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.

项目总结： 阿尔茨海默病(AD)是全球范围内日益严重的社会经济负担。寡聚化淀粉样蛋白-b 多肽(Ab)和过度磷酸化的tau是该病的病理标志，也是 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 全面描述目标的生化属性。这笔赠款的目标是将齐聚物结构 对毒性生物活性的影响，并描绘了阿尔茨海默病神经退行性级联反应的步骤。中环 假说是通过使用AD患者来源的诱导多能干细胞(IPSC)培养为大脑皮层 有机类化合物，我们可以足够近地重建AD利基，以产生类脑抗体和tau寡聚体，并 了解细胞类型对低聚物暴露的特定反应。我们的理论基础是，细胞类型中的一种蛋白质 会严重影响其功能，因此为了在体外全面总结AD，你必须对人脑进行建模。 我们的AD患者派生的IPSC模型将捕获重要的遗传和组织特异性元素以支持 AB/tau齐聚和细胞间通讯网络。我们的具体目标将检验以下几点 假设：(目的1)AD患者IPSC生长为大脑皮层器官样物质，重建AD利基并产生 生物活性抗体和tau寡聚体；(目标2)通过对iPSC来源的神经元和神经胶质细胞的毒性筛选 独立地，我们将发现对抗体和tau寡聚体暴露的初级和次级细胞应答； (目的3)并非每个细胞都直接暴露于抗体和tau寡聚体，但仍存在广泛的变性。 暴露在AD-有机化合物中的亚群低聚物将揭示细胞间通信网络 这就造成了这种现象。在结论中，我们将了解构成抗体和抗体的结构组件 Tau寡聚体的生物活性，响应细胞类型的主要寡聚体是什么，以及细胞间的通讯 当一些细胞暴露在低聚物时，会影响到壁龛中的所有细胞。这一贡献意义重大，因为 社区需要新的、可靠的和相关的抗体和tau寡聚体来源来进行大规模研究。 目前使用的尸检脑组织稀缺，且由于脂肪含量高，难以作为来源 材料。此外，通过生化定义生物活性抗体和tau寡聚体，治疗药物可以被设计成 专门针对它。这项研究还将确定疾病生态位中的哪些细胞类型是主要和 对抗体和tau寡聚体的次级应答。通过检测两个干细胞来源的脑中的寡聚体反应 在分离的细胞类型和有机类利基中，我们将建立神经毒性AD级联反应的步骤。这个 拟议的研究是创新的，因为我们将说明患者来源的干细胞的新用途是可靠的 生物活性低聚物的来源。这一发现可以推断为其他疾病，如帕金森氏症 及其相关的α-突触核蛋白聚集体。此外，我们还为RNA Drop设计了一种新用途 通过核苷酸条码进行多重寡聚体摄取跟踪和单细胞转录的测序 策略。