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

描述(申请人提供)：本项目的目标是研究淀粉样多肽(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，只有在比脑脊液和脑组织中测得的浓度高出1000倍以上的浓度时，A‘才会形成聚集体。尽管如此，A？斑块的形成是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性AD。这一明显的矛盾表明，一定存在以下两种情况：1)大脑中某处局部浓度较高的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，在淀粉样斑块第一次出现之前，A？就可以在神经细胞内的小泡中积聚。我们的初步数据表明，A？可以通过内切/溶酶体途径被内吞和运输。此外，我们还发现A？在晚期内切/溶酶体中聚集到支持A？聚集的水平。即使在细胞外加入与生理相关的低浓度的A？时，这种情况也会发生。此外，我们知道内切/溶酶体环境的某些特征，如低pH值，有利于A？聚集。这使得内切/溶酶体途径特别适合体内新生的A？聚集体的形成。这项工作要检验的假设是，体内A？聚集的最早阶段发生在内切/溶酶体成熟的神经细胞内。在这个方案中，我们将研究A？从细胞外空间的摄取以及沿着内切/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镜方法来开发沿着内切/溶酶体途径摄取、运输和聚集Aβ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，以询问内切/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A？的内吞作用和沿着内切/溶酶体途径的运输是质量控制和维持细胞外A？水平的正常途径。然而，内切/溶酶体环境的特点，如低pH和局部高蛋白质浓度，有利于A？聚集。沿内切/溶酶体途径的A？聚集和降解之间的平衡很可能是AD发病的关键决定因素。

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.