Affiliations: [a] Department of Pathology, University of Oklahoma Health Science Center, Oklahoma City, OK, 73104, USA | [b] Oklahoma Center for Neuroscience, University of Oklahoma Health Science Center, Oklahoma City, OK, 73104, USA | [c] Department of Biochemistry and Molecular Biology, University of Oklahoma Health Science Center, Oklahoma City, OK, 73104, USA
Corresponding author: Paula Grammas, Ph.D., Department of Pathology, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, BRC, Room 262, Oklahoma City, OK 73104, USA. Tel.: +1 405 271 3224; Fax: +1 405 271 6437; E-mail: [email protected].
Abstract: The multifunctional serine protease thrombin has been shown to be neurotoxic in vitro and in vivo and is demonstrable in the Alzheimer disease (AD) brain. We have documented that in AD the cerebral microvasculature is a source of inflammatory and neurotoxic proteins. The objective of this study was to determine if injured brain endothelial cells could be a source of neurotoxic thrombin. Brain endothelial cells were incubated with either sodium nitroprusside (SNP, 10 μM), inflammatory proteins (IL-1β, IL-6, TNFα, LPS, IFNγ) or the PKC inhibitor bisindolymaleimide (1 μM) for 24 h and conditioned media collected. Endothelial cell conditioned medium was incubated with purified apolipoprotein E4 (apoE4) for 24 h, and then analyzed for neurotoxic activity against primary cortical cultures and for apoE4 fragments by western blot. Endothelial cell conditioned medium collected after treatment with either SNP, inflammatory proteins, or the PKC inhibitor bisindolymaleimide, demonstrated a significant (p < 0.005) level of thrombin activity, the presence of apoE4 fragments, and was capable of evoking neuronal cell death. These data demonstrate that endothelial cell injury results in thrombin release and suggest that the brain microcirculation could be a source of neurotoxic factors in AD.
Keywords: thrombin, apolipoprotein E, neurotoxin, brain endothelium, Alzheimer's disease