Structures and Propagation of Pathologically Relevant Amyloids in Alzheimer's

 描述（由申请方提供）：42和40个残基的阿尔茨海默氏β-淀粉样蛋白（Aβ）肽的淀粉样原纤维代表老年斑的两种主要组分，老年斑是阿尔茨海默氏病（AD）的标志。由于通过Aβ自组装形成的淀粉样蛋白原纤维显示出显著的毒性，AD中的神经细胞死亡长期以来与Aβ淀粉样蛋白原纤维相关。最近，Aβ的可扩散亚原纤维聚集体被确定为AD的新怀疑因素，因为许多这些可扩散Aβ聚集体显示出比Aβ原纤维高得多的毒性。淀粉样纤维和Aβ扩散聚集体的分子结构引起了人们的极大关注。然而，传统方法如X射线晶体学或溶液NMR的应用已被限制到非结晶淀粉样蛋白聚集体。特别是，对于病理学上更相关的42-残基Aβ（Aβ42），由于其高聚集倾向而导致的众所周知的样品制备困难，因此仅知道少数原子水平的结构细节。类似地，对于与AD早发相关的Aβ突变体， 大多数致病性突变体如E22 G Aβ的结构，因为它们容易聚集成不均匀的聚集体，这不适合于结构分析。 通过这项研究，我们将揭示淀粉样蛋白原纤维和球形聚集体的原子细节，以突出不同淀粉样蛋白聚集体种类之间的结构关系和相互作用，特别是Aβ42，它被认为比更丰富的40-残基Aβ40更具致病性。作为淀粉样蛋白聚集体位点特异性结构分析的重要工具，我们将使用固态NMR（SSNMR），结合透射电子显微镜（TEM），MD模拟和其他生物化学/生物物理方法。 我们的研究涉及以下三个具体目标。(1)在目标1中，我们将建立用于Aβ42和致病突变体E22 G Aβ40结构分析的均匀淀粉样纤维的制备。对于Aβ42纤维，我们将通过先进的SSNMR光谱阐明原子水平的结构细节。AD中不同Aβ物质的相互作用将通过交叉接种实验进行建模，这使我们能够检查原纤维中两种不同Aβ物质的结构相容性。目的是定义Aβ42原纤维的结构和结构变异，同时深入了解结构如何影响AD进展。(2)在目标2中，我们将研究两种不同的可扩散Aβ42聚集体的原子细节。首先，我们将建立一个高毒性的亚纤维聚集体，称为淀粉样球蛋白（ASPD），这是在AD大脑中发现的合成重建。由于ASPD水平与AD的严重程度相关，本研究将首次揭示AD中病理相关的淀粉样蛋白寡聚体的原子细节。我们还将研究一种类似的称为SPA的球形组装体，它具有更大的尺寸（20 nm直径）和适度的毒性。(3)在目的3中，我们将描述脑源性Aβ42原纤维的结构和毒性，这些原纤维在AD脑中由Aβ42原纤维复制。通过比较~30个脑样本，目的将使我们能够比较AD中脑源性淀粉样纤维的结构和毒性。

 DESCRIPTION (provided by applicant): Amyloid fibrils of 42- and 40-residues Alzheimer's β-amyloid (Aβ) peptides represent two primary components of senile plaques, which are a hallmark of Alzheimer's disease (AD). As amyloid fibrils formed via self-assembly of Aβ show notable toxicity, neural cell deaths in AD have long been associated with Aβ amyloid fibrils. More recently, diffusible subfibrillar aggregates of Aβ were identified as a new suspect of AD since many of these diffusible Aβ aggregates show much higher toxicity than Aβ fibrils. Molecular structures of amyloid fibrils and diffusible aggregates of Aβ have attracted great attention. However, applications of traditional methods such as X-ray crystallography or solution NMR have been limited to non-crystalline amyloid aggregates. In particular, for more pathologically relevant 42-residue Aβ (Aβ42), only a few atomic-level structural details were known because of notoriously difficult sample preparation due to its high propensity to aggregate. Similarly, for Aβ mutants associated with early onset of AD, very little is known about structures of most pathogenic mutants such as E22G Aβ because they aggregate readily into heterogeneous aggregates, which are not suited for a structural analysis. Through this study, we will reveal atomic details of amyloid fibrils and spherical aggregates in order to highlight structural relationships and interactions between different amyloid aggregate species, in particular for Aβ42, which is considered to be more pathogenic than more abundant 40-residue Aβ40. As a vital tool for site- specific structural analysis for amyloid aggregates, we will use solid-state NMR (SSNMR), in a combination with transmission electron microscopy (TEM), MD simulations, and other biochemical/biophysical methods. Our studies entail the following three Specific Aims. (1) In Aim 1, we will establish preparation of homogenous amyloid fibrils optimized for structural analyses for Aβ42 and a pathogenic mutant E22G Aβ40. For the Aβ42 fibrils, we will elucidate atomic-level structural details by advanced SSNMR spectroscopy. The interactions of different Aβ species in AD will be modeled by cross-seeding experiments, which allow us to examine structural compatibility of two different Aβ species in fibrils. The aim will define structures and structural variations of Aβ42 fibrils while offering insight into how the structures can impact AD progression. (2) In Aim 2, we will examine atomic details of two distinctive diffusible Aβ42 aggregates. First, we will establish a synthetic reconstitute of highl toxic subfibrillar aggregate called amylospheroid (ASPD), which is found in AD brains. As the level of ASPD is correlated with the severity of AD, this study will reveal the first atomic detail a pathologically relevant amyloid oligomer in AD. We will also examine a similar spherical assembly called SPA, which has a larger size (20 nm diameter) and modest toxicity. (3) In Aim 3, we will profile structures and toxicity of brain-derived Aβ42 fibrils, which are replicated fro Aβ42 fibrils in AD brain. Through a comparison of ~30 brain samples, the aim will allow us to compare structures and toxicity of brain-derived amyloid fibrils in AD.