Probes to differentiate AD brain beta-amyloid from model beta-amyloid systems

描述(申请人提供)：阿尔茨海默病(AD)的动物模型概括了人类状况的早期阶段，但不是神经元死亡和痴呆的后期阶段。这些模型在组织学上相似，但可区分Aβ淀粉样蛋白病理。成像配体匹兹堡化合物B(PIB)与具有NM亲和力的人、动物和合成系统中的不溶性Aβ结合。然而，PIB与人AD脑结合的化学计量比大约是动物模型或合成Aβ纤维的500-10,000倍。其他Aβ配体，如硫代黄素S和刚果红，不区分人、动物和合成的Aβ纤维。我们假设，人脑和动物Aβ纤维之间PIB结合的差异是人类AD神经元死亡和痴呆机制所不可或缺的，并有助于在动物模型中缺乏对疾病的完整概括。初步数据表明，除了Aβ外，AD脑中PIB的高结合化学计量比还需要未知的生化成分，它位于一个抗变性的组装中。这项拟议的工作将为人类AβPIB结合部位产生光亲和探针，该探针也包含一个标签以实现亲和纯化，我们将利用这些探针通过质谱分析来亲和分离和鉴定AD脑中与PIB配体接触的蛋白质和脂质成分。具体目的1.合成和优化一系列对人AD脑内PIB结合位点具有选择性的光亲和标记手柄。具体目的2.用MR-PIB和质谱仪鉴定形成AD脑内PIB配体结合部位的蛋白质和脂类成分。该项目的长期目标是利用已识别的人类AD Aβ纤维及其PIB配体结合口袋的成分和结构来深入了解与AD有关的人类AD Aβ组件的独特性质。最终，我们将确定人类和AD动物模型Aβ组件之间的差异，以阐明AD相关的细胞和环境机制，这些机制将为动物模型的改进提供信息，并确定潜在的新药物靶点。

DESCRIPTION (provided by applicant): Animal models of Alzheimer's disease (AD) recapitulate early phases of the human condition, but not the later stages of neuronal death and dementia. These models develop histologically similar, but distinguishable A beta amyloid pathology. The imaging ligand Pittsburgh Compound B (PIB) binds insoluble A beta from human, animal and synthetic systems with nM affinity. However, the stoichiometry of PIB binding to human AD brain is approximately 500- to 10,000-fold greater than that in animal models or synthetic A beta fibrils. Other A beta ligands such as Thioflavin S and Congo Red do not discriminate between human, animal, and synthetic A beta fibrils. We hypothesize that the difference in PIB binding between human brain and animal A beta fibrils is integral to the mechanism of neuronal death and dementia in human AD, and contributes to the lack of complete recapitulation of the disease in animal models. Preliminary data indicate that uncharacterized biochemical components in addition to A beta are required for the high binding stoichiometry of PIB in AD brain, which resides in a denaturation-resistant assembly. The proposed work will generate photoaffinity probes for the human A beta PIB binding site that also contain a tag to enable affinity purification and we will utilize these probes to affinity isolate nd identify protein and lipid components contacting the PIB ligand in the AD brain by mass spectral analysis. Specific Aim 1. Synthesize and optimize a series of photoaffinity ligands with handles for affinity tags that are selective for the PIB binding site of human AD brain. Specific Aim 2. Identify the protein and lipid components that form the PIB ligand binding site of AD brain using MR-PIB and mass spectrometry. The long term goal of this project is to use the identified components and structure of the human AD A beta fibril and its PIB ligand binding pocket to provide insight into unique properties of human AD A beta assemblies that contribute to AD. Ultimately we will determine the differences between human and AD animal models A beta assemblies to elucidate AD-related cellular and environmental mechanisms that will inform the improvement of animal models and identify potential novel drug targets.