The neuroinflammatory role of Fyn‐PKC‐δ signaling pathway in the mouse kainate model of epileptogenesis
Authors: Sharma Shaunik 
Published: November 2023

Rationale

Chemoconvulsant‐induced status epilepticus in rodents can lead to the development of temporal lobe epilepsy (TLE). This may be due to sustained microglial activation causing neuro‐inflammation resulting in lipid peroxidation, reactive gliosis and hippocampal neurodegeneration. We unexpectedly found fyn kinase, a non‐receptor tyrosine kinase, upregulation in microglial cells in a mouse kainate (KA) model of TLE. We hypothesized that modulating fyn kinase levels will prevent seizure onset and decrease the severity of SE, and also reduces brain pathology associated with epileptogenesis.

Methods

We tested the hypothesis in the fyn knockout mice (fyn −/−), wildtype (fyn +/+), and in wildtype C57BL/6J mice treated with the fyn kinase inhibitor, Saracatinib (25 mg/kg) 4h prior to SE induction with KA (5mg/kg). 2h after the established SE, the behavioral SE was terminated with diazepam (10 mg/kg). The animals were euthanized with pentobarbitone (100 mg/kg) at 4h, 24h and 7d (for IHC & WB) and 4 weeks (video‐EEG group) post‐SE. The brain, serum, and cerebrospinal fluid (CSF) were collected for various analyses. We examined electroencephalographic (EEG) analysis and the time‐dependent activation of fyn in microglial cells and initiation of neuro‐inflammatory and neurodegenerative mechanisms in the hippocampus at 4h, 24h and 7day time points post‐SE.

Results

The behavioral studies revealed a significant reduction in the severity of the seizures and the time spent in convulsive motor seizures (CMS) stages in the fyn−/− mice and saracatinib treated mice when compared to the control groups. The mortality rate in the fyn−/− mice was lower than the fyn+/+ mice. The number of spontaneous electrographic non‐convulsive seizures, the duration of CMS, and the spike frequency were also reduced in the fyn−/− mice. The epileptiform spike rate was higher in fyn+/+ mice during the first 7 days, but they decreased thereafter. IHC analysis of brain sections revealed a significant increase in fyn, PKC‐δ, oxidative and nitrative stress markers in the microglia in the dentate gyrus, CA3 and CA1 at 4h and 24h post‐SE in fyn+/+ mice. These levels were significantly reduced in the fyn−/− mice. We also observed a significant increase in the fyn and PKC‐δ nuclear translocation in reactive microglia at 24h post SE in the fyn+/+ mice compared to the fyn−/− suggesting their role in the pro‐inflammatory responses. Quantitative RT‐PCR analysis revealed an increase in pro‐inflammatory cytokine mRNA levels in controls when compared with the fyn−/− mice. In the 24h group, hippocampal Western blots revealed downregulation of fyn, PKC‐δ, phospho Src‐416, phospho PKCδ‐507, GP‐91phox, 4‐HNE and caspase‐3 levels in fyn−/− mice compared to fyn+/+ mice. Moreover, a significant increase in the numbers of FJB‐positive neurons were also observed in CA3 region of hippocampus in fyn+/+ mice compared to fyn−/−.

Conclusions

Collectively, our data suggest that knocking out fyn gene or inhibiting its activity with a pharmacological inhibitor reduces the severity of SE. Fyn kinase absence, prevented neurodegeneration, perhaps by polarizing the microglia and by decreasing the expression of reactive oxygen and nitrogen species levels in the hippocampus.

Support or Funding Information

The fyn wild type & knockout mice were from Dr. Kanthasamy's lab. The work was funded by the startup funds to T. Thippeswamy from Iowa State University, Iowa, USA.