Fig 2: TrkB/Akt/Fyn signaling is required for the phosphorylation of NMDA receptors in vitro.a–e After normal or transfected HT22 cells were pretreated with brain-derived neurotrophic factor (BDNF) and Mn, the levels of phospho-Akt and phospho-Fyn, and Akt and Fyn expression were evaluated by western blotting. f–j After normal or transfected HT22 cells were pretreated with BDNF and Mn, the levels of phospho-GluN2B and phospho-GluN2A, and the expression of GluN2B and GluN2A were evaluated by western blotting. n = 4. ß-actin was used as a loading control. **P < 0.01, and *P < 0.05 compared to their control counterparts; ##P < 0.01 compared to their Mn-treated cells; ??P < 0.01, and ?P < 0.05 for comparison between their BDNF-pretreated and Mn-treated cells.

Fig 3: Downregulation of excitatory receptor and postsynaptic transmission after BPA exposure. A,B) Representative immunoblot and corresponding densitometric analysis showing the ratios of expression amounts of NMDA receptor subtypes (NR1, NR2A, and NR2B to GAPDH (#p > 0.05, *p < 0.05, and #p > 0.05, respectively) and AMPA receptor subtypes (GluR1 and GluR2) to GAPDH in control and juvenile BPA treated groups (n = 8 rats per group, *p < 0.05 and p > 0.05, respectively); C,D) Excitatory synaptic transmission in BPA-treated brain slices (10 × 10-6 m) was assessed by recording NMDA and AMPA receptor mediated current in the hippocampal CA1 areas, as shown by decreased EPSCNMDA, not EPSCAMPA, after acute BPA treatment (n = 8 rats per group, *p < 0.05 and p > 0.05, respectively).

Fig 4: KIF2C deficiency impairs synaptic transmission and plasticity.(A) mEPSCs recorded from WT and cKO CA1 pyramidal neurons. n = 21 neurons per group; ***p < 0.001, Student’s t-test; For the cumulative probability curves, n.s. p > 0.05, ****p < 0.0001, Kolmogorov-Smirnov test. (B) Paired-pulse stimuli evoked EPSPs with different interval. n = 9 neurons per group, p > 0.05. The data were analyzed in separate t-tests at each stimulus intensity. (C) AMPAR and NMDAR EPSCs in CA1 pyramidal neurons of WT and cKO mice. Sample sweeps illustrating NMDAR-EPSCs recorded at +40 mV and AMPAR-EPSCs recorded at -70 mV. Histograms show ratio of NMDAR-EPSCs amplitude / AMPAR-EPSCs amplitude (right). n = 10 neurons, *p < 0.05. Student’s t-test. (D) Sample traces and input-output curve of NMDA receptor-mediated EPSCs between WT and cKO mice (n = 6 neurons, p > 0.05). The data were analyzed in separate t-tests at each stimulus intensity. (E) Sample traces and input-output curve of AMPA receptor-mediated EPSCs between WT and cKO mice (n = 6 neurons, *p < 0.05). The data were analyzed in separate t-tests at each stimulus intensity. (F) Cell-surface biotinylation shows a significant increase in GluA1 and GluA2 subunit surface expression in cKO hippocampus compared with WT. Histograms show percent change of GluA1, GluA2, GluN1, GluN2A, GluN2B subunits in total and biotinylation fraction. Flotillin was the internal control. n = 7 experiment repeats, *p < 0.05, **p < 0.01, Student’s t-test. (G) Example fEPSP before (baseline) and after (t = 60 min) 1× HFS stimulation in WT and cKO CA1 slices. Time course of percentage changes of fEPSP slope in WT and cKO CA1 slices. WT, n = 9 slices from 7 mice; cKO, n = 7 slices from 6 mice. Histogram shows fEPSP slope of WT and cKO neurons (55–60 min). *p < 0.05, Student’s t-test. (H) Example fEPSP before (baseline) and after (t = 135 min) 4× HFS stimulation in WT and cKO CA1 slices. Time course of percentage changes of fEPSP slope in WT and cKO CA1 slices. WT, n = 4 slices from 4 mice; cKO, n = 5 slices from 4 mice. Histogram shows fEPSP slope of WT and cKO neurons (130–135 min). *p < 0.05, Student’s t-test. (I) Time course of percentage changes of fEPSP slope before (baseline) and after low frequency stimulus (LFS) in control and cKO CA1 slices. WT, n = 7 slices from 5 mice; cKO, n = 7 slices from 6 mice. Histogram shows fEPSP slope of WT and cKO neurons (55–60 min). p = 0.7642, Student’s t-test. Figure 5—source data 1.Values for mEPSCs of WT and cKO neurons.Values for PPR ratio; Values for input-output curve of AMPAR and NMDAR mediated EPSCs between WT and cKO neurons; Values for NMDAR and AMPAR subunit surface expression; Values for percentage changes of fEPSCs amplitudes in WT and cKO neurons. Figure 5—source data 2.Original files of blots with the relevant bands.

Fig 5: Recombinant ADAMTS 13 blocked tPA neurotoxicity by reducing exogenous tPA/NR2B interactions and the activation of NR2B.(A) Representative immunoblots and quantitative determinations of the levels of NR1, NR2A, and NR2B in mice treated with vehicle, tPA, tPA in combination with rADAMTS 13, and rADAMTS 13 24 hours after MCA occlusion. The gels have been run under the same experimental conditions. Values are mean ± SD (n = 5 per group). *P < 0.05. (B) Interaction between tPA and NR2B detected by immunoprecipitation analysis in ischemic brain of mice treated with vehicle, tPA, rADAMTS 13 in combination with tPA, and rADAMTS 13 24 hours after MCA occlusion. IP, immunoprecipitation; WB, Western blotting. Input, protein of the extracts without IP. The gels have been run under the same experimental conditions. Data are representative of three independent experiments. (C) Representative photographs of TTC stained coronal section and quantitative analysis of infarct volume in wild-type, ADMTS13-/- mice and ADMTS13-/- mice treated with tPA. Values are mean ± SD (n = 8 per group). *P < 0.05. (D) Representative photographs of TTC stained coronal section and quantitative analysis of infarct volume in mice treated with vehicle, tPA, tPA + rADAMTS 13, tPA + ifenprodil, and tPA + rADAMTS 13 + ifenprodil 48 hours after MCA occlusion. Values are mean ± SD (n = 8 per group). *P < 0.05.