Fig 2: Effects of LPM580098 on synaptic plasticity-associated proteins in the spinal dorsal horn after SNL. (A–C, E–G, I–N) were extracted from total protein S1; (D,H) were extracted from membrane fraction S3. The expression of (A) pY1472-NR2B, (B) pS1303-NR2B, (D) NR2B, (E) pT286-CaMKIIa, (F) pS831-GluR1, (H) GluR1, (I) PSD95, (K) pS71-Rac1, and (M) pS188-RhoA proteins significantly increased after SNL compared to the sham controls, whereas those was significantly decreased after LPM580098 treatment relative to the SNL group. No significant differences in total (C) NR2B, (E) CaMKIIa, (G) GluR1, (J) synaptophysin, (L) Rac1, and (N) RhoA levels were observed among groups. Quantification was performed on five blots for each protein (A'–N'). Graphs were plotted as mean ± SEM. #p < 0.05, ###p < 0.001 versus sham group, *p < 0.05, **p < 0.01 versus vehicle group. Un-paired Student’s t-test was used to assess statistical difference in the band intensity.

Fig 3: Importance of CaM acetylation in hippocampal LTP.A, reduced Ac-CaM, p-CaMKIIa, and p-GluR1 in 3KR/3KR hippocampal slices, in response to cLTP stimulation. B–D, quantification of Ac-CaM/CaM (B), p-CaMKIIa/CaMKIIa (C), and p-GluR1/GluR1 (D) in panel A. Data were represented as mean ± SD. ***p < 0.0001, two-way ANOVA followed by Tukey’s multiple comparisons test, n = 6, data were normalized to WT slices under control condition. E, diagram showing field EPSP recording at SC-CA1 synapses from WT and 3KR/3KR mice. F, comparable paired pulse facilitation (PPF) at SC-CA1 synapses between WT and 3KR/3KR mice. Data were represented as mean ± SD. F (1,19) = 0.304, p = 0.5876, two-way ANOVA, n = 10 slices from four WT, n = 11 slices from four 3KR/3KR mice. G, normalized fEPSP amplitudes were plotted every 1 min for hippocampal slices from WT and 3KR/3KR mice. H, reduced TBS-induced LTP at SC-CA1 synapses in 3KR/3KR hippocampal slices, compared with WT. Data in panel G were quantified. Data were represented as mean ± SD. ** p = 0.0021, t test, n = 11 slices from six WT mice, n = 15 slices from eight 3KR/3KR mice. I, Coomassie blue staining of 10 µg GST-tagged WT, 3KR, and 3KQ-CaM proteins purified from bacteria. J, diagram showing whole-cell recording of eEPSC in hippocampal CA1 pyramidal neurons. Recording pipettes were infused with GST-WT, 3KR, or 3KQ-CaM. K, normalized eEPSC amplitudes were plotted every 1 min for CA1 pyramidal neurons infused with 100 nM GST-WT, 3KR, or 3KQ-CaM. L, quantification of LTP in panel K. Data were represented as mean ± SD. NS, not significant, ***p = 0.001, one-way ANOVA, n = 9 cells from nine mice for WT and 3KQ-CaM, n = 10 cells from ten mice for 3KR-CaM.

Fig 4: L-SPD rescues the decreased phosphorylation and surface expression of GluA1-containing AMPA receptors in the hippocampus of APP/PS1 mice. (a) Surface expression of GluA1 and GluA2 subunits were decreased, whereas S-GluA3 expression was not changed in APP/PS1 mice treated with vehicle. L-SPD significantly improved the levels of surface expression of GluA1 and GluA2 in APP/PS1 mice. (GluA1: n=4 in each group, GluA2: n=5 in each group, GluA3: n=4 in each group). (b) Total expression of GluA1, GluA2 and GluA3 subunits were not changed (n=4 in each group). (c) Phosphorylated levels of both pS845 and pS831 of GluA1 were decreased in APP/PS1 mice treated with vehicle. L-SPD could improve the levels of both pS845 and pS831 (n=4 in each group). (d) Phosphorylated levels of pS880 of GluA2 was not changed and L-SPD had no effect on pS880 in APP/PS1 mice (n=3 in each group). (e) Phosphorylated levels of CaMKIIα was increased in APP /PS1 mice treated with vehicle. L-SPD had no effect on p-CaMKIIα (n=6 in each group). *P<0.05, **P<0.01, ***P<0.001 versus corresponding WT group; #P<0.05, ##P<0.01 versus APP/vehicle group. Data are represented as mean±S.E.M

Fig 5: Role of cPKCγ in the MD-induced increase of phosphorylation levels of GluR1 at Ser831 in contralateral visual cortex. (A) Representative western blot images show the pGluR1 (Ser831) levels in ipsilateral visual cortex from cPKCγ+/+ and cPKCγ−/− mice after MD. (B) Statistical results of western blot analysis demonstrate pGluR1 (Ser831) levels in ipsilateral visual cortex of cPKCγ+/+ and cPKCγ−/− mice after MD. (C) Representative western blot images show pGluR1 (Ser831) levels in contralateral visual cortex from cPKCγ+/+ and cPKCγ−/− mice after MD. (D) Statistical results of western blot analysis show the changes of pGluR1 (Ser831) levels in contralateral visual cortex of cPKCγ+/+ and cPKCγ−/− mice after MD. ***P < 0.001 compared with the cPKCγ+/+ mice group; ###P < 0.001 compared with the cPKCγ+/++MD group; &&P < 0.01 compared with the cPKCγ−/− mice group (n = 6 per group).