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，我们已经确定了独特的LS异常，其在有退化风险的神经元中早期发展，随着疾病进展，并且在家族性AD类型中加重。本项目的总体目标是验证AD中神经元LS功能障碍导致细胞萎缩并促进修饰小鼠和神经元细胞培养模型的假设。使用LS功能的特异性免疫探针和共聚焦显微镜应用于AD早期阶段的特征良好的人脑，我们将建立LS变化与Abeta，β-淀粉样蛋白沉积和神经系统病变之间的时空关系，并检查ApoE基因型的可能影响。这些关系将在表达突变早老素1（PS1）和/或突变淀粉样前体蛋白（APP）的转基因小鼠中进行前瞻性研究，这些小鼠表现出类似于早期AD的神经元LS异常。结合EM和LM形态计量学和细胞生物学方法，我们将检查的前因的LS异常的转基因小鼠和FAD成纤维细胞表达突变PS1的内吞途径和表征，第一次在大脑中，其他两个途径溶酶体自噬和直接转换的内质网到溶酶体。 将在AD组织中证实这些发现的疾病相关性。为了建立改变的LS功能、神经变性和体内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与胱抑素B敲除小鼠杂交来加重转基因小鼠的阿尔茨海默病。为了解决溶酶体反应的起源，我们将鉴定可能刺激培养物中的神经元（或内皮细胞）中的这种反应的损伤形式，包括缺血、葡萄糖剥夺、缺氧、氧化应激、营养消耗和Abeta毒性。最后，为了定义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以启动细胞死亡，并将表征导致死亡的事件级联及其与AD的相关性。总之，这些研究将阐明LS在AD神经变性中的重要性，产生AD病理学的改进模型，并确定治疗干预的新策略。

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.