The Effects of Neuronal Fyn Knockdown in the Hippocampus in the Rat Kainate Model of Temporal Lobe Epilepsy

Abstract

Previous studies have demonstrated neuronal and microglial Fyn, a Src family kinase (SFK), and how its interactions with tau contribute to epileptogenesis. Saracatinib, a Fyn/SFK inhibitor, modifies disease progression in rat kainate (KA) epilepsy models. In this study, we investigated neuronal-specific fyn knockdown effects on Fyn-tau signaling, neurodegeneration, and gliosis using a calcium/calmodulin-dependent protein kinase II (CaMKII)-promoter-driven adeno-associated viral vector (AAV9)-mediated fyn-shRNA injection in the rat hippocampus. Eight days following AAV administration, rats received repeated low-dose KA injections intraperitoneally to induce status epilepticus (SE). Both fyn-shRNA and control groups showed comparable SE severity, indicating inadequate neuronal fyn knockdown at this timepoint. Two weeks post fyn-shRNA injection, hippocampal Fyn significantly decreased, alongside reductions in NR2B, pNR2B^Y1472, PSD95, and total tau. There was also a compensatory activation of SFK (pSFK^Y416:Fyn) and tau hyperphosphorylation (AT8:total tau), negatively correlating with NeuN expression. Proximity ligation assay indicated unchanged Fyn-tau interactions, suggesting tau interactions with alternative SH3 domain proteins. Persistent neuronal loss, astrogliosis, and microgliosis suggested limited effectiveness of neuronal-specific fyn knockdown at this timepoint. An extended-duration fyn knockdown study, or using broad SFK inhibitors such as saracatinib or tau-SH3 blocking peptides, may effectively prevent SE-induced epileptogenesis.

Keywords: Src family kinase; epilepsy; fyn knockdown; neurodegeneration; neuroinflammation.

Figure 1

Experimental design and SE characteristics after KA-induced SE. (A) Experimental design. Created in BioRender https://BioRender.com/d74m332 (accessed on 15 May 2025). (B,C) There were no significant differences in the latency to the onset of convulsive seizures (CS) nor in the number of repeated low-dose KA injections required to induce SE in the fyn knockdown versus SCR-shRNA (scramble) groups. (D) After the onset of SE, there were no differences in SE severity scores between the groups. (B,C) Unpaired t-test; (D) Two-way ANOVA (Šídák’s multiple comparisons test). n = 6–7. Data represented as mean ± SEM.

Figure 2

Stereotaxic injection coordinates. Rat brain schematics (not to scale) showing the sites for stereotaxic injection of AAV vectors in the hippocampus (highlighted green). Representative images of eGFP expression in the rostral and caudal hippocampus of the rat brains injected with SCR-CMV-mir30 (scramble) or fyn-shRNA-CamKII-mir30 (fyn knockdown) at 8 days post-SE.

Figure 3

Western blot analysis of the whole hippocampal lysates probed for Fyn–tau interacting molecules. (A) Representative Western blots of the hippocampal whole cell lysates probed for pNR2B, NR2B, nNOS, PSD95, pSFK, Fyn, c-Src, pTau (AT8), and total tau and beta-actin. (B) Densitometric analysis of the Western blots. (C) Ratios of relative proteins of the markers probed in the Western blots. (D) There was a significant negative correlation between the hyperphosphorylated tau (AT8) and NeuN positive neurons. (B,C) One-way ANOVA (Tukey’s multiple comparisons test) or Kruskal–Wallis test (Dunn’s multiple comparisons test), n = 6–7. Data represented as mean ± SEM. (D) Spearman correlation with simple linear regression. The dotted lines represent the 95% confidence interval of the mean, n = 6–7. * p < 0.05, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01 vs. KA + SCR. Data represented as mean ± SEM.

Figure 4

Gliosis and neurodegeneration in the hippocampus. (A) Representative images of the CA3 region of the hippocampus of rats from different treatment groups immunostained for NeuN (neurons), GFAP (astrocytes), and IBA1 (microglia). Scalebar 100 μm. (B) Representative Western blots of the whole hippocampal lysates of rats from different treatment groups at 8 days post-SE. (C) Fyn knockdown, compared to the control, exacerbated KA-induced neurodegeneration by reducing NeuN expression. Fyn knockdown did not mitigate KA-induced astrogliosis (GFAP expression) and microgliosis (IBA1 expression) in the hippocampus. (D) Representative images of the DG region of the hippocampus of control and AAV9-vector-injected rats (without KA) immunostained for GFAP, IBA1, and DAPI. Scalebar 100 μm. (E) Injection of AAV9 vectors alone did not cause significant increases in gliosis compared to the control. (C) One-way ANOVA (Tukey’s multiple comparisons test), n = 6–7, * p < 0.05, ** p < 0.01 vs. control.; (E) Unpaired t-test, n = 4 per group. Data represented as mean ± SEM.

Figure 5

Fyn–tau interactions at 8 days post-SE. (A) Representative PLA images of Fyn–tau interactions in the CA3 region of the hippocampus of KA-treated rats. (B) Fyn knockdown did not significantly reduce Fyn-tau interactions versus the scramble-treated rats.(B) Ordinary one-way ANOVA (Tukey’s multiple comparisons test), n = 4–7. * p < 0.05 vs. control. Data represented as mean ± SEM.