ABSTRACT Aging and expression of apolipoprotein E ε4 (APOE4) gene alleles represent two major risk factors for Alzheimer’s disease. Recent studies have shown that aging leads to impaired brain fluid drainage by the bona fide lymphatic vascular network that is found in the mammalian meningeal dura. Interestingly, early loss of meningeal lymphatic drainage has been linked to worse amyloid-β (Aβ) pathology, vascular and microglial activation, and spatial memory deficits in Alzheimer’s disease mouse models of brain amyloidosis. However, little is known about the effects of APOE4 expression on meningeal lymphatic function. Our preliminary data shows that expression of APOE4 in humanized knock-in mice leads to a deleterious remodeling of meningeal lymphatic vessels and reduced drainage of cerebrospinal fluid (CSF) into the cervical lymph nodes. Interestingly, we also show that meningeal peri-lymphatic myeloid cells are a main local source of apoE protein and become activated in middle-aged APOE4 mice. These observations led us to raise the overarching hypothesis that expression of APOE4 disturbs the crosstalk between meningeal innate myeloid and lymphatic endothelial cells, which will subsequently lead to reduced CSF drainage by meningeal lymphatics, and exacerbated neuroinflammation and brain amyloidosis in Alzheimer’s disease. In Aim 1, we plan to use constitutive or conditional knockout mouse models to investigate the cell autonomous and non-autonomous effects of APOE4 on meningeal lymphatic dysfunction at different ages, determine the underlying innate immune transcriptional signatures, and identify alterations in lymphatic growth factors and/or cytokines. In Aim 2, we will test the hypothesis that an early impairment in meningeal lymphatic drainage promotes a faster cognitive decay in APOE4 mice, and that fine-tuning meningeal lymphatics in aged APOE4 mice ameliorates neuronal function and cognition in sex-dependent manner. To assess this, will modulate meningeal lymphatic drainage, using adeno- associated viruses that promote lymphatic vessel ablation or expansion, and evaluate the downstream effects on the neuronal transcriptomes, synaptic plasticity, and behavioral performances of aged female and male APOE humanized mice. In Aim 3, the therapeutic outcomes of manipulating meningeal lymphatic function will be tested in a knock-in Alzheimer’s disease mouse model of brain amyloidosis, the APP-SAA, expressing APOE3 or APOE4. After inducing meningeal lymphatic vessel loss or expansion in 2- and 10-month-old mice, we will evaluate different experimental outcomes including behavioral performance, neuronal activity, brain interstitial fluid Aβ (by in vivo microdialysis), soluble and insoluble Aβ aggregates, and the profiles of innate and adaptive immune cell activation in the brain and its border tissues. Altogether, this proposal will allow us to uncover novel mechanisms of meningeal lymphatic decline that will serve as cornerstones for future project...