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

摘要 美国有超过500万例阿尔茨海默病（AD）病例，预计病例数将接近1000万。 在未来15年内翻一番，每年的经济损失总额超过2 250亿美元。尽管如此， 目前尚无有效的治疗AD的方法。β淀粉样蛋白长期以来一直与AD有关，但对 β淀粉样蛋白在AD中的作用仍然存在，并且靶向β淀粉样蛋白的治疗取得了有限的成功。 淀粉样斑块是AD的标志性特征，但斑块负荷与AD进展并不相关， 研究表明，低聚β淀粉样蛋白形式而不是纤维状形式是相关的神经毒性物质， 在AD中。在体内，淀粉样β蛋白是一种非常异质的蛋白质， 变体存在于人类AD脑中。由于蛋白质构象与功能直接相关， 关键A β变体的构象对于理解其在AD中的机制作用至关重要。然而，3- 参与AD神经变性的相关寡聚淀粉样蛋白β变体的D结构仍然难以捉摸。这 这可能是由于不同的寡聚淀粉样蛋白β变体仅以非常低的水平存在于 人AD样品使得结构分析困难。拟议的研究直接集中在这一点上 问题：在这里，我们将产生和表征在人类AD中发现的关键寡聚淀粉样β聚集体 大脑，但不是认知正常的脑组织使用抗体衍生工具（Fab）在我们的实验室产生。C6T Fab选择性识别细胞内产生的淀粉样蛋白-β的关键寡聚体， 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物化学 在AD脑组织中发现的淀粉样蛋白-β的毒性寡聚体形式的表征。C6T Fab将用于 以通过免疫沉淀分离细胞内产生的毒性淀粉样β变体。质谱将 然后用于确定所产生的聚集体的蛋白质组成。由于聚集体产生的 来自人脑组织的蛋白质可能是相当异质的，这是由于蛋白质的广泛翻译后修饰， 在人脑中的β淀粉样蛋白，我们也将类似地分离和表征产生β淀粉样蛋白的毒性细胞 在哺乳动物细胞系7PA2中产生的聚集体。这些研究将为今后的工作提供框架。 这一关键淀粉样蛋白β变体的三维表征研究。我们组建了一个有着独特能力的团队 在Fab开发、3-D结构测定和蛋白质建模中进行这些研究。结构 不同的寡聚淀粉样蛋白β种类的信息对于我们理解如何 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 可以容易地应用于涉及神经退行性疾病的其他关键蛋白变体， 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.