Purification and Characterization of a toxic AD associated intracellularly generated amyloid beta fragment

摘要 美国有500多万阿尔茨海默病(AD)病例，预计病例数量接近 在接下来的15年里翻一番，每年的总经济成本将超过225亿美元。尽管如此，还是有 仍然没有有效的治疗AD的方法。淀粉样β蛋白长期以来一直被认为与阿尔茨海默病有关，但人们对此感到困惑 淀粉样β蛋白在阿尔茨海默病中的作用仍然存在，针对淀粉样β蛋白的治疗效果有限。 淀粉样斑块是AD的标志性特征，但斑块负荷与AD进展没有很好的相关性，并且 研究表明，相关的神经毒性物质是低聚淀粉样β蛋白，而不是纤维样蛋白。 在公元后。在体内，淀粉样β蛋白是一种非常不同的蛋白质，并且有许多截然不同的单体和聚集态 人类阿尔茨海默病的大脑中存在变异体。由于蛋白质构象与功能直接相关，因此确定 关键的Aβ变异体的构象对于理解它们在AD中的机制作用至关重要。然而，3- 与阿尔茨海默病神经变性有关的相关寡聚体淀粉样β变异体的D结构仍然难以捉摸。这 可能是由于不同的寡聚体淀粉样β变异体只在很低的水平上存在于 人类AD样本使结构分析变得困难。拟议的研究直接集中在这一点上 问题：在这里，我们将产生并表征在人类阿尔茨海默病中发现的关键的低聚淀粉样β聚集体 使用我们实验室开发的抗体衍生工具(Fabs)，大脑但不是认知正常的脑组织。C6T FAB选择性地识别存在的关键的细胞内产生的淀粉样β蛋白的寡聚聚体 在AD脑组织中，而不是来自认知正常的年龄匹配的对照脑组织的组织中。C6T Fab将 作为拟议研究的基础，该研究旨在使完整的结构和生化 阿尔茨海默病脑组织中发现的有毒低聚体淀粉样β蛋白的特征。将使用C6T FAB 通过免疫沉淀分离有毒的细胞内产生的淀粉样β变异体。质谱学将 然后用来确定产生的聚集体的蛋白质组成。由于生成的聚合 由于广泛的翻译后修饰，来自人脑组织的可能是相当不同的 人类大脑中的淀粉样β蛋白，我们也将类似地分离和鉴定有毒细胞产生的淀粉样β蛋白 在哺乳动物细胞系7PA2中产生的聚集体。这些研究将提供框架，以便进一步 这一关键的淀粉样β变异体的三维特性研究。我们已经组建了一支具有独特能力的团队 在FAB的开发中，3D结构确定和蛋白质建模来执行这些研究。结构性 不同的寡聚体淀粉样β蛋白物种的信息对于我们理解 它们与不同的细胞靶点相互作用，并帮助设计适当的治疗策略。这些方法 可以很容易地应用于其他与神经退行性疾病有关的关键蛋白质变体，包括tau， α-突触核蛋白和TDP-43。

ABSTRACT There are over 5 million cases of Alzheimer’s disease (AD) in the US, the number of cases is expected to nearly double over the next 15 years, and the total annual economic costs are over $225 billion. Despite this, there is still no effective treatment for AD. The protein amyloid-beta has long been linked to AD, but much confusion over the role of amyloid-beta in AD still exists and therapeutics targeting amyloid-beta have had limited success. Amyloid plaques are hallmark features of AD, but plaque loads do not correlate well with AD progression, and studies suggest that oligomeric amyloid-beta forms rather than fibrillar forms are the relevant neurotoxic species in AD. In vivo, amyloid-beta is a very heterogeneous protein, and many distinct monomeric and aggregated variants are present in human AD brain. Since protein conformation is directly related to function, determining the conformations of key Aβ variants is crucial to understanding their mechanistic roles in AD. However, the 3- D structures of relevant oligomeric amyloid-beta variants involved in AD neurodegeneration remain elusive. This is likely due to the fact that the different oligomeric amyloid-beta variants are present at only very low levels in human AD samples making structural analysis difficult. The proposed research is focused directly on this problem: here we will generate and characterize a key oligomeric amyloid-beta aggregates found in human AD brain but not cognitively normal brain tissue using antibody derived tools (Fabs) generated in our lab. The C6T Fab selectively recognizes a key intracellularly generated oligomeric aggregates of amyloid-beta that is present in AD brain tissue but not tissue from cognitively normal age-matched control brain tissue. The C6T Fab will serve as the foundation of the proposed research, which seeks to enable the complete structural and biochemical characterization of the toxic oligomeric forms of amyloid-beta found in AD brain tissue. The C6T Fab will be used to isolate the toxic intracellularly generated amyloid-beta variant via immunoprecipitation. Mass spectrometry will then be used to determine the protein composition of the generated aggregates. Since the aggregates generated from human brain tissue may be quite heterogeneous due to the extensive post-translational modifications of amyloid-beta in human brain, we will also similarly isolate and characterize the toxic cell generated amyloid-beta aggregate as generated in the mammalian cell line, 7PA2. These studies will provide the framework for further 3-D characterization studies of this key amyloid-beta variant. We have assembled a team with unique capabilities in Fab development, 3-D structure determination and protein modeling to perform these studies. Structural information of the different oligomeric amyloid-beta species will be vitally important to our understanding of how they interact with different cellular targets and to help devise appropriate therapeutic strategies. These methods could be easily applied to other key protein variants implicated in neurodegenerative diseases including tau, alpha-synuclein and TDP-43.