Fig 2: HIP1R knockdown reduces expression of NMDARs, AMPARs and PSD95, but not GABAA. Cultured hippocampal neurons were infected with HIP1R-shRNA or Scrambled lentivirus at DIV6 and analyzed by western blotting at DIV13. (A) A representative western blot for total GluN2A, GluN2B, GluA1 and GABAA-a1 expression. (B) Quantification of blots from repeated independent experiments showed that the total expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control with 0.52 ± 0.11, n = 7 vs. 1.02 ± 0.18, n = 7 for GluN2A; 0.46 ± 0.07, n = 6 vs. CTL, 1.00 ± 0.20, n = 6 for GluN2B; and 0.52 ± 0.06, n = 6 vs. 1.00 ± 0.08, n = 6 for GluA1, respectively. (C) A representative western blot for surface GluN2A, GluN2B, GluA1 and GABAA-a1 expression. (D) The ratio of surface to total expression levels of GluN2A, GluN2B, GluA1 and GABAA-a1 did not differ between the HIP1R-KD group and the control. (E) Surface expression levels of GluN2A, GluN2B and GluA1 were significantly reduced in the HIP1R-KD group compared to the control, when normalized for GABAA-a1. There was no significant change in the total and surface expression levels of GABAA-a1 in the HIP1R-KD group. The ratio of HIP1R-KD vs. the control: 0.47 ± 0.05, n = 4 for GluN2A, 0.66 ± 0.06, n = 3 for GluN2B; 0.36 ± 0.01, n = 5 for GluA1 vs. 1.00 ± 0.05, n = 5 for GABAA-a1, respectively. (F) Representative images of dendrites immunostained using anti-PSD95 antibody at DIV16 in cultured hippocampal neurons transfected at DIV6 with HIP1R-shRNA and Scrambled vectors, respectively. (G) Quantification showed a significant decrease in the cluster density of PSD95 in the HIP1R-KD group compared to the control with 0.12 ± 0.01, n = 55 vs. 0.48 ± 0.02, n = 55, respectively. (H) Representative western blots for PSD95 of lysates from cultured hippocampal neurons at DIV13 6 days after infection with HIP1R-shRNA or Scrambled lentivirus. (I) Quantification of western blots from three independent experiments showed decreased expression of PSD95 in the HIP1R-KD group compared to the control with 0.68 ± 0.06, n = 3 vs. 1.00 ± 0.07, n = 3, respectively. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, #P < 0.0001. Uncropped images of blots are shown in Supplementary Figure S1.5A,C,H.

Fig 3: In vitro phosphorylation of NMDA receptors using a purified recombinant Cyclin B1/CDK1 complex.a Cartoon illustrating the NMDA subunit combinations purified from cells in interphase. The cylinders represent the subunits of the NMDAR. The triangles simulate the phosphorus given by the cyclin B1/CDK1 complex (green circle/yellow square). The subunits with the alanine mutant (mA) are not phosphorylated by the complex. c, d Purified NR1wt/NR2Awt, NR1mA/NR2Awt and NR1wt/NR2AmA were incubated with a purified recombinant CyclinB1/CDK1 complex (see “Materials and methods” section) for an in vitro phosphorylation assay. b Blots using the MPM2 antibody. NR1wt blot shows in lanes 1–2 replicas of NR1wt from the combination NR1wt/NR2Awt; and lanes 3–4 replicas of NR1wt from combination NR1wt/NR2mA. NR1mA blot shows in lanes 1–4 replicas from the NR2A purified from the combination NR1mA/NR2Awt. NR2Awt blot shows in lanes 1–2 replicas of NR1wt from the combination NR1wt/NR2Awt; and lanes 3–4 replicas from the NR2A purified from the combination NR1mA/NR2Awt. Finally, NR2AmA blot shows in lanes 1–4 replicas from the NR2A purified from the combination NR1wt/NR2AmA. c Blots show in the same order the total protein content identified by Coomassie staining. d Blots show the total protein (left) and western blot using the MPM2 antibody (right) from the combination NR1wt/NR2Awt which was not incubated with the phosphorylation cocktail containing the recombinant purified Cyclin B1/CDK1 complex. Lanes 1–2 replicas from NR1wt and lanes 3–4 replicas from NR2Awt. This was used to assess the background phosphorylation of NMDAR purified from cells in interphase.

Fig 4: Calcium entry via NMDA receptors is reduced in mitotic astrocytes and mitotic HEK293 cells.a Representative traces of cytosolic calcium measurements in rat astrocytes for 300 s using Fura Red. Pink trace: a mitotic astrocyte. Blue trace: an astrocyte in interphase. The upper bars indicate the time when the glutamate, glycine and calcium ion were applied. b Summary plot of area under the curve (arbitrary units, AU) from the calcium addition to astrocytes comparing interphase (n = 32 cells) and mitotic astrocytes (n = 31 cells). c Confocal images of an interphase astrocyte (above) and a mitotic astrocyte (below). Left: DNA stain with Syto-59. Right: bright field (BF). d Representative traces of cytosolic calcium measurements in HEK293 cells transfected with wild-type NR1 and NR2A subunits of NMDAR. e Summary plot of area under the curve from the calcium addition to HEK293 cells comparing interphase (n = 19 cells) and mitotic HEK293 cells (n = 6 cells). f Confocal images of HEK293 cells. NMDAR-subunits were identified by the florescence of GFP. The scale bar on all confocal images is 5 µm. Data represent mean ± standard deviation with significance set at ***p < 0.0001, analyzed by two-tailed Student’s t-test in b and e.

Fig 5: Intraperitoneal injection of O-1602 reverses forced swimming-induced anxiety-like behavior. a In the EPM, administration of O-1602 increased the number of entries in the open arms and the time into the open arms did not change significantly as compared to the vehicle group. CID16020046 and O-1602 decreased the frequency in the open arms and had no significant changes on the duration in the open arms as compared to the O-1602 group. *p < 0.05, **p < 0.01 versus control group; ## p < 0.01 versus vehicle group; && p < 0.01 versus O-1602 group. b In the OFT, administration of O-1602 significantly increased the time spent in the central area and had no effect on the total distance traveled. CID16020046 and O-1602 treatment decreased the time spent in the central area, while it had no significant effect on the total distance traveled. *p < 0.05 versus control group; ## p < 0.01 vehicle group; && p < 0.01 versus O-1602 group. c Administration of O-1602 reversed stress-induced expression of GluA1 and GluN2A, but not GluN2B. CID16020046 and O-1602 treatment abolished O-1602-mediated decrease in GluA1 and GluN2A expression, while had no effect on the expression of GluN2B. **p < 0.01 versus control group; ## p < 0.01 versus vehicle group; && p < 0.01 versus O-1602 group. Each group contains 6–8 mice. Data are from three independent experiments