Fig 2: Fibrinogen (Fg)-induced astrocyte activation causes apoptosis of co-cultured neurons (A) Representative images of neuronal apoptosis assessed by Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay. TUNEL assay is a method for detecting DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis. Neurons were co-cultured with astrocytes treated with Fg for 24 h. Astrocytes were treated with medium alone (control), 4 mg/mL of Fg (Fg4), 4 mg/mL of Fg in the presence of a function-blocking peptide against cellular prion protein (Fg4/block PrPC) and 4 mg of Fg in the presence of function-blocking antibody against intercellular adhesion molecule-1 (Fg4/block ICAM-1). For positive control, lipopolysaccharide (LPS) was used at 1µg/mL with co-stimulation with 20 ng of a murine interferon gamma (IFNγ). Immunostaining of apoptotic (red) neurons with 4′,6-diamidino-2-phenylindole (DAPI, blue) nuclear stain is shown. Arrows indicate some of the apoptotic cells (red). (B) Summary of image analyses for the detection of apoptotic neurons presented as a percent of a total number of cells. p < 0.05 in all; *—vs. Control, †—vs. Fg4; n = 4.

Fig 3: Fibrinogen (Fg)-induced expression of pro-inflammatory cytokines in astrocytes. (A) Gene expression of astrocyte pro-inflammatory cytokines interleukin 6 (IL-6), C-X-C motif chemokine 10 (CXCL10), C-C motif chemokine 2 (CCL2), intercellular adhesion molecule-1 (ICAM-1) and anti-inflammatory cytokine interleukin 10 (IL-10) in response to treatment overnight (17 hr) were detected with quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Cells were treated with medium alone (control), 2 mg/mL or 4 mg/mL of Fg (Fg4), 4 mg/mL of Fg in the presence of PrPC function-blocking peptide (Fg4/block PrPC) and 4 mg/mL of Fg in the presence of function-blocking antibody against ICAM-1 (Fg4/block ICAM-1). Lipopolysaccharide (LPS, 1 µg/mL), with or without co-stimulation with 20 ng/mL of a murine interferon gamma (IFNγ), was used as a positive control. Data were presented as a gene fold normalized to 18S, a housekeeping gene. (B) Content of the astrocytic IL-6 and CXCL-10 proteins in astrocyte conditioned media was measured by enzyme-linked immunosorbent assay. Cells were treated with medium alone (control), 2 mg/mL or 4 mg/mL of Fg, 4 mg/mL of Fg in the presence of a PrPC function-blocking peptide (Fg4/block PrPC), and 4 mg/mL of Fg in the presence of a function-blocking antibody against ICAM-1 (Fg4/block ICAM-1). p < 0.05 in all; *—vs. Control, †—vs. Fg4 and ‡—vs. Fg4/block PrPC; n = 6.

Fig 4: Interaction of fibrinogen (Fg) with its astrocytic receptors, intercellular adhesion molecule 1 (ICAM-1) and cellular prion protein (PrPC) detected by proximity ligation assay (PLA). (A) Representative images of in situ PLA signal (shown in red) depicting an interaction between Fg and astrocyte ICAM-1. Cells were treated with medium alone (control), 0.5 mg/mL of Fg (Fg 0.5), 1 mg/mL of Fg (Fg 1), and 1 mg/mL of Fg in the presence of function-blocking antibody against ICAM-1 (Fg1/block ICAM-1) for 17 h. The blocker of ICAM-1 function was added to cells 2 h prior to Fg treatment. Fg and ICAM-1 were detected using anti-Fg and anti-ICAM-1 antibodies, respectively. Cellular nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue). (B) PLA signals for Fg and ICAM-1 interactions were quantified and normalized per number of cells in each an image. (C) Representative images of in situ PLA signal (shown in red) depicting an interaction between Fg and astrocyte PrPC. Cells were treated with medium alone (control), 0.5 mg/mL of Fg (Fg 0.5), 1 mg/mL of Fg (Fg 1), and 1 mg/mL of Fg in the presence of PrPC function-blocking peptide (Fg1/block PrPC) for 17 h. The blocker of PrPC function was added to cells 2 h prior to Fg treatment. Fg and PrPC were detected using anti-Fg and anti-PrPC antibodies, respectively. Cellular nuclei were labeled with DAPI (blue). (D) PLA signals for Fg and PrPC interactions were quantified and normalized per number of cells in each image. p < 0.05 in all; *—vs. Control, †—vs. Fg1; n = 4.

Fig 5: The fibrinogen (Fg)-induced increase in expression of interleukin 6 (IL-6) in neurons. The cells were treated with medium alone (control), 0.5 mg/mL, 1 mg/mL of Fg, 1 mg/mL of Fg in the presence of a cellular prion protein (PrPC)-blocking peptide (Fg1/block PrPC), or 1 mg/mL of Fg in the presence of a function-blocking antibody against intercellular adhesion molecule-1 (ICAM-1) (Fg1/block ICAM-1) for 24 h. The experimental group treated with tumor necrosis factor alpha (TNFα) co-stimulated with interferon gamma (IFNγ) was used as a positive control. The treated cells were kept for 24 h in 5% CO2 at 37 °C. Hirudin was present in all experimental groups to prevent the conversion of Fg to fibrin. (A) IL-6 mRNA expression analyzed by quantitative real-time PCR (qRT-PCR). The relative expression of cytokine RNA was normalized to the 18S housekeeping gene. (B) The level of IL-6 protein was measured in the medium from neurons using an enzyme-linked immunosorbent assay (ELISA). p < 0.05 in all; ∗—vs. Control, †—vs. Fg0.5, ‡—vs. Fg1; n = 3.