Identifying the role of notch3 in brain pericyte function in health and Alzheimer's disease

摘要 血脑屏障作为血液和大脑之间的信号和运输接口，其极低的 通透性和丰富的分子运输系统，血脑屏障有助于调节细胞外成分 大脑的一部分。虽然脑微血管内皮细胞(BMECs)具有这些血脑屏障功能，但 血脑屏障受到与神经血管单位(NVU)支持细胞的相互作用的很大影响，如 星形胶质细胞、周细胞和神经元。最近的研究表明，中枢神经系统周细胞在血脑屏障中的重要性 形成和维持，周细胞触发减少的跨细胞作用，减少白细胞的表达 BMEC中的黏附分子和适当的紧密连接组织。周细胞-内皮细胞丢失 相互作用和血脑屏障功能障碍被认为是阿尔茨海默病发病机制的关键。 尽管有潜在的重要性，但驱动脑周细胞调节血脑屏障的分子机制在 健康和阿尔茨海默氏症在很大程度上是未知的，特别是在人类身上。在这项建议中，我们的目标是进一步 检查脑周细胞被指定并随后影响血脑屏障功能的机制。一个 探索人类周细胞发育和功能的强大而创新的途径是利用人类 多能干细胞(HPSC)技术模拟脑周细胞。然而，当前的差异化战略 脑周细胞导致缺乏关键脑周细胞特征的细胞。我们设计了一种方案，让大脑 周细胞可以通过激活神经棘中的Notch3信号从hPSCs中分化出来，从而获得改善 活体脑周细胞的传真件。在这里，我们将进一步探讨Notch3信号对周细胞的影响 发展以及随后对健康和疾病中血脑屏障的诱导和维持的影响。这个 Notch3激活对hPSC来源的神经脊的影响将通过评估脑周细胞的命运来评估。 共同培养的骨髓间充质细胞的周细胞功能和诱导血脑屏障特性的能力。并行地，使用 基因组学方法，我们已经确定了一个由Notch3激活直接调控的转录网络 并发现这个网络在阿尔茨海默病患者的脑周细胞中下调。 为了阐明Notch3激活驱动周细胞规范和发育的机制，我们将 利用CRISPR编辑的hPSC系和发育小鼠模型的补充工具，系统地 调控Notch3转录网络，确定对周细胞发育和血脑屏障的影响 编队和维护。最后，我们将评估Notch信号在小鼠模型中的上调 治疗阿尔茨海默病可以改善周细胞功能障碍对血脑屏障相关病理生理的影响 患有阿尔茨海默氏症。综上所述，了解Notch3信号对周细胞的影响 发展和血脑屏障功能可以产生许多关于人类血脑屏障诱导和 维护并开辟恢复阿尔茨海默病患者血脑屏障功能的新途径。

ABSTRACT The BBB acts as a signaling and transport interface between the blood and brain, and with its very low permeability and a wealth of molecular transport systems, the BBB helps regulate the extracellular composition of the brain. While brain microvascular endothelial cells (BMECs) are possessive of these BBB functions, the BBB is greatly influenced by interactions with supporting cells of the neurovascular unit (NVU) such as astrocytes, pericytes and neurons. Recent studies have indicated the importance of CNS pericytes in BBB formation and maintenance, with pericytes triggering reduced transcytosis, reduced expression of leukocyte adhesion molecules and proper tight junction organization in BMECs. Loss of pericyte-endothelial cell interactions and BBB dysfunction are thought to be critical for the pathogenesis of Alzheimer’s disease. Despite the potential importance, the molecular mechanisms driving brain pericyte regulation of the BBB in health and Alzheimer’s disease are largely unknown, particularly in humans. In this proposal, we aim to further examine the mechanisms by which brain pericytes are specified and subsequently impact BBB function. A powerful and innovative approach to explore human pericyte development and function is the use of human pluripotent stem cell (hPSC) technology to model brain pericytes. However, current strategies for differentiating brain pericytes result in cells that lack key brain pericyte hallmarks. We have devised a protocol where brain pericytes can be differentiated from hPSCs by activation of Notch3 signaling in neural crest, yielding improved facsimiles of in vivo brain pericytes. Here, we will further explore the impact of Notch3 signaling on pericyte development and the subsequent effects on BBB induction and maintenance in health and disease. The impact of Notch3 activation in hPSC-derived neural crest will be evaluated by assessing brain pericyte fate, pericyte functionality and the ability to induce BBB properties in co-cultured BMECs. In parallel, using genomics approaches, we have identified a transcriptional network directly regulated by Notch3 activation in pericytes, and have found that this network is downregulated in brain pericytes of Alzheimer’s disease patients. To elucidate the mechanism by which Notch3 activation drives pericyte specification and development, we will use complementary tools of CRISPR-edited hPSC lines and developmental mouse models to systematically regulate the Notch3 transcriptional network and determine impacts on pericyte development and BBB formation and maintenance. Finally, we will assess whether upregulation of Notch signaling in a mouse model of Alzheimer’s disease can ameliorate the effects of pericyte dysfunction on BBB pathophysiology associated with Alzheimer’s disease. Taken together, understanding the impact of Notch3 signaling on pericyte development and BBB function could yield many new mechanistic insights about human BBB induction and maintenance and open new avenues for restoring BBB function in Alzheimer’s disease.