The Effect of TGF-β1 Reduced Functionality on the Expression of Selected Synaptic Proteins and Electrophysiological Parameters: Implications of Changes Observed in Acute Hepatic Encephalopathy

Abstract

Decreased platelet count represents a feature of acute liver failure (ALF) pathogenesis. Platelets are the reservoir of transforming growth factor 1 (TGF-β1), a multipotent cytokine involved in the maintenance of, i.a., central nervous system homeostasis. Here, we analyzed the effect of a decrease in TGF-β1 active form on synaptic proteins levels, and brain electrophysiology, in mice after intraperitoneal (ip) administration of TGF-β1 antibody (anti-TGF-β1; 1 mg/mL). Next, we correlated it with a thrombocytopenia-induced TGF-β1 decrease, documented in an azoxymethane-induced (AOM; 100 mM ip) model of ALF, and clarified the impact of TGF-β1 decrease on blood-brain barrier functionality. The increase of both synaptophysin and synaptotagmin in the cytosolic fraction, and its reduction in a membrane fraction, were confirmed in the AOM mice brains. Both proteins' decrease in analyzed fractions occurred in anti-TGF-β1 mice. In turn, an increase in postsynaptic (NR1 subunit of N-methyl-D-aspartate receptor, postsynaptic density protein 95, gephyrin) proteins in the AOM brain cortex, but a selective compensatory increase of NR1 subunit in anti-TGF-β mice, was observed. The alterations of synaptic proteins levels were not translated on electrophysiological parameters in the anti-TGF-β1 model. The results suggest the impairment of synaptic vesicles docking to the postsynaptic membrane in the AOM model. Nevertheless, changes in synaptic protein level in the anti-TGF-β1 mice do not affect neurotransmission and may not contribute to neurologic deficits in AOM mice.

Keywords: LTP; acute liver failure; blood–brain barrier; glutamatergic transmission; synaptic proteins; transforming growth factor β1.

Figure 1

Analysis of peripheral blood parameters. (A) Representative images of blood morphotic elements in control (C; upper) and azoxymethane (AOM) (bottom) mice (from left: thrombocytes indicated with arrows; lymphocytes; neutrophils). (B) Platelet and lymphocyte count in the blood of control and AOM mice. Results are mean ± SEM. n = 4; * p < 0.01 vs. control, t-test.

Figure 2

The effect of transforming growth factor 1 (TGF-β1) neutralizing antibody on the endothelium. (A) Endothelial cells monolayer (in vitro model of blood-brain barrier) permeability for 40 kDa fluorescein-5-isothiocyanate (FITC)-dextran (left) and integrin β1 mRNA expression level (right) after exposure for 24 h to TGF-β1 neutralizing antibody (Anti-TGF-β1; 2.5 µg/mL) and/or ammonium chloride (ammonia; 5 mM); n = 5, * p < 0.05 vs. control, one-way ANOVA, Dunnett’s post hoc test. (B) Protein levels of occludin, ZO-1, and claudin-5 in the frontal cortex homogenates from anti-TGF-β1 and AOM mice with representative immunoblots. Results are the mean ± SEM. n = 4; * p < 0.05 vs. control, one-way ANOVA, Dunnett’s post hoc test.

Figure 3

The TGF-β1 signaling parameters in serum and frontal cortex homogenates from anti-TGF-β1 and AOM mice. Results are the mean ± SEM. n = 5–6, n = 4 for SMAD3 P425 Western blot experiment; * p < 0.05 vs. control, one-way ANOVA, Dunnett’s post hoc test.

Figure 4

Expression and distribution of presynaptic proteins in frontal cortex from AOM, and anti-TGF-β1 mice. (A–C) Level of synaptophysin, synaptotagmin (in cytosolic and membrane fraction), and VAMP 1/2 (in membrane fraction) in frontal cortex from mice after TGF-β1 neutralization and AOM injection with representative immunoblots. Results are the mean ± SEM. n = 6 for synaptophysin and synaptotagmin, n = 4 for VAMP 1/2, * p < 0.05 vs. control, one-way ANOVA, Dunnett’s post hoc test.

Figure 5

Expression of post-synaptic proteins in frontal cortex from AOM, and anti-TGF-β1 mice. (A,B) Level of NR1 subunit, PSD-95, GABAR1α, and gephyrin in membrane fraction in frontal cortex from mice after TGF-β1 neutralization and AOM injection with representative immunoblots. Results are the mean ± SEM. n = 6 for NR1 and PSD-95, n = 4 for GABAR1α and gephyrin, * p < 0.05 vs. control, one-way ANOVA, Dunnett’s post hoc test.

Figure 6

Electrophysiology of cerebrocortical slices from anti-TGF-β1 mice. (A) The amplitudes of field potentials of control and anti-TGF-β1 mice. (B) Summary quantification of the average paired pulse ratio. (C) Long-term potentiation (LTP) analysis, arrows denote the theta burst insets, 18 measurements for control and 15 for anti-TGFβ1. Results are the mean ± SEM. n = 4.

Figure 7

Graphical representation of experimental protocol.