Structures and Toxicity of Amyloid Protein Assemblies in Alzheimer's

描述（由申请人提供）：本研究的长期目标是通过研究阿尔茨海默病（AD）中常见的三种阿尔茨海默病-淀粉样蛋白(A)的高毒性蛋白组装体的分子细节，确定阿尔茨海默病（AD）中观察到的神经细胞死亡的起源。大脑中老年斑的形成是阿尔茨海默病（AD）的标志；斑块的主要成分是由A¿组成的纤维组装体。由于A¿通过自组装成更大的聚集形式表现出毒性（即单体A¿无毒），长期以来人们一直怀疑A¿的错误折叠导致原纤维的形成，并通过自组装引起A¿的结构变化，从而引发AD的毒性和神经功能障碍。事实上，聚集的A¿的毒性受到组装A¿的形态、序列的细微差异以及特定配体（如Cu(II)）的存在的极大调节。在本研究中，我们将通过固态核磁共振（SSNMR）检查三种不同形式的A¿有毒淀粉样蛋白聚集体的原子水平结构，固态核磁共振已被用作淀粉样蛋白原纤维结构分析的主要工具，包括A¿的结构。在目标1中，我们将检验一个流行的假设，即a¿的毒性与Cu（II）结合到a¿聚集体有关。人们普遍认为，Cu（II）结合的淀粉样蛋白聚集体可能催化反应产生H2O2，而H2O2对神经细胞有毒性。然而，由于原纤维本身的非均质性，传统的结构生物学方法如x射线晶体学和溶液核磁共振等对金属结合结构的分析并不有效。利用SSNMR，我们将研究Cu（II）与A¿结合的位置和模式，以及由于结合而引起的任何结构变化。在Aim 2中，我们针对A¿（1-42）的淀粉样蛋白原纤维和弥漫性纤维下聚集体的结构，由于样品制备的极端困难，尽管它们具有致病重要性，但它们的特征却很差。这些淀粉样原纤维和A¿（1-42）中间体的分子结构引起了广泛的关注，因为这些结构为设计新疗法和早期检测AD提供了见解。我们将通过使用一种新的灵敏度增强方法来克服样品制备问题，该方法可以最大限度地减少SSNMR所需的样品量。这些研究将通过SSNMR揭示高神经毒性淀粉样蛋白纤维和A¿（1-42）中间体的第一个位点特异性结构细节。在Aim 3中，我们表征了E22G A¿（1-40）错误折叠的结构和动力学，这是一种独特的致病突变，促进了纤维下聚集体的形成。我们的研究旨在首次揭示E22G A¿（1-40）原纤维和亚原纤维聚集体的位点特异性构象。相关淀粉样蛋白聚集体的神经毒性将在小鼠PC12细胞上进行检测。

DESCRIPTION (provided by applicant): The long-term objective of this research is to identify the origin of neural cell deaths observed in Alzheimer's disease (AD) through investigating molecular details of three types of highly toxic protein assemblies of Alzheimer's ¿-amyloid (A¿), which are commonly observed in AD. The formation of senile plaques in a brain is a hall mark of Alzheimer's disease (AD); the primary component of the plaque is fibrillar assemblies comprised of A¿. Because A¿ exhibits toxicity through self-assembly into larger aggregated forms (i.e. monomeric A¿ is non-toxic), it has been long suspected that misfolding of A¿ resulting in the fibril formation and structural changes of A¿ via the self-assembly trigger the onset of the toxicity and the neural dysfunctions in AD. Indeed, the toxicity of the aggregated A¿ is greatly modulated by morphologies of the assembled A¿, subtle difference in the sequence, and the presence of particular ligands such as Cu(II). In this research, we will examine atomic-level structures of three distinctive forms of toxic amyloid aggregates for A¿ by solid-state NMR (SSNMR), which has been used as a primary tool in structural analysis for amyloid fibrils, including those for A¿. In Aim 1, we will examine a popular hypothesis that the toxicity of A¿ is associated with Cu(II) binding to A¿ aggregates. It is widely believed that Cu(II)-bound amyloid aggregates may catalyze reactions producing H2O2, which is toxic to neural cells. However, because of the intrinsic heterogeneity of the fibrils, traditional methods in structural biology such as X-ray crystallography and solution NMR are not effective for analysis of the metal-binding structures. With SSNMR, we will examine the location and the mode of Cu(II) binding to A¿ as well as any structural changes due to binding. In Aim 2, we target the structure of amyloid fibrils and diffusible subfibrillar aggregates for A¿(1-42), which have been poorly characterized, despite their pathogenic importance, because of the extreme difficulties in sample preparation. Molecular structures of these amyloid fibrils and intermediates for A¿(1-42) have attracted broad attention since the structures offer insights into designs of new therapies and early detection for AD. We will overcome the sample preparation problems by using a novel sensitivity enhancement method, which minimizes sample amount required for SSNMR. The studies will reveal the first site-specific structural details of highly neurotoxic amyloid fibril and intermediate for A¿(1-42) by SSNMR. In Aim 3, we characterize structure and kinetics in misfolding for E22G A¿(1-40), which is a unique pathogenic mutant that promotes formation of subfibrillar aggregates. Our study aims to reveal site-specific conformations for E22G A¿(1-40) fibrils and subfibrillar aggregates for the first time. The neural toxicity of the relevant amyloid aggregates will be examined on mouse PC12 cells. PUBLIC HEALTH RELEVANCE: This project provides detailed molecular structure for neurotoxic proteins, which are likely to play central roles in development of Alzheimer's disease. The research will provide insights into rational designs of drugs for Alzheimer's.