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

阿尔茨海默氏症是一场全国性的危机，这种可怕疾病的负担正在上升。之后 几十年来未能产生一种治疗阿尔茨海默病的疾病改良方法，这是至关重要的 我们确定的药物靶点与不同的 阿尔茨海默病的神经病理基础。我们的研究主要集中在它的功能上。 轴突结构中的溶酶体称为营养不良的轴突。营养不良的神经突起形成时 轴突节段通过β-淀粉样斑块附近，变得膨胀和堵塞 溶酶体和相关细胞器。Tau聚集物也存在于某些营养不良 神经突起，所以这种病理似乎与阿尔茨海默氏症的主要病理特征有关 疾病。因为溶酶体对蛋白质的动态平衡至关重要，我们假设 这些溶酶体的功能是形成有毒蛋白质聚集体的关键步骤。我们 显示了这一溶酶体池的功能变化模式，这可能是 由酸化失败解释，因此在本方案中，我们将评估1)溶酶体是否在 营养不良的神经突起能够酸化，失败的分子机制可能是什么 酸化，2)溶酶体酸化的丧失是否导致自噬缺陷和 病理性自噬中间产物在营养不良轴突内的积聚和3) 一种新的阿尔茨海默病遗传风险因子RBFOX1对改变的影响程度 神经元溶酶体的自噬和pH调节。对于其中的每一个目标，我们都利用 先进的体外方法，包括成年模型动物的器官型脑片培养 从诱导的多能干细胞衍生的三维人类神经元培养 生长在定制设计的水凝胶基质中。利用实时成像，我们将定量测量 营养不良性轴突中溶酶体的pH值。我们将进行详细的神经病理研究 同时在人类和小鼠模型系统中识别空泡分布的变化 ATPase亚基，ARL1和Golgin A4，自噬的关键调节因子，以及RBFOX1。 此外，我们还想了解每种疾病的认知和神经病理联系。 导致酸化失败、自噬缺陷的分子途径的组成部分 和tau聚合，所以我们将利用RNAseq和来自大型临床研究的蛋白质组数据， 特别是宗教秩序研究/记忆和阿尔茨海默氏症项目，以确保这些 路径在功能上很重要。这些协同的数据流将汇聚到强大的 建立营养不良患者溶酶体酸化丢失的机制和后果 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.