PROJECT 3 – PROJECT SUMMARY/ ABSTRACT Dysfunctionin the blood-brain barrier (BBB) and each of the cellular components of the neurovascular unit (NVU) – vascular cells, glial cells, and neurons – has been linked to Alzheimer’s disease (AD) evolution in experimental, imaging, pathological, and epidemiological studies. These findings have led to an emerging ‘neurovascular hypothesis’ of AD which holds that cerebrovascular dysfunction contributes to cognitive decline and dementia in AD. Project 3 supports the overall theme of P01 focused on apolipoprotein E (APOE) gene, the major genetic risk factor for AD, by interrogating the BBB/neurovascular hypothesis experimentally at the cellular, molecular and systems levels and in an isoform-specific and gender-specific fashion using novel APOE3 and APOE4knock in (KI)flox/flox mice (E3F; E4F) with and without apoE deletion from astrocytes and pericytes (the key sources of BBB-associated apoE), and with and without Aβ and tau pathology. Based on our pilot and published data we hypothesize that 1) Disrupted blood-brain barrier (BBB) protein-protein interaction (PPI) signaling networks underlying endothelial barrier disruption and BBB dysfunction will precede and predict synaptic and neuronal dysfunction and behavioral changes driven by APOE4 relative to APOE3 in new APOE KIF lines both without and with AD pathology; and 2) 3K3A-activated protein C (APC), a cell signaling analog of APC, will correct dysregulated BBB and PSD95 synaptic PPI signaling networks, will protect BBB and neuronal function, and diminish AD pathology, thereby delaying onset and slowing down disease progression in APOE/AD models. To test our hypothesis, we will use novel E3F and E4F mice alone and crossed i) with Cre lines that specifically express Cre recombinase in astrocytes and pericytes to determine the effects of cell-specific deletion of the key BBB-associated apoE source(s) and ii) with APP/PS1-21 mice and P301S tau mice to investigate interactions with Aβ and tau pathways. To evaluate BBB/vascular dysfunction we will use: 1) advanced molecular assessment of the BBB by simultaneously measured BBB/NVU cell-specific and Aβ and tau biomarkers in CSF and plasma; 2) a novel BBB proteomics analysis; 3) advanced MRI assessment of BBB and CBF; and 4) validation of biofluid biomarkers by tissue analysis. To evaluate synaptic and neuronal dysfunction we will use: 5) PSD95 interactomeanalysis; and 6) viral tract-tracing of hippocampal pathways in the Papez circuit/DMN; and 6) behavior and 7) neuropathology analysis. We will evaluate apoE-allele-specific effects on BBB and neuronal dysfunction by biofluid, tissue and imaging biomarkers, BBB and PSD95 PPI analysis, hippocampal viral tract-tracing, and behavior in APOE KIF mice(male, female) at different ages with and without apoE deletion from astrocytes and pericytes (AIM 1), crossed with APP/PS1-21 and P301S tau micein relation to AD pathology (AIM 2), and will evaluate 3K3A-APC therapy in AP...