PREPARATION AND CHARACTERIZATION OF AB-DERIVED ACTIVE LIGANDS

该计划项目旨在测试假设，非纤维 β淀粉样蛋白（AB）的聚集体，我们称之为 A β衍生的活性配体（ADAL）负责触发 导致神经胶质活化、神经元可塑性的过程 阿尔茨海默氏症中的功能障碍、突触变性和细胞死亡 疾病 扩展这一假设，提出特定的神经胶质细胞 蛋白质调节竞争性Abeta组装过程-以增强 ADAL编队。 在这个项目中，我们将准备和 描述ADAL的特征，定义促进其 形成，我们将确定激活的具体反应， 神经元、神经胶质、混合培养物和器官型脑切片中的ADAL。 这些研究将与组织化学研究协调， 确定ADAL与AD病理学的相关性。 的焦点 项目1是ADAL的制备和表征， 调查其形成的条件。 我们将 确定体外可重复形成的最佳条件 ADAL，制备足够量的结构、生化和 生物物理特征研究，以及对其进行研究 与项目2和项目3合作进行生物反应。 的 ADAL形成的机制将被调查， 强调所选择的胶质辅因子蛋白所起的作用 与AD中的Abeta相关。 我们将扩大我们的研究， 原子力显微镜（AFM）提供更详细的分析， ADAL形态和分布，我们将使用技术，如 作为ADAL的场流分级和天然凝胶电泳 洁净. 我们将采用激光散射，分析 超浓缩荧光偏振和能量传递 研究ADAL组装机制的技术。 主要 在这个项目中要解决的问题是：什么实验 条件有利于ADAL的形成， 隔离的ADAL结构的均匀制备？ 什么因素决定了不同ADAL结构的形成， 无活性纤维状或可溶性Abeta组装体？ 有哪些 ADALs的生物化学和生物物理学特征？ 哪些具体 Abeta 1-42一级结构的特征对于 组装成不同的ADAL和纤维结构？ 什么氨基 酸残基对ADAL的生物活性很重要， 神经元？在神经胶质细胞中？将这些研究与广泛的 本计划项目中的生物能力将定义 导致炎症的分子和细胞过程 发生的病理学、突触变性和神经元细胞死亡 老年痴呆症

This Program Project seeks to test the hypothese that non-fibrillar aggregated assemblies of amyloid beta (AB), which we have term Abeta-derived active ligands (ADALs), are responsible for triggering processes leading to the glial activation, neuronal plasticity malfunction, synaptic degeneration and cell death in Alzheimer's disease. Extending this hypothesis, it is proposed that particular glial proteins modulate competing Abeta assembly process-to enhance ADAL formation. In this program project we will prepare and characterize ADALs, definign conditions that promote their formation, and we will identify the specific responses activated by ADALs in neurons, glia, mixed cultures and organotypic brain slices. These studies will be coordinated with histochemical studies to establish the relevance of ADALs to AD pathology. The focus of project 1 is the preparation and characterization of ADALs and investigation of conditions reponsible for their formation. We will define optimal in vitro conditions for reproducible formation of ADALs,preparing sufficient quantities for structural, biochemical and biophysical characterization studies, and for investigations of their biological responses in collaboration with Projects 2 and 3. The mechanisms governing ADAL formation will be investigated, with an emphasis on the role played by selected glial co-factor proteins associated wit Abeta in AD. We will extend our studies involving atomic force microscopy (AFM) to provide more detailed analysis of ADAL morphology and distribution, and we will use techniques such as field flow fractionation and native gel electrophoresis for ADAL purification. We will employ laser light scattering, analytical ultracentrifugation and fluroescence polarization and energy transfer techniques to investigate ADAL assembly mechanisms. The major questions to be addressed in this project are: What experimental conditions favor the formation of ADALs and what techniques enable the isolation of homogeneous preparation of ADAL structures? What factors govern the formation of different ADAL structures, and inactive fibrillar or soluble Abeta assemblies? What are the biochemical and biophysical characteristics of ADALs? What specific features of the Abeta 1-42 primary structure are important for assembly into different ADAL and fibrillar structures? What amino acid residues are important for the biological activity of ADALs in neurons? in glia? The integration of these studies with the extensive biological capabilities within this Program Project will define the molecular and cellular processes that lead to the inflammatory pathology, synaptic degeneration and neuronal cell death that occurs in Alzheimer's disease.