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. 2021 Jan 13;24(2):102058. doi: 10.1016/j.isci.2021.102058

Figure 5.

Figure 5

Reduction of GSK-3β leads to lower levels of p-Tau tau in synapses

(A) Representative western blot images using homogenates from WT and GSK-3β-HK primary neurons after treatment with hTau (AAV added to culture media). Immunoblots were probed with antibodies for GSK-3β, total tau, and PHF-1 (pS396, pS404). GAPDH was used as loading control.

(B) Protein quantification showed that despite significantly lower levels of GSK-3β in total cell homogenates, GSK-3β-HK neurons displayed similar overall levels of total tau and p-Tau (PHF-1) compared with WT neurons. Data are presented as mean ± SEM, n = 10: 5WT, 5HK. Two-tailed Student's t test, ∗∗p < 0.001.

(C) Horizontal brain section showing an illustrative outline of the EC where AAV-transduced neurons can be easily identified by the presence of GFP labeling.

Brain sections of WT and GSK-3β-HK-injected mice were co-labeled for GFP and specific tau-phosphorylation sites targeted by GSK-3β: CP13: pS202; PHF-1: pS396/pS404, and Alz50: misfolded tau.

(D) Quantification of GFP + neurons that were also positive for CP13, PHF-1, and Alz50. About 27% of the transduced neurons were positive for CP13, 15% were positive for PHF-1, and only 6% were positive for Alz50. There were no significant differences between the number of neurons positive for any of the three markers between GSK-3β-HK and WT mice. Data are presented as mean ± SEM, n = 14: 7WT, 7HK, 3 sections per mouse. Two-tailed Student's t test, p > 0.05.

(E) Representative images of cytosolic (C) and synaptoneurosome (S) fractions on western blots probed for PSD95 (confirming appropriate separation of cytosolic and synaptic compartments), GSK-3β, total tau (ms + h), p- GSK-3β-Tyr216 (active form), p-GSK-3β-Ser9 (inhibited form), and p-Tau (PHF-1 antibody).

(F) There was a lower accumulation of GSK-3β and p-Tau in synapses in the EC of GSK-3β-HK compared with WT mice in the presence of equal amounts of tau. Lower levels of total GSK-3β in GSK-3β-HK mice led to redistribution of active and inhibited GSK-3β forms (with lower synaptic levels of active GSK-3β (Tyr216) and equivalent levels of inhibited GSK-3β (Ser9) compared with WT mice. Significantly lower levels of p-Tau (as reported by PHF-1 antibody) were detected in the synapses of GSK-3β-HK mice compared with WT. GAPDH on the same membrane was used for normalization. Data are presented as mean ± SEM, n = 14: 7WT, 7HK, one-way ANOVA, Holm-Sidak post-hoc multiple comparisons, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bars: 500 μm (C, EC image) and 20 μm (C, phospho-tau).