GLYX-13 Ameliorates Schizophrenia-Like Phenotype Induced by MK-801 in Mice: Role of Hippocampal NR2B and DISC1

Abstract

Background: Evidence supports that the hypofunction of N-methyl-D-aspartate receptor (NMDAR) and downregulation of disrupted-in-schizophrenia 1 (DISC1) contribute to the pathophysiology of schizophrenia. N-Methyl D-aspartate receptor subtype 2B (NR2B)-containing NMDAR are associated with cognitive dysfunction in schizophrenia. GLYX-13 is an NMDAR glycine-site functional partial agonist and cognitive enhancer that does not induce psychotomimetic side effects. However, it remains unclear whether NR2B plays a critical role in the GLYX-13-induced alleviation of schizophrenia-like behaviors in mice. Methods: The effect of GLYX-13 was tested by observing changes in locomotor activity, novel object recognition ability, and prepulse inhibition (PPI) induced by dizocilpine (known as MK-801) in mice. Lentivirus-mediated NR2B knockdown in the hippocampus was assessed to confirm the role of NR2B in GLYX-13 pathophysiology, using Western blots and immunohistochemistry. Results: The systemic administration of GLYX-13 (0.5 and 1 mg/kg, i.p.) ameliorates MK-801 (0.5 mg/kg, i.p.)-induced hyperlocomotion, deficits in memory, and PPI in mice. Additionally, GLYX-13 normalized the MK-801-induced alterations in signaling molecules, including NR2B and DISC1 in the hippocampus. Furthermore, we found that NR2B knockdown produced memory and PPI deficits without any changes in locomotor activity. Notably, DISC1 levels significantly decreased by NR2B knockdown. However, the effective dose of GLYX-13 did not alleviate the memory and PPI dysfunctions or downregulation of DISC1 induced by NR2B knockdown. Conclusion: Our results suggest GLYX-13 as a candidate for schizophrenia treatment, and NR2B and DISC1 in the hippocampus may account for the molecular mechanisms of GLYX-13.

Keywords: GLYX-13; N-methyl D-aspartate receptor subtype 2B; N-methyl-D-aspartate receptor; disrupted-in-schizophrenia 1; schizophrenia.

FIGURE 1

Experimental time line and effects of GLYX-13 on MK-801-induced hyperactivity in the open-field test (OFT). (A) Mice received daily administration of vehicle 1, MK-801 (0.5 mg/kg, i.p.), for 14 days. Vehicle 2, GLYX-13 (0.01, 0.5, and 1 mg/kg, i.v.), treatment began on the 7th day after the start of MK-801 administration and continued until the end of the treatment. Twenty-four hours after the last drug treatment animals completed the OFT, novel object recognition task (NORT), and prepulse inhibition (PPI) test between day 1 and day 4. Immediately after the PPI, the hippocampus was removed and processed to assess immunohistochemical changes in NR2B and DISC1 expression, and changes in protein expression by western blotting. (B,C) After 14 days of treatment with MK-801, hyperactivity was induced in mice and compared to the control (vehicle 1 and vehicle 2) groups. Horizontal locomotor activity was examined as the distance traveled (cm) in 30 min. The distances traveled in 5 min intervals (B) and the total distance traveled in 30 min (C) are shown. Data represent the mean ± SEM (n = 12 per group; ^∗∗∗P < 0.001, versus the vehicle 1 + vehicle 2-treated group; ^#P < 0.05 and ^###P < 0.001, versus the MK-801 + vehicle 2-treated group).

FIGURE 2

Effects of GLYX-13 on MK-801-induced object recognition deficits in mice through NORT. The recognition index of the familiar objects and the discrimination index of the novel object are shown. Data represent the mean ± SEM (n = 12 per group; ^∗∗∗P < 0.001, versus the vehicle 1 + vehicle 2-treated group; ^###P < 0.001, versus the MK-801 + vehicle 2-treated group).

FIGURE 3

GLYX-13 significantly attenuated MK-801-induced impairments in PPI but not the startle response. (A) MK-801 significantly increased the startle amplitude. (B) Effect of GLYX-13 on PPI inhibition of the startle response at different prepulses (4, 8, and 12 dB above background) presented before the startle pulse intensity (120 dB). Data represent the mean ± SEM (n = 12 per group; ^∗∗∗P < 0.001, versus the vehicle 1 + vehicle 2-treated group; ^#P < 0.05 and ^###P < 0.001, versus the MK-801 + vehicle 2-treated group).

FIGURE 4

GLYX-13 restored the decrease in NR2B and DISC1 expressions in the dentate gyrus. (A) Confocal microscopy images show double staining for NR2B (red) or DISC1 (green) and merged images in hippocampal slices. Scale bar = 200 μm. (B) Quantitative analyses of NR2B and DISC1-immunopositive cells in the DG. (C,D) Representative images of immunoblots using antibodies against NR2B and DISC1 along with quantitative analyses. Data are expressed as the mean ± SEM (n = 6 per group for immunofluorescent analysis and n = 3 per group for western blotting; ^∗∗∗P < 0.001 compared with the vehicle 1 + vehicle 2 group; ^###P < 0.001 compared with the MK-801 + vehicle 2 group).

FIGURE 5

Lentivirus-mediated NR2B knock-down in the dentate gyrus. (A) Experimental procedure for the test schedule. NC siRNA or NR2B siRNA were microinjected into the hippocampus following a 13-day acclimatization. (B) NC siRNA or NR2B siRNA were well-expressed in the hippocampal DG regions as indicated by the merged images, which include nuclear staining with DAPI (blue) and green fluorescent protein (GFP, green) observed under a fluorescence microscope. Scale bars = 200 μm. (C,D) The expression of NR2B was significantly decreased by NR2B siRNA in the hippocampus of mice. Data are expressed as means ± SEM (n = 3; ^∗∗P < 0.01 compared with NC siRNA group).

FIGURE 6

GLYX-13 did not change the locomotor activity NR2B siRNA knockdown mice. (A) Experimental time line. (B) Horizontal locomotor activity was examined as the distance traveled (cm) in 30 min. The distances traveled in 5 min intervals (B) and the total distance traveled in 30 min (C) are shown. Data are expressed as means ± SEM (n = 12).

FIGURE 7

GLYX-13 did not reverse the object recognition deficits induced by NR2B siRNA in the mice during the NORT. The recognition index of the familiar objects and the discrimination index for the novel object are shown. Data represent the mean ± SEM (n = 12 per group; ^∗∗∗P < 0.001, versus the NC siRNA + vehicle-treated group).

FIGURE 8

GLYX-13 did not attenuate the NR2B siRNA-induced impairments in PPI of mice. (A) NR2B siRNA significantly increased the startle amplitude. (B) Effect of GLYX-13 on PPI inhibition induced by NR2B siRNA in mice. Data represent the mean ± SEM (n = 12 per group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001, versus the NC siRNA + vehicle-treated group).

FIGURE 9

GLYX-13 did not reverse the downregulation of DISC1 induced by NR2B siRNA in mice. (A,B) Representative images of immunoblots using antibodies against NR2B and DISC1 along with quantitative analyses. Data are expressed as the mean ± SEM (n = 4 per group for western blotting; ^∗∗P < 0.01 and ^∗∗∗P < 0.001 compared with the NC siRNA + vehicle-treated group).