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

细胞内生成的有毒 AD 相关的β淀粉样蛋白片段的纯化和表征

• 批准号: 10511148
• 负责人: MICHAEL R SIERKS
• 金额: $ 43.18万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10511148
• 关键词: 3-Dimensional; Age; Alzheimer' s Disease; Alzheimer' s disease brain; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Antibodies; Autopsy; Binding; Biochemical; Brain; Cell Culture Techniques; Cell Line; Cells; Characteristics; Clinical Trials; Cognitive; Confusion; Cryoelectron Microscopy; Dementia; Development; Disease; Disease Progression; Elements; Failure; Foundations; Future; Generations; Goals; Health; Heterogeneity; Human; Immunoglobulin Fragments; Immunoprecipitation; Inflammation; Link; Literature; Mammalian Cell; Mass Spectrum Analysis; Methods; Microtubule-Associated Proteins; Modeling; Molecular Conformation; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Pathologic; Pathology; Pharmaceutical Preparations; Play; Post-Translational Protein Processing; Process; Protein Conformation; Proteins; Reproducibility; Research; Role; Sampling; Senile Plaques; Structure; Therapeutic; Toxic effect; Variant; abeta accumulation; alpha synuclein; biophysical analysis; brain tissue; cellular targeting; direct application; economic cost; effective therapy; extracellular; improved; in vivo; mind control; neurotoxic; novel; oAβ; overexpression; protein TDP-43; protein aggregation; proteostasis; success; symptom treatment; targeted treatment; tau Proteins; therapeutic target; three dimensional structure; tool

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.

抽象的 在美国，有超过500万例阿尔茨海默氏病（AD），预计几乎 在接下来的15年中，两倍，年度经济成本超过2250亿美元。尽管如此，还有 仍然没有有效的AD治疗方法。蛋白质淀粉样蛋白β长期以来一直与AD联系在一起，但混乱了很多 淀粉样蛋白β在AD中的作用仍然存在，靶向淀粉样蛋白β的治疗的成功有限。 淀粉样蛋白斑是AD的标志性特征，但斑块负载与AD的进展不太相关，并且 研究表明，低聚物淀粉样蛋白β形式而不是原纤维形式是相关的神经毒性物种 在广告中。在体内，淀粉样蛋白β是一种非常异质的蛋白质，许多独特的单体和聚集 人类广告大脑中存在变体。由于蛋白质构象与功能直接相关，因此 关键Aβ变体的构象对于理解其在AD中的机械作用至关重要。但是，3- d参与AD神经变性的相关寡聚淀粉样β变异的结构仍然难以捉摸。这 可能是由于以下事实：不同的寡聚淀粉样蛋白-beta变体仅存在很低的水平 人类广告样品使结构分析变得困难。拟议的研究直接集中于此 问题：在这里，我们将生成和表征人类广告中发现的关键低聚淀粉样蛋白β骨料 大脑但不使用实验室中产生的抗体衍生工具（FAB）的认知正常脑组织。 C6T 晶圆厂有选择地识别存在的关键细胞内产生的淀粉样蛋白β的寡聚聚集体 在AD脑组织中，而不是来自认知正常年龄匹配的对照脑组织的组织。 C6T Fab将 作为拟议研究的基础，该研究旨在实现完整的结构和生化 在AD脑组织中发现的淀粉样蛋白β的有毒寡聚形式的表征。 C6T Fab将使用 通过免疫沉淀分离有毒细胞内产生的淀粉样蛋白β变体。质谱将 然后用于确定生成的聚集体的蛋白质组成。由于生成的聚集体 由于广泛的翻译后修饰，从人类脑组织中可能是异质的 人脑中的淀粉样蛋白β，我们还将类似地分离并表征有毒细胞产生的淀粉样蛋白β 在哺乳动物细胞系中产生的骨料，7PA2。这些研究将为进一步提供框架 该关键淀粉样蛋白β变体的3-D表征研究。我们已经组建了一个具有独特功能的团队 在FAB开发中，进行这些研究的3-D结构测定和蛋白质建模。结构 不同寡聚淀粉样β种的信息对于我们对如何理解的理解至关重要 它们与不同的细胞靶标相互作用，并帮助制定适当的治疗策略。这些方法 可以轻松地应用于在包括tau的神经退行性疾病中实现的其他关键蛋白质变异体 α-核蛋白和TDP-43。