the Religious Orders Study/Memory and Alzheimer’s Project, to ensure these pathways are functionally important. These synergistic streams of data will converge to robustly establish the mechanism and consequence of loss of lysosome acidification in dystrophic neurites and identify molecular targets for future drug discovery efforts.