Lysosomal Dysreg & Neurodeg in Alzheimer's Disease

遗传性人类疾病中溶酶体系统（LS）的功能障碍通常与神经变性和认知能力下降有关。在阿尔茨海默病中，我们已经发现了独特的LS异常，这些异常发生在有退化风险的神经元早期，随着疾病的发展，并在家族性AD类型中加剧。本项目的总体目标是验证阿尔茨海默病神经元LS功能障碍导致细胞萎缩的假设，并促进修饰小鼠和神经元细胞培养模型的建立。我们将利用特异性免疫探针检测LS功能，并利用共聚焦显微镜对阿尔茨海默病早期的人类大脑进行研究，建立LS变化与Abeta、β -淀粉样蛋白沉积和神经原纤维病变之间的时空关系，并探讨ApoE基因型可能的影响。这些关系将在表达早老素1 （PS1）突变体和/或淀粉样前体蛋白（APP）突变体的转基因小鼠中进行前瞻性研究，这些小鼠表现出与早期AD相似的神经元LS异常。我们将结合EM和LM形态测定和细胞生物学方法，通过评估内吞途径，并首次在大脑中表征溶酶体的另外两种途径——自噬和内质网直接转化为溶酶体，来研究转基因小鼠和表达突变体PS1的FAD成纤维细胞中LS异常的前因。这些发现的疾病相关性将在AD组织中得到证实。为了在体内建立LS功能改变、神经退行性变和Abeta产生/清除之间的关系，我们建议通过调节LS组织蛋白酶抑制剂胱抑素B （cystatin B敲除小鼠的杂交）来强化转基因小鼠的阿尔茨海默病病理。为了解决溶酶体反应的起源，我们将确定可能刺激培养神经元（或内皮细胞）中这种反应的损伤形式，包括缺血、葡萄糖剥夺、缺氧、氧化应激、营养物质消耗和Abeta毒性。最后，为了确定LS功能障碍如何促进或触发细胞死亡，我们将通过靶向光氧化选择性地破坏溶酶体膜来启动细胞死亡，并将描述导致死亡的级联事件及其与AD的相关性。总之，这些研究将阐明LS在阿尔茨海默病神经退行性变中的重要性，生成改进的阿尔茨海默病病理模型，并确定新的治疗干预策略。

Dysfunction of the lysosomal system (LS) in inherited human disease is usually associated with neurodegeneration and cognitive decline. In Alzheimer's disease, we have identified distinctive LS abnormalities that develop early in neurons at risk to degenerate, progress with the disease, and are accentuated in types of familial AD. The overall objectives of this project are to test the hypothesis that neuronal LS dysfunction in AD leads to cell atrophy and promotes modified mice, and neuronal cell culture models. Using specific immunological probes of LS function and confocal microscopy applied to well-characterized human brains in the earliest stages of AD, we will establish the temporal and spatial relationship between LS changes, and Abeta, beta-amyloid deposition, and neurofibrillary lesions and examine the possible influences of ApoE genotype. These relationships will be studied prospectively in transgenic mice expressing mutant presenilin 1 (PS1) and/or mutant amyloid precursor protein (APP), which exhibit neuronal LS abnormalities resembling those in early AD. Combining EM and LM morphometry and cell biological approaches, we will examine the antecedents of the LS abnormalities in the transgenic mice and in FAD fibroblasts expressing mutant PS1 by evaluating the endocytic pathway and characterizing, for the first time in brain, two other routes to lysosomes-autophagy and the direct conversion of endoplasmic reticulum to lysosomes. Disease relevance of these findings will be confirmed in AD tissue. To establish relationships between altered LS function, neurodegeneration, and Abeta production/clearance in vivo, we propose to accentuate Alzheimer pathology in transgenic mice by modulating LS cathepsin inhibitor, cystatin B, in crosses with cystatin B knock-out mice. To address the origins of the lysosomal response, we will identify forms of injury that may stimulate this response in neurons in culture (or endothelial cells) including ischemia, glucose deprivation, hypoxia, oxidative stress, nutrient depletion, and Abeta toxicity. Finally, to define how LS dysfunction promotes or triggers cell death, we will selectively disrupt lysosomal membranes by targeted photo-oxidation to initiate cell death and will characterize the resulting cascade of events leading to death and their relevance to AD. Together, these studies will clarify the importance of the LS in neurodegeneration in AD, generate improved models of AD pathology, and identify novel strategies for therapeutic intervention.