Fig 2: The percentage of monasters in monastrol-arrested cells is the same between cells transfected with wild-type NMDAR and phosphomimetic mutants (mE).a Representative images of cells arrested as monasters after 4 h treatment with monastrol. Scale bar: 5 µm. b Quantification of percentage of monasters with respect to the total number of cells. There is no significant difference between wild-type NMDAR-subunits (NR1wt and NR2Awt), phosphomimetic mutants (NR1mE and NR2AmE) and alanine mutants (NR1mA and NR2AmA) transfected cells. Each symbol represents a field of view of 100 cells from three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparisons tests; data are presented as mean ± standard deviation with no significance.

Fig 3: NMDA receptors are phosphorylated during mitosis.a A sequence alignment of all wild-type rat NMDAR-subunits showing a consensus site of phosphorylation by CDK. The consensus site of phosphorylation is highlighted in purple with the Serine that is phosphorylated by CDK highlighted in dark purple and underlined with a pink line. The amino acids of helix 2 (M2) and part of helix 1 (M1) are highlighted in blue. The alignment was performed with the Clustal W 2.0.12 software. b Topological diagram of NMDAR; cylinders represent alpha-helices and the purple line the intracellular loop that connects M1–M2 helices. The pink dotted lines indicate the residues that form the M1–M2 loop of the wild-type NR1 (top) and NR2A (bottom) subunits. Numbers before and after the sequences denote the position number in the whole sequence. Putative phosphorylation sites for CDK are serines S584 for NR1wt and S580 for NR2Awt (dark purple and underlined with a pink line). c) Western blots of NR1wt subunit (left) and NR2Awt subunit (right) immunoprecipitated from lysates of asynchronous-HEK293 cultures (AS) or arrested-cells in mitosis (M). First, immunoprecipitates were analyzed with the anti-GFP antibody to reveal total protein (NR1wt and NR2Awt), then, the blot was stripped and re-revealed with an anti-MPM2 antibody to detect total phosphorylated protein. Full scans of these blots are shown in Supplementary Fig. 1. d Western blots of immunoprecipitated from lysates of arrested-cells in mitosis and transfected with NMDAR-subunits mutated to prevent phosphorylation (alanine mutant, mA) and wild-type NMDAR-subunits (wt). Cells expressed combinations of NR1wt/NR2Awt, NR1mA/NR2Awt, and NR1wt/NR2AmA. The left blot shows total protein and the right blot shows phosphorylated protein.

Fig 4: Increased mEPSC amplitude and levels of excitatory postsynaptic receptors in model rats. (A) Diagram showing model generation and patch clamp/western blot analysis. (B) Representative mEPSC (left) and mIPSC (right) traces recorded in the ACC. (C) Statistical analysis indicating significantly increased amplitude but not frequency of mEPSCs in model rat ACC (frequency: Control, 1.92 ± 0.06 Hz, PP, 1.71 ± 0.124 Hz, p = 0.136; amplitude: Control, 12.26 ± 0.48 pA, PP, 14.22 ± 0.55 pA, p = 0.012; Control, n = 21 cells of five rats; PP, n = 18 cells of five rats). (D) Identical mIPSC frequency and amplitude in control and model ACC (frequency: Control, 2.00 ± 0.11 Hz, PP, 1.95 ± 0.11 Hz, p = 0.20; amplitude: Control,13.06 ± 0.59 pA, PP, 11.67 ± 0.55 pA, p = 0.09; Control, n = 22 cells of five rats; PP, n = 22 cells of five rats). (E) Example of immunoblots of ACC extracts probed with anti-GluR1, GluR2, GluR4, NR1, and NR2B antibodies and quantification of the immunoblots revealing significant increases in levels of GluR1 (p = 0.014) and NR1 (p = 0.024), but not GluR2, GluR4 and NR2B. (F) Representative mEPSC (left) and mIPSC (right) traces in the HIPP. (G) Statistical analysis showing significantly increased mEPSC amplitude, but not frequency, in model rats (frequency: Control, 1.36 ± 0.12 Hz, p = 0.23; PP, 1.31 ± 0.15 Hz, p = 0.23; amplitude: Control, 10.89 ± 0.78 pA, PP, 12.67 ± 0.84 pA, p = 0.02; Control, n = 21 cells of five rats; PP, n = 20 cells of five rats). (H) Identical mIPSC frequency and amplitude in HIPP of control and model rats (frequency: Control, 0.98 ± 0.14, PP, 0.76 ± 0.18, p = 0.43; amplitude: Control, 10.71 ± 0.53, PP, 9.82 ± 0.59, p = 0.36; Control, n = 22 cells of 5 rats; PP, n = 22 cells of five rats). (I) Example of immunoblots probed with anti-GluR1, GluR2, GluR4, NR1, and NR2B antibodies and quantification of the immunoblots revealing a significant increase in levels of NR1 (p = 0.029). Values represent mean ± SEM. One-way ANOVA was used for mEPSCs and mIPSCs, and two-sample t-test for western blotting. *p < 0.05, ***p < 0.001.

Fig 5: Schematic diagram of the mechanism of cognitive impairment in 8-month-old APP/PS1 mice. In hippocampal CA1 area of APP/PS1 mice, impaired function of NMDAR, reduced expression of synaptic proteins, abnormal neuronal morphology, and decreased dendritic spine density may give rise to weak synaptic plasticity, which mediates age-related cognitive dysfunction.