Zhenglin Gu

Regulation of NMDA Receptors by Neuregulin Signaling in Prefrontal Cortex

Recent linkage studies have identified a significant association of the neuregulin gene with schizophrenia, but how neuregulin is involved in schizophrenia is primarily unknown. Aberrant NMDA receptor functions have been implicated in the pathophysiology of schizophrenia. Therefore, we hypothesize that neuregulin, which is present in glutamatergic synaptic vesicles, may affect NMDA receptor functions via actions on its ErbB receptors enriched in postsynaptic densities, hence participating in emotional regulation and cognitive processes that are impaired in schizophrenia. To test this, we examined the regulation of NMDA receptor currents by neuregulin signaling pathways in prefrontal cortex (PFC), a prominent area affected in schizophrenia. We found that bath perfusion of neuregulin significantly reduced whole-cell NMDA receptor currents in acutely isolated and cultured PFC pyramidal neurons and decreased NMDA receptor-mediated EPSCs in PFC slices. The effect of neuregulin was mainly blocked by application of the ErbB receptor tyrosine kinase inhibitor, phospholipase C (PLC) inhibitor, IP3 receptor (IP3R) antagonist, or Ca2+ chelators. The neuregulin regulation of NMDA receptor currents was also markedly attenuated in cultured neurons transfected with mutant forms of Ras or a dominant-negative form of MEK1 (mitogen-activated protein kinase kinase 1). Moreover, the neuregulin effect was prevented by agents that stabilize or disrupt actin polymerization but not by agents that interfere with microtubule assembly. Furthermore, neuregulin treatment increased the abundance of internalized NMDA receptors in cultured PFC neurons, which was also sensitive to agents affecting actin cytoskeleton. Together, our study suggests that both PLC/IP3R/Ca2+ and Ras/MEK/ERK (extracellular signal-regulated kinase) signaling pathways are involved in the neuregulin-induced reduction of NMDA receptor currents, which is likely through enhancing NR1 internalization via an actin-dependent mechanism.

Serotonin 5-HT1A Receptors Regulate NMDA Receptor Channels through a Microtubule-Dependent Mechanism

The serotonin system and NMDA receptors (NMDARs) in prefrontal cortex (PFC) are both critically involved in the regulation of cognition and emotion under normal and pathological conditions; however, the interactions between them are essentially unknown. Here we show that serotonin, by activating 5-HT1A receptors, inhibited NMDA receptor-mediated ionic and synaptic currents in PFC pyramidal neurons, and the NR2B subunit-containing NMDA receptor is the primary target of 5-HT1A receptors. This effect of 5-HT1A receptors was blocked by agents that interfere with microtubule assembly, as well as by cellular knock-down of the kinesin motor protein KIF17 (kinesin superfamily member 17), which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Inhibition of either CaMKII (calcium/calmodulin-dependent kinase II) or MEK/ERK (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) abolished the 5-HT1A modulation of NMDAR currents. Biochemical evidence also indicates that 5-HT1A activation reduced microtubule stability, which was abolished by CaMKII or MEK inhibitors. Moreover, immunocytochemical studies show that 5-HT1A activation decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Together, these results suggest that serotonin suppresses NMDAR function through a mechanism dependent on microtubule/kinesin-based dendritic transport of NMDA receptors that is regulated by CaMKII and ERK signaling pathways. The 5-HT1A-NMDAR interaction provides a potential mechanism underlying the role of serotonin in controlling emotional and cognitive processes subserved by PFC.

Regulation of NMDA receptors by dopamine D4 signaling in prefrontal cortex.

Increasing evidence has suggested that the interaction between dopaminergic and glutamatergic systems in prefrontal cortex (PFC) plays an important role in normal mental functions and neuropsychiatric disorders. In this study, we examined the regulation of NMDA-type glutamate receptors by the PFC dopamine D4 receptor (one of the principal targets of antipsychotic drugs). Application of the D4 receptor agonist PD168077 caused a reversible decrease of the NMDA receptor (NMDAR)-mediated current in acutely isolated and cultured PFC pyramidal neurons, an effect that was blocked by selective D4 receptor antagonists. Furthermore, application of PD168077 produced a potent reduction of the amplitude (but not paired-pulse ratio) of evoked NMDAR EPSCs in PFC slices. The D4 modulation of NMDA receptors in PFC involved the inhibition of protein kinase A, activation of protein phosphatase 1 and the ensuing inhibition of active Ca2+–calmodulin-dependent kinase II (CaMKII). Moreover, PD168077 reduced the surface expression of NMDARs and triggered the internalization of NMDARs in a manner dependent on CaMKII activity. These results identify a mechanistic link between D4 and NMDA receptors in PFC pyramidal neurons, suggesting that D4 receptors may play an important role in modulating synaptic plasticity and thus cognitive and emotional processes in PFC circuits.

β-Amyloid Impairs AMPA Receptor Trafficking and Function by Reducing Ca2+/Calmodulin-dependent Protein Kinase II Synaptic Distribution

A fundamental feature of Alzheimer disease (AD) is the accumulation of β-amyloid (Aβ), a peptide generated from the amyloid precursor protein (APP). Emerging evidence suggests that soluble Aβ oligomers adversely affect synaptic function, which leads to cognitive failure associated with AD. The Aβ-induced synaptic dysfunction has been attributed to the synaptic removal of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs); however, it is unclear how Aβ induces the loss of AMPARs at the synapses. In this study we have examined the potential involvement of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a signaling molecule critical for AMPAR trafficking and function. We found that the synaptic pool of CaMKII was significantly decreased in cortical neurons from APP transgenic mice, and the density of CaMKII clusters at synapses was significantly reduced by Aβ oligomer treatment. In parallel, the surface expression of GluR1 subunit as well as AMPAR-mediated synaptic response and ionic current was selectively decreased in APP transgenic mice and Aβ-treated cultures. Moreover, the reducing effect of Aβ on AMPAR current density was mimicked and occluded by knockdown of CaMKII and blocked by overexpression of CaMKII. These results suggest that the Aβ-induced change in CaMKII subcellular distribution may underlie the removal of AMPARs from synaptic membrane by Aβ.

Glycogen Synthase Kinase 3 Regulates N-Methyl-d-aspartate Receptor Channel Trafficking and Function in Cortical Neurons

Emerging evidence has suggested that glycogen synthase kinase 3 (GSK-3) is a key regulatory kinase involved in a plethora of processes in the nervous system, including neuronal development, mood stabilization, and neurodegeneration. However, the cellular mechanisms underlying the actions of GSK-3 remain to be identified. In this study, we examined the impact of GSK-3 on the N-methyl-d-aspartate (NMDA) receptor channel, a central player involved in cognitive and emotional processes. We found that application of various structurally different GSK-3 inhibitors caused a long-lasting reduction of NMDA receptor-mediated ionic and synaptic current in cortical pyramidal neurons. Cellular knockdown of GSK-3β in neuronal cultures with a small interfering RNA led to smaller NMDA receptor current and loss of its regulation by GSK-3 inhibitors. The NR2B subunit-containing NMDA receptor was the primary target of GSK-3, but the GSK-3 modulation of NMDAR current did not involve the motor protein kinesin superfamily member 17-based transport of NR2B-containing vesicles along microtubules. Combined electrophysiological, immunocytochemical, and biochemical evidence indicated that GSK-3 inhibitors induced the down-regulation of NMDAR current through increasing the Rab5-mediated and PSD-95-regulated NMDAR internalization in a clathrin/dynamin-dependent manner.

Serotonin facilitates long‐term depression induction in prefrontal cortex via p38 MAPK/Rab5‐mediated enhancement of AMPA receptor internalization

The serotonin system in prefrontal cortex (PFC) is critically involved in the regulation of cognition and emotion. To understand the cellular mechanisms underlying its physiological actions, we investigated the role of serotonin in regulating synaptic plasticity in PFC circuits. We found that tetanic stimuli coupled to bath application of serotonin induced long‐term depression (LTD) at excitatory synapses of PFC pyramidal neurons. This effect was mediated by 5‐HT2A/C receptors and was independent of NMDA receptor activation. A group I metabotropic glutamate receptor (mGluR) antagonist blocked the LTD induction by serotonin + tetani, and co‐application of a group I mGluR agonist and serotonin, but not application of either drug alone, induced LTD without tetani. The effect of serotonin on LTD was blocked by selective inhibitors of p38 mitogen‐activated protein kinase (MAPK), but not p42/44 MAPK. Biochemical evidence also indicated that serotonin and a group I mGluR agonist synergistically activated p38 MAPK in PFC slices. The serotonin‐facilitated LTD induction was prevented by blocking the activation of the small GTPase Rab5, as well as by blocking the clathrin‐dependent internalization of AMPA receptors with postsynaptic injection of a dynamin inhibitory peptide, while it was unaffected by manipulating the cytoskeleton. Interestingly, in animals exposed to acute stress, the LTD induction by serotonin + tetani was significantly impaired. Taken together, these results suggest that serotonin, by cooperating with mGluRs, regulates synaptic plasticity through a mechanism dependent on p38 MAPK/Rab5‐mediated enhancement of AMPA receptor internalization in a clathrin/dynamin‐dependent manner. It provides a potential mechanism underlying the role of serotonin in controlling emotional and cognitive processes that are mediated by synaptic plasticity in PFC neurons.

RGS4 Modulates Serotonin Signaling in Prefrontal Cortex and Links to Serotonin Dysfunction in a Rat Model of Schizophrenia

Regulator of G protein signaling 4 (RGS4) has recently been identified as one of the genes linked to the susceptibility of schizophrenia. However, the functional roles of RGS4 and how it may be involved in the pathophysiology of schizophrenia remain largely unknown. In this study, we investigated the possible impact of RGS4 on the function of serotonin and dopamine receptors, two main targets for schizophrenia treatment. Activation of serotonin 5-HT1A receptors or dopamine D4 receptors down-regulates the function of NMDA receptor (NMDAR) channel, a key player controlling cognition and emotion, in pyramidal neurons of prefrontal cortex (PFC). Blocking RGS4 function significantly potentiated the 5-HT1A regulation of NMDAR current; conversely, overexpression of RGS4 attenuated the 5-HT1A effect. In contrast, the D4 regulation of NMDAR current was not altered by RGS4 manipulation. Moreover, the 5-HT1A regulation of NMDA receptors was significantly enhanced in a subset of PFC pyramidal neurons from rats treated with subchronic phencyclidine, an animal model of schizophrenia, which was found to be associated with specifically decreased RGS4 expression in these cells. Thus, our study has revealed an important coupling of RGS4 to serotonin signaling in cortical neurons and provided a molecular and cellular mechanism underlying the potential involvement of RGS4 in the pathophysiology of schizophrenia.

Group II metabotropic glutamate receptors enhance NMDA receptor currents via a protein kinase C-dependent mechanism in pyramidal neurones of rat prefrontal cortex

The action of glutamate in CNS is mediated by the activation of metabotropic and ionotropic receptors. The metabotropic glutamate receptors (mGluRs) are highly enriched in prefrontal cortex (PFC) – a brain region critically involved in the regulation of cognition and emotion. Emerging evidence has suggested that mGluRs are viable drug targets for neuropsychiatric disorders associated with PFC dysfunction. However, the mGluR‐mediated signalling in PFC remains unclear. To understand the physiological functions of postsynaptic group II mGluRs (mGluR2/3) in PFC neurones, we investigated the molecular and cellular mechanisms underlying the regulation of NMDA receptor channels by group II mGluRs. We found that APDC, a highly selective and potent group II mGluR agonist, reversibly increased NMDAR currents in acutely dissociated PFC pyramidal neurones. Selective group II mGluR antagonists, but not group I mGluR antagonists, blocked APDC‐induced enhancement of NMDAR currents, suggesting the mediation by mGluR2/3 receptors. The APDC effect on NMDAR currents was independent of Mg2+ block or membrane voltages, and primarily targeted NR2A subunits containing NMDARs. While changing protein kinase A levels was without effect, inhibiting protein kinase C (PKC) or dialysis with Ca2+ chelators largely blocked the mGluR2/3 modulation of NMDAR currents. In contrast, inhibiting protein tyrosine kinases, cyclin‐dependent kinase 5, Ca2+/calmodulin‐dependent kinase II or the Ca2+/calmodulin‐dependent phosphatase calcineurin failed to do so. Moreover, treatment of PFC slices with APDC significantly increased the PKC activity and PKC phosphorylation of NMDA receptors. These findings suggest that activation of mGluR2/3 receptors potentiates NMDAR channel functions in PFC through a PKC‐dependent mechanism. This modulation may be relevant for developing novel mGluR‐related pharmacological agents for the treatment of mental illnesses.

Calpain regulation of AMPA receptor channels in cortical pyramidal neurons

AMPA receptors (AMPARs) are the principal glutamate receptors mediating fast excitatory synaptic transmission in neurons. Aberrant extracellular glutamate has long been recognized as a hallmark phenomenon during neuronal excitotoxicity. Excessive glutamate triggers massive Ca2+ influx through NMDA receptors (NMDARs), which in turn can activate Ca2+‐dependent protease, calpain. In the present study, we found that prolonged NMDA treatment (100 μm, 10 min) caused a sustained and irreversible suppression of AMPAR‐mediated currents in cortical pyramidal neurons, which was largely blocked by selective calpain inhibitors. Biochemical and immunocytochemical studies demonstrated that in cortical cultures, prolonged glutamate or NMDA treatment reduced the level of surface and total GluR1, but not GluR2, subunits in a calpain‐dependent manner. Consistent with the in vitro data, in animals exposed to transient ischaemic insults, calpain was strongly activated, and the AMPAR current density and GluR1 expression level were substantially reduced. Moreover, calpain inhibitors blocked the ischaemia‐induced depression of AMPAR currents, and the NMDAR‐induced, calpain‐mediated depression of AMPA responses was occluded in ischaemic animals. Taken together, our studies show that overstimulation of NMDARs reduces AMPAR functions in cortical pyramidal neurons through activation of endogenous calpain, and calpain mediates the ischaemia‐induced synaptic depression. The down‐regulation of AMPARs by calpain provides a negative feedback to dampen neuronal excitability in excitotoxic conditions like ischaemia and epilepsy.

Regulation of N-Methyl-d-aspartic Acid (NMDA) Receptors by Metabotropic Glutamate Receptor 7

Background: The metabotropic glutamate receptors (mGluRs) are potential novel targets for mental disorders. Results: Activation of mGluR7 significantly reduced NMDAR-mediated currents and NMDAR surface expression via an actin-dependent mechanism. Conclusion: mGluR7, by affecting the cofilin/actin signaling, regulates NMDAR trafficking and function. Significance: It provides a potential mechanism for understanding the role of mGluR7 in mental health and disorders. Emerging evidence suggests that metabotropic glutamate receptors (mGluRs) are potential novel targets for brain disorders associated with the dysfunction of prefrontal cortex (PFC), a region critical for cognitive and emotional processes. Because N-methyl-d-aspartic acid receptor (NMDAR) dysregulation has been strongly associated with the pathophysiology of mental illnesses, we examined the possibility that mGluRs might be involved in modulating PFC functions by targeting postsynaptic NMDARs. We found that application of prototypical group III mGluR agonists significantly reduced NMDAR-mediated synaptic and ionic currents in PFC pyramidal neurons, which was mediated by mGluR7 localized at postsynaptic neurons and involved the β-arrestin/ERK signaling pathway. The mGluR7 modulation of NMDAR currents was prevented by agents perturbing actin dynamics and by the inhibitor of cofilin, a major actin-depolymerizing factor. Consistently, biochemical and immunocytochemical results demonstrated that mGluR7 activation increased cofilin activity and F-actin depolymerization via an ERK-dependent mechanism. Furthermore, mGluR7 reduced the association of NMDARs with the scaffolding protein PSD-95 and the surface level of NMDARs in an actin-dependent manner. These data suggest that mGluR7, by affecting the cofilin/actin signaling, regulates NMDAR trafficking and function. Because ablation of mGluR7 leads to a variety of behavioral symptoms related to PFC dysfunction, such as impaired working memory and reduced anxiety and depression, our results provide a potential mechanism for understanding the role of mGluR7 in mental health and disorders.