Project Summary Increased blood content of fibrinogen (Fg), e.g. hyperfibrinogenemia (HFg) is a risk factor for Alzheimer's disease (AD). As it occurs during neuroinflammation, it is a risk factor for AD & related disorders (ADRD), including vascular cognitive impairment & dementia (VCID). Our goal is to characterize the role of a novel mechanistic pathway, namely Fg-sphingolipid-caveolae nexus during traumatic brain injury (TBI) as an example of ADRD, which is accompanied with HFg. Our data indicate that cortical contusion injury (CCI)-induced HFg enhances cerebrovascular permeability mainly via caveolar protein transcytosis leading to Fg deposition in extravascular space and resulting in greater formation of Fg and cellular prion protein complexes resulting in short-term memory (STM) reduction, typically occurring in AD. We showed that HFg increased Fg binding to endothelial intercellular adhesion molecule-1 (ICAM-1) enhancing formation of functional caveolae & exocytosis via activation of ERK-1/2 and caveolin-1 (Cav-1). We found that HFg upregulated de novo sphingolipid synthesis pathway & caveolar transcytosis using mitochondrial ATP. A selective inhibitor of sphingolipid synthesis pathway ameliorated the HFg- triggered caveolar protein transcytosis in mouse brain endothelial cells, & importantly, reduced cerebrovascular protein transcytosis after CCI. Based on these results, we hypothesize that at elevated levels, Fg, through binding to its endothelial receptor ICAM-1, activates sphingolipid production resulting in increased caveolar protein transcytosis in ECs that is supported by ATP generated in mitochondria. A corollary hypothesis is that an enhanced caveolar Fg transcytosis contributes to STM reduction similar to that during AD. Specific aims are: (1) Determine whether the increased interaction of Fg with endothelial ICAM-1 enhances formation of functional caveolae via production of Cer, GlcCer, and SPM sphingolipids and thereby increases caveolae-mediated protein transcytosis. (2) Determine whether increased interaction of Fg with ECs enhances mitochondrial activity using up energy for caveolar protein transcytosis. (3) Determine whether inhibition of de novo sphingolipid synthesis attenuates HFg- induced increased formation of functional caveolae decreasing caveolar protein transcytosis and thus, ameliorates the STM reduction during TBI. To test mechanisms of HFg-induced caveolar transcytosis via sphingolipid signaling we will use WT, HFg, and endothelium-specific serine palmitoyltransferase long chain-2 gene knockout (Sptlc2endo-/-) and HFg/Sptlc2endo-/- mice generated by Cre/lox method, with or without CCI. The dual-tracer probing method will be used to define changes in caveolar transcytosis. Loss-of-function strategy (specific siRNAs against ICAM-1 & Cav-1) and specific inhibitors of sphingolipid synthesis will be used. Immunohistochemistry and intravital, confocal, electron, total internal reflection fluorescence, and fluorescenc...