Regulation of N-methyl-D-aspartate receptors by disrupted-in-schizophrenia-1

Abstract

Background: Genetic studies have implicated disrupted-in-schizophrenia-1 (DISC1) as a risk factor for a wide range of mental conditions, including schizophrenia. Because N-methyl-D-aspartate receptor (NMDAR) dysfunction has been strongly linked to the pathophysiology of these conditions, we examined whether the NMDAR is a potential target of DISC1.

Methods: DISC1 was knocked down with a small inference RNA. NMDAR-mediated currents were recorded and NMDAR expression was measured.

Results: We found that cellular knockdown of DISC1 significantly increased NMDAR currents in cortical cultures, which were accompanied by an increase in the expression of NMDAR subunit, GluN2A. NMDAR-mediated synaptic response in prefrontal cortical pyramidal neurons was also increased by DISC1 knockdown in vivo. The effect of DISC1 knockdown on NMDAR currents in cortical cultures was blocked by protein kinase A (PKA) inhibitor, occluded by PKA activator, and prevented by phosphodiesterase 4 inhibitor. Knockdown of DISC1 caused a significant increase of cyclic adenosine monophosphate response element-binding protein (CREB) activity. Inhibiting CREB prevented the DISC1 deficiency-induced increase of NMDAR currents and GluN2A clusters.

Conclusions: Our results suggest that DISC1 exerts an important impact on NMDAR expression and function through a phosphodiesterase 4/PKA/CREB-dependent mechanism, which provides a potential molecular basis for the role of DISC1 in influencing NMDAR-dependent cognitive and emotional processes.

Keywords: CREB; DISC1; GluN2A; NMDA receptors; PKA; schizophrenia.

Fig. 1

Knockdown of DISC1 increases NMDAR current density and synaptic response in vitro. A, B, Immunoblots and quantification analysis of DISC1 (A: anti-DISC1 440; B: anti-rDISC1 C-term) in rat cortical cultures infected with control shRNA or DISC1 shRNA lentivirus. *: p < 0.01. C, Representative NMDA (100 μM)-elicited current traces in cultured cortical neurons transfected with control shRNA, DISC1 shRNA or DISC1 shRNA plus DISC1-FL^R, a full-length DISC1 rescue construct that is insensitive to the DISC1 shRNA. Scale bar: 100 pA, 1 s. D, Cumulative data (mean ± SEM) of NMDAR current density, charge transfer and decay time constant in neuronal cultures with different transfections. *: p < 0.01. #: p < 0.05. E, Representative NMDAR current traces in the absence or presence of ifenprodil (10 μM, a GluN2B blocker) in cultured cortical neurons transfected with control shRNA or DISC1 shRNA. Scale bar: 100 pA, 1 s. F, Cumulative data (mean ± SEM) of GluN2A and GluN2B components in neuronal cultures with different transfections. *: p < 0.01.

Fig. 2

Knockdown of DISC1 increases NMDAR-EPSC in PFC pyramidal neurons in vivo. A, Immunoblots and quantification analysis of DISC1 (detected with anti-DISC1 440) in rat PFC slices taken from animals with stereotaxical injections of control shRNA or DISC1 shRNA lentivirus. #: p < 0.05. B, Dot plots showing the amplitude of NMDAR-EPSC in control shRNA or DISC1 shRNA lentivirus-infected PFC pyramidal neurons. The average data are also shown. *: p < 0.01. Inset: representative NMDAR-EPSC traces. Scale bar: 100 pA, 200 ms. C, Summarized input-output curves of NMDAR-EPSC in pyramidal neurons from rats with the PFC injection of control shRNA or DISC1 shRNA lentivirus. *: p < 0.01, #: p < 0.05. D, Representative NMDAR-EPSC traces in the absence or presence of ifenprodil (10 μM) in PFC pyramidal neurons infected with control shRNA or DISC1 shRNA lentivirus. Scale bar: 50 pA, 100 ms. E, Cumulative data (mean ± SEM) of total NMDAR-EPSC amplitude, GluN2A or GluN2B components, decay time constant and charge transfer in neurons with different viral infections. #: p < 0.05.

Fig. 3

Knockdown of DISC1 increases NMDAR subunit GluN2A expression. A, Immunoblots and quantification analysis of GluN1, GluN2A, GluN2B, GABA_AR β2/3 and actin in cultured cortical neurons infected with control shRNA or DISC1 shRNA lentivirus. *: p < 0.01. B, Immunoblots and quantification analysis of surface GluN1, GluN2A, and GluN2B in cultured cortical neurons infected with control shRNA or DISC1 shRNA lentivirus. Actin was used as a control. *: p < 0.05. C, Immunocytochemical images of NMDAR GluN2A subunits and MAP2 in cortical cultures transfected with a control shRNA or DISC1 shRNA. D, Quantitative analysis of GluN2A clusters (density, intensity, size) along the dendrites in control shRNA or DISC1 shRNA-transfected neurons. *: p < 0.01, #: p < 0.05. E, F, Immunocytochemical images (E) and quantitative analysis (F) of synaptic GluN2A clusters (synaptophysin co-localized, yellow puncta) in cortical cultures transfected with control shRNA or DISC1 shRNA. *: p < 0.01.

Fig. 4

PKA activation is required for DISC1 regulation of NMDARs. A, Representative whole-cell NMDAR current traces in cultured cortical neurons transfected with a control shRNA and DISC1 shRNA in the absence or presence of PKI (0.2 μM, a PKA inhibitor), 8-cpt-cAMP (50 μM, a PKA activator), rolipram (0.1 μM, a PDE4 inhibitor), and Bisindolylmaleimide I (Bis I, 0.5 μM, a PKC inhibitor). Scale bar: 100 pA, 1 s. B, Cumulative data (mean ± SEM) of NMDAR current density in transfected neurons with different treatments. *: p < 0.05.

Fig. 5

DISC1 knockdown induces an increase of CREB activity. A, Immunoblots and quantification showing the level of p-CREB in cortical cultures infected with control shRNA or DISC1 shRNA lentivirus. Tubulin was used as a control. *: p < 0.01. B, Immunocytochemical images and quantitative analysis of p-CREB in PFC cultures transfected with a control shRNA or DISC1 shRNA. MAP2 was co-stained. *: p < 0.01. C, D, Immunoblots and quantification showing the level of p-CREB and CREB in cortical cultures infected with control shRNA or DISC1 shRNA lentivirus in the presence of vehicle, PKI (0.2 μM) or PD98059 (20 μM, an ERK inhibitor). *: p < 0.001.

Fig. 6

Inhibiting CREB blocks DISC1 regulation of NMDA receptors. A, Western blots in HEK293 cells transfected with FLAG-tagged CREB in the absence or presence of a control siRNA or CREB siRNA. B, Representative whole-cell NMDAR current traces in cultured cortical neurons transfected with a control shRNA or DISC1 shRNA in the absence or presence of CREB siRNA or DN-CREB. Scale bar: 100 pA, 1 s. C, Cumulative data (mean ± SEM) showing NMDAR current density in neurons with different transfections. *: p < 0.05. D, Immunocytochemical images of GluN2A subunits and MAP2 in cortical cultures transfected with a control shRNA or co-transfected DN-CREB with DISC1 shRNA. E, Quantitative analysis of GluN2A clusters (density, intensity, size) along the dendrites in transfected neurons.