Structures and Propagation of Pathologically Relevant Amyloids in Alzheimer's

 描述(申请人提供)：42-和40-残基阿尔茨海默氏症β-淀粉样多肽的淀粉样纤维(Aβ)是老年斑的两个主要成分，这是阿尔茨海默病(AD)的标志。由于通过Aβ自组装形成的淀粉样纤维显示出显著的毒性，AD的神经细胞死亡长期以来一直与Aβ淀粉样纤维有关。最近，由于许多可扩散的Aβ聚集体比Aβ纤维显示出更高的毒性，Aβ的扩散性亚纤维聚集体被确认为AD的新嫌疑人。淀粉样蛋白原纤维和Aβ扩散性聚集体的分子结构引起了人们的极大关注。然而，X射线结晶学或溶液核磁共振等传统方法的应用仅限于非晶态淀粉样聚集体。特别是，对于病理上更具相关性的42-残基Aβ(Aβ42)，由于其高度聚集的倾向，众所周知，样品制备困难，因此只知道几个原子级的结构细节。同样，对于与早发阿尔茨海默病相关的Aβ突变，人们知之甚少 大多数致病突变体，如E22G Aβ的结构，因为它们很容易聚集成异质聚集体，这不适合结构分析。通过这项研究，我们将揭示淀粉样纤维和球形聚集体的原子细节，以突出不同淀粉样聚集体物种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个残基的Aβ40更具致病性。作为淀粉样蛋白聚集体特定部位结构分析的重要工具，我们将使用固态核磁共振(SS核磁共振)，并结合透射电子显微镜(TEM)、分子动力学模拟和其他生化/生物物理方法。我们的研究包括以下三个具体目标。(1)在目标1中，我们将建立针对Aβ42和致病突变体E22G Aβ40的结构分析优化的均一淀粉样纤维的制备。对于Aβ42纤维，我们将通过先进的固体核磁共振波谱来阐明原子级结构细节。不同Aβ物种在AD中的相互作用将通过交叉种子实验来模拟，这使得我们能够检查两个不同Aβ物种在纤维中的结构兼容性。目的将定义Aβ42原纤维的结构和结构变化，同时提供对这些结构如何影响AD进展的洞察。(2)在目标2中，我们将研究两个不同的可扩散Aβ42聚集体的原子细节。首先，我们将建立高毒性亚纤维聚集体的合成重建，称为淀粉球蛋白(ASPD)，它存在于AD大脑中。由于ASPD水平与AD的严重程度相关，本研究将首次揭示AD中与病理相关的淀粉样寡聚体的原子细节。我们还将研究一种类似的球形组件，称为SPA，它具有更大的尺寸(直径20 nm)和适度的毒性。(3)在目标3中，我们将描述脑源性Aβ42纤维的结构和毒性，这些纤维是从AD大脑中复制的Aβ42纤维。通过对大约30个大脑样本的比较，目的将使我们能够比较阿尔茨海默病患者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.