The combinatorial effects of familial mutations, oxidative stress, and aging on BBB dysfunction in Alzheimer's disease

项目摘要 阿尔茨海默氏症是一种影响数百万美国人的神经退化和衰老疾病， 在没有进一步重大突破的情况下，预计会影响更多的人。1然而，大部分病因 阿尔茨海默氏症的进展仍然不清楚，特别是考虑到许多不同的 与表型结果相关的分子、细胞和环境线索。一个新兴的焦点 在阿尔茨海默病的研究中，脑血管系统在阿尔茨海默病的发生、发展和恶化中所起的作用 症状性疾病2-4血脑屏障的破坏，严密地控制着 体循环和脑组织，已表现在尸检和活体研究的晚期 阿尔茨海默病，表现为微出血、葡萄糖转运功能障碍和毒素外排障碍；额外5例 动物研究表明，一些血管功能障碍先于神经元变性。 6，7因此，为了了解阿尔茨海默病的驱动因素和进展， 希望找到治疗的突破点，必须研究血脑屏障功能障碍的作用。 为此，我们建议使用具有高度时空特性的血脑屏障组织工程模型。 在与阿尔茨海默氏症相关的三种关键扰动类别下评估其功能障碍的决议 疾病。这些干扰将跨越与阿尔茨海默氏症(APP(瑞氏)和阿尔茨海默病(APP)相关的细胞固有突变 PSEN(M146V)，氧化应激的外在信号(过氧化氢暴露)，以及 老年人血液成分(接触老年人与年轻人的血清)。我们假设这个组合 该方法将最好地概括阿尔茨海默病患者的人类BBB表型，并允许对每种表型进行模块化研究 贡献者。这些干扰将从转录、蛋白质组和功能上进行比较。 将通过批量RNA测序和基因集浓缩分析来研究转录变化 突出与已发表的人类数据集的相似性，识别受阿尔茨海默氏症影响的标志性路径 提示，并激励功能分析设计，以便在组织工程模型中进一步验证。蛋白质组学 功能评估包括屏障功能、细胞身份和通路验证的变化 转录本分析显示。这项研究将提供对血液作用的更深层次的理解- 阿尔茨海默病进展中的脑障碍，并强调未来干预的候选目标。 该项目和相关的研究培训将在约翰斯的Peter Searson博士的指导下进行 霍普金斯大学和纳米生物技术研究所。技能包括功能分析设计、干细胞 分化、显微制造和RNA测序分析将得到教育资源的支持 在机构内可用。研究金培训期的其他目标将包括专业人员 未来事业成功的发展，重点是沟通、指导和领导技能。

Project Abstract Alzheimer’s disease is a disease of neurodegeneration and aging that affects millions of Americans, and is expected to impact millions more without further significant breakthroughs.1 However, much of the etiology and progression of Alzheimer’s is still unclear, especially given the complex interactions of many different molecular, cellular, and environmental cues that are correlated with phenotypic outcomes. An emerging focus in Alzheimer’s study is the role of the cerebrovasculature in the initiation, progression, and exacerbation of symptomatic disease.2-4 Disruption of the blood-brain barrier, which tightly controls any exchange between systemic circulation and brain tissue, has manifested in post-mortem and in vivo studies of late-stage Alzheimer’s disease as microbleeds, dysfunctional glucose transport, and impaired efflux of toxins;5 additional animal studies have indicated that some vascular dysfunction precedes neuronal degeneration in the progression of the disease.6, 7 Thus, to understand the drivers and progression of Alzheimer’s disease in hopes of identifying therapeutic breakpoints, the role of blood-brain barrier dysfunction must be investigated. To do so, we propose using a tissue-engineered model of the blood-brain barrier with high spatiotemporal resolution to assess its dysfunction under three key categories of perturbation associated with Alzheimer’s disease. These perturbations will span cell-intrinsic mutations associated with Alzheimer’s (APP(Swe) and PSEN(M146V)), extrinsic cues of oxidative stress (hydrogen peroxide exposure), and the systemic influence of aged blood components (exposure to aged vs. young human serum). We hypothesize that this combinatorial approach will best recapitulate human BBB phenotype in Alzheimer’s, and allow for modular study of each contributor. These perturbations will be compared transcriptomically, proteomically, and functionally. Transcriptomic changes will be studied through bulk RNA-sequencing and gene set enrichment analysis to highlight similarities to published human datasets, identify hallmark pathways that are impacted by Alzheimer’s cues, and motivate functional assay design for further validation in the tissue-engineered model. Proteomic and functional assessments include changes to barrier function, cell identity, and validation of pathways implicated by transcriptomic analysis. This study will provide a deeper understanding of the role of the blood- brain barrier in Alzheimer’s progression and emphasize candidate targets for future intervention. This project and related research training will be conducted under the guidance of Dr. Peter Searson at Johns Hopkins University and the Institute for Nanobiotechnology. Skills including functional assay design, stem cell differentiation, microfabrication, and RNA-sequencing analysis will be supported by the educational resources available within the institution. Additional goals of the fellowship training period will incorporate professional development for future career success, with an emphasis on communication, mentorship, and leadership skills.