Modulation of Amyloid-Beta Aggregation in the Endosomal/Lysosomal Pathway

描述（由申请人提供）：本项目的目标是研究淀粉样肽（A）在大脑中形成聚集体的潜在机制。这与阿尔茨海默病（AD）的发病机制直接相关。体外研究表明，只有在浓度比脑脊液和脑组织中测量的浓度高出一千倍以上时，A？才会形成聚集体。即便如此，A？斑块形成是AD的特征，遗传学证据表明，增加斑块聚集倾向的突变导致早发性AD。这种明显的矛盾表明，必须有：1）大脑中某处的高局部浓度A <$，或2）细胞和/或生化机制，降低A <$聚集所需的浓度。越来越多的证据表明，在淀粉样斑块首次出现之前，A？可以在神经元内囊泡中积累。我们的初步数据表明，A？可以通过内吞/溶酶体途径被内吞和运输。此外，我们已经发现，A <$是集中在晚期内/溶酶体的水平，将支持A <$聚集。即使在细胞外加入低浓度的生理相关浓度的A？，也会发生这种情况。此外，我们知道内/溶酶体环境的某些特征，如低pH值，有利于A？聚集。这使得内/溶酶体途径对于体内新生A？聚集体形成特别有吸引力。在这项工作中要测试的假设是，体内A？聚集的最早阶段发生在神经细胞内的内/溶酶体成熟期间。在这项提案中，我们将调查从细胞外空间的A ²摄取和沿着内/溶酶体途径的运输对其聚集状态的影响。我们将使用现代荧光光谱和显微镜的方法来开发一个详细的时间过程中的摄取，运输和聚集的A沿着内/溶酶体途径。这些实验将与现代细胞生物学工具相结合，以询问内/溶酶体途径的特定环境因素如何有助于A？的聚集。我们推测，内吞作用和运输的A沿着内/溶酶体途径代表了一个正常的途径，质量控制和维持细胞外的A水平。然而，内/溶酶体环境的特征，如低pH和局部高蛋白浓度，有利于A？聚集。很有可能，A沿着内/溶酶体途径的聚集和降解之间的平衡是AD发病机制中的关键决定因素。

DESCRIPTION (provided by applicant): The goal of this project is to investigate a potential mechanism by which amyloid-¿ peptides (A¿) form aggregates in the brain. This is directly relevant to the pathogenesis of Alzheimer's disease (AD). In vitro studies have shown that A¿ forms aggregates only at concentrations that are more than one thousand times higher than those measured in cerebrospinal fluid and in brain tissue. Even so, A¿ plaque formation is characteristic for AD and genetic evidence demonstrates that mutations that increase A¿ aggregation propensity lead to early onset AD. This apparent contradiction suggests that there must either be: 1) high local concentrations of A¿ somewhere in the brain, or 2) cellular and/or biochemical mechanisms which lower the concentration required for A¿ to aggregate. Growing evidence suggests that A¿ can accumulate in intraneuronal vesicles well before the first appearance of amyloid plaques. Our preliminary data suggest that A¿ can be endocytosed and trafficked through the endo/lysosomal pathway. Moreover, we have found that A¿ is concentrated in late endo/lysosomes to levels that would support A¿ aggregation. This occurs even when low, physiologically relevant concentrations of A¿ are added to the outside of the cell. Also, we know that certain features of the endo/lysosomal environment, such as low pH, favor A¿ aggregation. This makes the endo/lysosomal pathway particularly attractive for nascent A¿ aggregate formation in vivo. The hypothesis to be tested in this work is that the earliest stages of A¿ aggregation in vivo occur within neural cells during endo/lysosome maturation. In this proposal, we will investigate A¿ uptake from the extracellular space and the effect of trafficking along the endo/lysosomal pathway on its aggregation state. We will use modern fluorescence spectroscopic and microscopic methods to develop a detailed time course of the uptake, trafficking, and aggregation of A¿ along the endo/lysosomal pathway. These experiments will be combined with modern cell biology tools to interrogate how specific environmental factors of the endo/lysosomal pathway contribute to the aggregation of A¿. We speculate that endocytosis and trafficking of A¿ along the endo/lysosomal pathway represents a normal pathway for quality control and maintenance of extracellular A¿ levels. However, characteristics of the endo/lysosomal environment, such as low pH and locally high protein concentrations, favor A¿ aggregation. It is quite possible that the balance between A¿ aggregation and degradation along the endo/lysosomal pathway is a critical determinant in AD pathogenesis.