Lysosomal Dysreg & Neurodeg in Alzheimer's Disease

在遗传性人类疾病中，溶酶体系统(LS)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，我们发现了独特的LS异常，这些异常在有退化风险的神经元中早期发展，随着疾病的进展，并在家族性AD类型中加重。该项目的总体目标是检验AD中神经元LS功能障碍会导致细胞萎缩并促进改良的小鼠和神经细胞培养模型的假设。在AD的早期阶段，使用LS功能的特异性免疫探针和共聚焦显微镜，我们将建立LS变化与Abeta、β-淀粉样蛋白沉积和神经纤维病变之间的时间和空间关系，并检查ApoE基因可能的影响。这些关系将在表达突变的早老素1(PS1)和/或突变的淀粉样前体蛋白(APP)的转基因小鼠中进行前瞻性研究，这些转基因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，我们将通过评估细胞内途径并首次在脑内表征另外两条溶酶体途径--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PS1的FAD成纤维细胞LS异常的前因。这些发现与疾病的相关性将在AD组织中得到证实。为了在体内建立LS功能改变、神经变性和Abeta产生/清除之间的关系，我们建议在与cystatin B基因敲除小鼠杂交的情况下，通过调节LS组织蛋白抑制剂cystatin B来加重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，我们将确定在培养的神经元(或内皮细胞)中可能刺激这种反应的损伤形式，包括缺血、葡萄糖剥夺、缺氧、氧化应激、营养耗竭和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以启动细胞死亡，并将表征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AD神经退行性变中的重要性，产生AD病理的改进模型，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.