Fibrinogen activates IκBα signaling in neurons via binding to its receptors, ICAM‐1 and PrP C

Abstract

Mild traumatic brain injury (TBI) is the most common type of brain injury that is a major health issue. The goal of the current study was to extend our previous observations that during TBI, extravasated fibrinogen (Fg) deposited in vasculo/astrocyte interfaces and was associated with neurodegeneration and short-term memory (STM) reduction. We have previously shown that an elevated level of Fg, called hyperfibrinogenemia, causes astrocyte activation via interactions with intercellular adhesion molecule-1 (ICAM-1) and cellular prion protein (PrPC). However, the direct effect of Fg on neurons is not known. Primary mouse brain cortical neurons from C57BL/6 mice (Lonza) were grown in Primary Neural Basal Medium and PNGMTM Single QuotsTM. Twenty-four well plates, with #1 glass-bottomed coverslips coated with Poly-D-Lysine (30 µg/ml) and laminin (200 µg/ml) were used for seeding neurons at the density of 200,000 cells/well. After the required 7-day growth in complete medium, neurons were treated with various doses of Fg for 24 hours unless otherwise indicated. Each experimental group contained hirudin (1 U/ml) to inhibit the conversion of Fg to fibrin via thrombin. In parallel, experiments to test neuronal viability using a live/dead assay were performed without the presence of hirudin. Interactions of Fg with neuronal ICAM-1 and PrPC were tested using a proximity ligation assay (PLA), which allows highly specific and sensitive immunofluorescent detection to be visualized and consequently protein-protein interactions in situ to be objectively quantified. Real time PCR was performed to investigate the expression of mRNAs for interleukin 6 (IL-6) and inhibitor of kappa Bα (IκBα). The levels of IκBα mRNA reflect stimulation of nuclear factor-kappa B (NF-κB) transcription. PLA confirmed Fg-ICAM-1 and Fg-PrPC interactions on the surface of neurons. An increased expression of neuronal IκBα and IL-6 was induced after treatment with Fg for 2hr or 24 hr, respectively. Data showed that Fg, in the presence or absence of hirudin, caused increased neuronal death. Thus, Fg binding to its neuronal receptors, ICAM-1 and PrPC may activate NF-κB, triggered by IκBα signaling, that leads to neuroinflammation and increased neuronal death. This study is a first attempt to define the molecular mechanisms by which Fg causes neuroinflammation and neurodegeneration that resulted in the cognitive decline previously seen during mild-to-moderate TBI in our studies.