Fig 2: Fyn or tau or both KO attenuated PTZ-induced abnormal tau hyperphosphorylation and Fyn activation. (A) Representative hippocampal WB images showed increased tau hyperphosphorylation at AT8 and pY18 epitopes, and activated Fyn (measured by pSFK) in WT+PTZ mice, but not in any KOs. (B,C) Quantification of AT8 and pY18 normalized to total tau (Tau5). Compared to WT+PTZ, Fyn KO+PTZ mice showed a significant decrease in tau hyperphosphorylation at AT8 and Y18. (D) Quantification of pSFK normalized to β-Actin showed a significant increase in pSFK in WT+PTZ mice relative to WT+Vehicle mice, while all KOs significantly decreased their levels. (E,F) Quantification of total tau (Tau 5) or Fyn normalized to β-Actin showed no differences between groups (n = 4–5; ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant; two-way ANOVA with Tukey's post-hoc test for multiple comparisons). Data presented as a mean ± SEM.

Fig 3: Absence of Fyn or tau or both reduced PTZ-induced microgliosis at 24 h post-seizure. (A) WT, fyn−/−, tau−/−, and DKO mouse brain sections were labeled with IBA1 (red) for microglia and counterstained with DAPI (blue). The large inset square at the upper left corner of each panel represents the enlarged area of the small square in dotted lines (representative images were shown). Scale bar, 100 μm. (B) Microglial cell quantification from CA1, CA3, DG, and ENT. The number of microglia was significantly increased in WT group as compared with its control in all regions. Both DKO and fyn−/− demonstrated a significant reduction of microgliosis in CA3 and CA1 relative to WT. There was also a significant reduction in microgliosis in CA3 and DG in tau−/− and DKO but not in fyn−/−. Only in DKO, microgliosis was significantly reduced in all four areas. (C) Linear mixed-effects analysis to measure index change of microglia count from fyn−/−, tau−/−, and DKO mice relative to WT. DKO showed more reduction in microgliosis relative to WT as compared to fyn−/− and tau−/− (n = 4–5, 4 sections/animal; ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant; two-way ANOVA with Tukey's post-hoc test for multiple comparisons). Data presented as mean ± SEM.

Fig 4: Fyn and tau loss prevented the polarization of microglia after PTZ-induced seizures. (A) A representative microglial skeletal analysis showing the images of IBA1 positive cells was captured with 40x objective. The cell body and the processes of microglia were measured. The number of endpoints (indicated by the arrows) and the two longest process paths (red processes) per microglia were quantified from >50 cells/group for statistical comparison across the groups. Scale bar 10 μm. (B) Cell body area was significantly increased in the WT and significantly reduced in DKO mice. (C) A significantly reduced number of microglial endpoints was found in the WT. Both tau−/− and DKO, but not fyn−/−, showed significantly more endpoints than WT. (D) The length of the longest microglial processes was significantly reduced in WT and tau−/−, but they were unaffected in fyn−/− and DKO (n = 4–6, 4 sections/animal; ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant; two-way ANOVA with Tukey's post-hoc test for multiple comparisons). Data presented as mean ± SEM.

Fig 5: Increased expression of Fyn, AT8 (phospho-tau), and Fyn-tau complexes after PTZ-induced seizures in the WT mice. (A) Double immunostaining of AT8 (green) and Fyn (red) in WT mice at 24 h post-PTZ showed a significant increase of AT8+Fyn double-labeled cells (yellow) in the CA3 of the hippocampus and ENT (representative images are shown). Arrowheads indicate the co-localization of AT8 with Fyn in neurons. Sections were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Fyn immunopositive cell quantification in the hippocampus (CA1, CA3, and DG) and ENT. A significant increase of Fyn expressing cells was observed in CA3 region. (C) AT8/Fyn double labeled cell quantification in the hippocampus and ENT. In response to PTZ, AT8 expression was significantly upregulated in CA3, CA1, and ENT but not in DG (n = 4–5; 4 sections/animal; *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant; repeated measure two-way ANOVA with Sidak's post-hoc for multiple comparisons). (D) Fyn-tau complexes, detected by PLA (red), and MAP2 labeling (green) were observed in the vehicle, and PTZ treated WT mice, showing an increase of Fyn-tau PLA punctates in CA3 (magnified photograph). Scale bar 100 μm. (E) Quantitative analysis of Fyn-tau PLA punctates showed a significant increase of PLA complexes in the hippocampus of WT+PTZ as compared to WT+Vehicle (n = 4, 4 sections/animal; *p < 0.05; unpaired Student's t-test). Data presented as mean ± SEM.