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

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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.