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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

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos 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

项目摘要

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.

摘要