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

描述（由申请人提供）：该项目的目标是研究淀粉样蛋白肽 (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，A¿ 仅在浓度比脑脊液和脑组织中测量的浓度高一千倍以上时才会形成聚集体。即便如此，A¿ 斑块形成​​是 AD 的特征，遗传证据表明，增加 A 聚集倾向的突变会导致早发 AD。这种明显的矛盾表明，一定存在：1）大脑某处的 A 局部浓度较高，或 2）细胞和/或生化机制降低了 A 聚集所需的浓度。越来越多的证据表明，A¿ 可以在淀粉样斑块首次出现之前就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/溶酶体途径被内吞和运输。此外，我们发现 A¿ 集中在晚期内切酶/溶酶体中，达到支持 A¿ 聚集的水平。即使在细胞外部添加低的生理相关浓度的 A 时，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（例如低 pH）有利于 A¿ 聚集。这使得内切/溶酶体途径对于体内新生A¿聚集体形成特别有吸引力。这项工作要测试的假设是，体内 A¿ 聚集的最早阶段发生在神经细胞内，在内/溶酶体成熟过程中。在本提案中，我们将研究 A¿ 从细胞外空间的摄取以及沿内/溶酶体途径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方法来开发 A¿ 沿内/溶酶体途径的摄取、运输和聚集的详细时间过程。这些实验将与现代细胞生物学工具相结合，以探究内切/溶酶体途径的特定环境因素如何促进 A¿ 的聚集。我们推测 A¿ 沿着内/溶酶体途径的内吞作用和运输代表了质量控制和维持细胞外 A¿ 水平的正常途径。然而，内/溶酶体环境的特征，例如低 pH 值和局部高蛋白质浓度，有利于 A¿ 聚集。沿着内质/溶酶体途径的 A¿ 聚集和降解之间的平衡很可能是 AD 发病机制的关键决定因素。

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.