Exploiting Alzheimer's disease patient-derived stem cells to biochemically define tau and amyloid-beta oligomer toxic features and their downstream cellular effects

利用阿尔茨海默氏病患者来源的干细胞来生化定义 tau 和淀粉样蛋白-β 寡聚物的毒性特征及其下游细胞效应

• 批准号: 10670985
• 负责人: Alison Linsley O'Neil
• 金额: $ 32.86万
• 依托单位: WESLEYAN UNIVERSITY
• 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 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670985
• 关键词: 3-Dimensional; Affect; Alzheimer' s Disease; Alzheimer' s disease patient; American; Amyloid beta-Protein; Animals; Astrocytes; Autopsy; Bar Codes; Biochemical; Biological Assay; Brain; Cell Survival; Cells; Circular Dichroism Spectroscopy; Clinic; Communities; Complementary DNA; Complex; Data Set; Development; Disease; Dissociation; Drops; Drug Screening; Early Intervention; Economic Burden; Elements; Exposure to; Genetic; Genetic Transcription; Goals; Grant; Health; Human; Human Genetics; Image; In Vitro; Induced pluripotent stem cell derived neurons; Interneurons; Length; Life; Link; Lipids; Mass Spectrum Analysis; Measures; Mentorship; Methodology; Methods; Microglia; Modeling; Molecular Sieve Chromatography; Mutation; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neuroglia; Neurons; Nucleotides; Organoids; Parkinson Disease; Pathologic; Patients; Population; Post-Translational Protein Processing; Production; Proteins; Public Health; RNA; Reproducibility; Research; Research Personnel; Source; Structure; Structure-Activity Relationship; Symptoms; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxic effect; abeta accumulation; abeta oligomer; alpha synuclein; brain cell; brain tissue; cell type; design; early onset; experimental study; gray matter; human disease; hyperphosphorylated tau; imaging modality; improved; in vitro Model; indexing; induced pluripotent stem cell; induced pluripotent stem cell technology; innovation; insight; intercellular communication; light scattering; neuronal cell body; neurotoxic; new therapeutic target; operation; protein aggregation; protein oligomer; research study; response; screening; single-cell RNA sequencing; socioeconomics; stem cell model; stem cells; success; targeted treatment; tau Proteins; tau aggregation; therapeutic development; tool; transcriptomics; uptake

Project Summary: Alzheimer's disease (AD) is an ever-growing socio-economic burden across the globe. Oligomerized amyloid-b peptide (Ab) and hyper-phosphorylated tau are the pathological hallmarks of the disease and a key piece of the neurodegenerative cascade that leads to symptoms. To develop effective oligomer targeting therapies, we must fully describe the biochemical attributes of the target. The objective of this grant is to link oligomer structural components to toxic bioactivity and delineate the steps in the AD neurodegenerative cascade. The central hypothesis is that by using AD patient derived induced pluripotent stem cells (iPSC) cultured as cerebrocortical organoids, we can recreate the AD niche closely enough to produce brain-like Ab and tau oligomers and to understand cell type specific responses to oligomer exposure. Our rationale is that the cell type in which a protein is made heavily impacts its function and so to fully recapitulate AD in vitro, you must model the human brain. Our AD patient derived iPSC model will capture the important genetic and tissue specific elements to support Ab/tau oligomerization and intercellular communication networks. Our specific aims will test the following hypotheses: (Aim 1) AD patient iPSC grown as cerebrocortical organoids recreate the AD niche and produce bioactive Ab and tau oligomers; (Aim 2) Through toxicity screening on iPSC derived neurons and glia independently, we will discover the primary and secondary cellular responders to Ab and tau oligomer exposure; (Aim 3) Not every cell is exposed to Ab and tau oligomers directly and yet there is wide spread degeneration. Subpopulation oligomer exposure in an AD-organoid will uncover the intercellular communication networks that causes this phenomenon. Upon conclusion, we will understand the structural components that make Ab and tau oligomers bioactive, what the main oligomer responding cell types are, and how intercellular communication upon oligomer exposure of some cells, effects all the cells in the niche. This contribution is significant because a new, reliable, and relevant source of Ab and tau oligomers is needed by the community for large scale studies. Currently used post-mortem brain tissue is scarce and due to the high lipid content, difficult to use as a source material. Further, by biochemically defining a bioactive Ab and tau oligomer, therapeutics can be designed to specifically target it. This research will also establish which cell types in the disease niche are the primary and secondary responders to Ab and tau oligomers. By examining oligomer response in both stem cell derived brain cell types in isolation and in the organoid niche, we will establish the steps in the neurotoxic AD cascade. The proposed research is innovative because we will illustrate a new use for patient derived stem cells as a reliable source for bioactive oligomers. This finding can be extrapolated to other diseases such as Parkinson's disease and its related a-synuclein protein aggregates. Additionally, we have designed a new use for RNA drop sequencing to multiplex oligomer uptake tracking and single cell transcriptomics through a nucleotide barcoding strategy.

项目概要： 阿尔茨海默病 (AD) 是全球范围内日益严重的社会经济负担。低聚淀粉样蛋白-b 肽 (Ab) 和过度磷酸化的 tau 蛋白是该疾病的病理标志，也是该疾病的关键部分。 导致症状的神经退行性级联反应。为了开发有效的寡聚体靶向疗法，我们必须 充分描述目标的生化属性。这笔赠款的目的是将低聚物结构连接起来 有毒生物活性的成分并描述 AD 神经退行性级联反应的步骤。中央 假设是通过使用 AD 患者来源的诱导多能干细胞 (iPSC) 培养为脑皮质 类器官，我们可以足够紧密地重建 AD 生态位，以产生类似大脑的 Ab 和 tau 寡聚体，并 了解细胞类型对寡聚物暴露的特异性反应。我们的基本原理是，蛋白质所在的细胞类型 其制造会严重影响其功能，因此要在体外完全重现 AD，您必须对人脑进行建模。 我们的 AD 患者衍生的 iPSC 模型将捕获重要的遗传和组织特异性元素来支持 Ab/tau 寡聚化和细胞间通讯网络。我们的具体目标将测试以下内容 假设：（目标 1）AD 患者 iPSC 生长为脑皮质类器官，重建 AD 生态位并产生 生物活性 Ab 和 tau 寡聚体； （目标 2）通过 iPSC 来源的神经元和神经胶质细胞的毒性筛选 我们将独立地发现 Ab 和 tau 寡聚体暴露的主要和次要细胞反应； （目标 3）并非每个细胞都直接暴露于 Ab 和 tau 寡聚物，但存在广泛的变性。 AD 类器官中的亚群寡聚物暴露将揭示细胞间通讯网络 导致这种现象的发生。得出结论后，我们将了解制造 Ab 和 tau 寡聚体具有生物活性，主要的寡聚体响应细胞类型是什么，以及细胞间通讯如何 当某些细胞暴露于寡聚物时，会影响生态位中的所有细胞。这一贡献意义重大，因为 大规模研究界需要新的、可靠的、相关的 Ab 和 tau 寡聚物来源。 目前使用的死后脑组织稀缺，且由于脂质含量高，难以用作来源 材料。此外，通过生化定义生物活性 Ab 和 tau 寡聚体，可以设计治疗方法 专门针对它。这项研究还将确定疾病生态位中哪些细胞类型是主要的和 Ab 和 tau 寡聚体的次要反应者。通过检查干细胞来源的大脑中的寡聚物反应 分离的细胞类型和类器官生态位中的细胞类型，我们将建立神经毒性 AD 级联的步骤。这 拟议的研究具有创新性，因为我们将说明患者来源的干细胞作为可靠的来源的新用途 生物活性低聚物的来源。这一发现可以推广到帕金森病等其他疾病 及其相关的α-突触核蛋白聚集体。此外，我们还设计了 RNA drop 的新用途 通过核苷酸条形码进行多重寡聚体摄取追踪和单细胞转录组学测序 战略。