PROJECT SUMMARY/ABSTRACT There is an unmet medical need to develop new therapeutic strategies for Alzheimer’s disease (AD). Brain lymphatic vasculature is (re)discovered at the interface between skull and brain (meninges) and is a pathogenic factor for AD. We recently found that meningeal lymphatic dysfunction drives neurocognitive defects in craniosynostosis disorder and established three independent approaches to activate brain lymphatics (Cell, 2021, PMID: 33417861; Cell Stem Cell, 2023, PMID: 37863055). The goal of this proposal is to determine whether craniosynostosis is a risk factor for AD and how our multiplex brain lymphatic activation approaches can be re-deployed to mitigate AD. These three approaches include the transcranial delivery of VEGF-C recombinant protein, intra-cisterna magna (i.c.m.) injection of AAV1-VEGF-C gene, and MSC implantation. They are derived from our investigation of skull-brain communication via skull mesenchymal stem cells (MSCs) and meningeal lymphatic endothelial cells (LECs) crosstalk in health and craniosynostosis. Craniosynostosis is a major craniofacial skull disorder characterized by the premature skull bone fusion due to suture MSC loss coupled with neurocognitive defects. Our meningeal lymphatic investigation in Twist1+/- craniosynostosis mice provides a foundational framework for structural and functional studies of brain lymphatics. Further mechanistic studies revealed that skull MSCs directly promote LEC growth via VEGF-C signaling. Preliminary data identified an abnormal buildup of amyloid β in Twist1+/- mouse brain, which is further exacerbated in the background of 5xFAD mouse model of AD. We are in a unique position to re-deploy these discoveries and multiplex tools to promote brain lymphatics as a therapeutic treatment for AD. We hypothesize that craniosynostosis is a risk factor for AD due to its impaired meningeal lymphatics, and lymphatic activation by multiplex approaches can promote brain fluid homeostasis and waste clearance, mitigating AD-like pathologies and behaviors. The successful outcome of this study will determine the efficacy of individual brain lymphatic activation approaches in promoting brain fluid homeostasis and waste clearance to mitigate AD pathogenesis.