NMDA receptor hypofunction for schizophrenia revisited: Perspectives from epigenetic mechanisms.

Schizophrenia (SZ) is a neurodevelopmental disorder with cognitive deficits manifesting during early stages of the disease. Evidence suggests that genetic factors in combination with environmental insults lead to complex changes to glutamatergic, GABAergic, and dopaminergic systems. In particular, the N-methyl-d-aspartate receptor (NMDAR), a major glutamate receptor subtype, is implicated in both the disease progression and symptoms of SZ. NMDARs are critical for synaptic plasticity and cortical maturation, as well as learning and memory processes. In fact, any deviation from normal NMDAR expression and function can have devastating consequences. Surprisingly, there is little evidence from human patients that direct mutations of NMDAR genes contribute to SZ. One intriguing hypothesis is that epigenetic changes, which could result from early insults, alter protein expression and contribute to the NMDAR hypofunction found in SZ. Epigenetics is referred to as modifications that alter gene transcription without changing the DNA sequence itself. In this review, we first discuss how epigenetic changes to NMDAR genes could contribute to NMDAR hypofunction. We then explore how NMDAR hypofunction may contribute to epigenetic changes in other proteins or genes that lead to synaptic dysfunction and symptoms in SZ. We argue that NMDAR hypofunction occurs in early stage of the disease, and it may consequentially initiate GABA and dopamine deficits. Therefore, targeting NMDAR dysfunction during the early stages would be a promising avenue for prevention and therapeutic intervention of cognitive and social deficits that remain untreatable. Finally, we discuss potential questions regarding the epigenetic of SZ and future directions for research.

Juvenile treatment with mGluR2/3 agonist prevents schizophrenia-like phenotypes in adult by acting through GSK3ß.

Prodromal memory deficits represent an important marker for the development of schizophrenia (SZ), in which glutamatergic hypofunction occurs in the prefrontal cortex (PFC). The mGluR2/3 agonist LY379268 (LY37) attenuates excitatory N-methyl-D-aspartate receptor (NMDAR)-induced neurotoxicity, a central pathological characteristic of glutamatergic hypofunction. We therefore hypothesized that early treatment with LY37 would rescue cognitive deficits and confer benefits for SZ-like behaviors in adults. To test this, we assessed whether early intervention with LY37 would improve learning outcomes in the Morris Water Maze for rats prenatally exposed to methylazoxymethanol acetate (MAM), a neurodevelopmental SZ model. We found that a medium dose of LY37 prevents learning deficits in MAM rats. These effects were mediated through postsynaptic mGluR2/3 via improving GluN2B-NMDAR function by inhibiting glycogen synthase kinase-3ß (GSK3ß). Furthermore, dendritic spine loss and learning and memory deficits observed in adult MAM rats were restored by juvenile LY37 treatment, which did not change prefrontal neuronal excitability and glutamatergic synaptic transmission in adult normal rats. Our results provide a mechanism for mGluR2/3 agonists against NMDAR hypofunction, which may prove to be beneficial in the prophylactic treatment of SZ.

Epigenetic mechanisms underlying NMDA receptor hypofunction in the prefrontal cortex of juvenile animals in the MAM model for schizophrenia.

Schizophrenia (SCZ) is characterized not only by psychosis, but also by working memory and executive functioning deficiencies, processes that rely on the prefrontal cortex (PFC). Because these cognitive impairments emerge prior to psychosis onset, we investigated synaptic function during development in the neurodevelopmental methylazoxymethanol (MAM) model for SCZ. Specifically, we hypothesize that N-methyl-D-aspartate receptor (NMDAR) hypofunction is attributable to reductions in the NR2B subunit through aberrant epigenetic regulation of gene expression, resulting in deficient synaptic physiology and PFC-dependent cognitive dysfunction, a hallmark of SCZ. Using western blot and whole-cell patch-clamp electrophysiology, we found that the levels of synaptic NR2B protein are significantly decreased in juvenile MAM animals, and the function of NMDARs is substantially compromised. Both NMDA-mEPSCs and synaptic NMDA-eEPSCs are significantly reduced in prelimbic PFC (plPFC). This protein loss during the juvenile period is correlated with an aberrant increase in enrichment of the epigenetic transcriptional repressor RE1-silencing transcription factor (REST) and the repressive histone marker H3K27me3 at the Grin2b promoter, as assayed by ChIP-quantitative polymerase chain reaction. Glutamate hypofunction has been a prominent hypothesis in the understanding of SCZ pathology; however, little attention has been given to the NMDAR system in the developing PFC in models for SCZ. Our work is the first to confirm that NMDAR hypofunction is a feature of early postnatal development, with epigenetic hyper-repression of the Grin2b promoter being a contributing factor. The selective loss of NR2B protein and subsequent synaptic dysfunction weakens plPFC function during development and may underlie early cognitive impairments in SCZ models and patients. Read the Editorial Highlight for this article on page 264.

Sigma receptor type 1 knockout mice show a mild deficit in plasticity but no significant change in synaptic transmission in the CA1 region of the hippocampus.

The sigma-1 receptor (σ-1R) is a chaperone protein located at the endoplasmic reticulum (ER) mitochondrial interface with roles in neuroprotection and cognition. Increasing evidence suggests that loss of σ-1R function could contribute to neurological disease states making it a target for therapeutic intervention. Our objective was to elucidate the consequences to synaptic transmission and plasticity when σ-1R is absent. We utilized a knockout mouse in which the gene encoding for σ-1R was deleted (σ-1R-KO mouse). Using whole-cell patch-clamp recordings from CA1 pyramidal neurons in the hippocampus, we examined neuronal excitability and glutamatergic synaptic function. Surprisingly, we detected no significant change in action potential firing and basic cellular characteristics. Furthermore, we found no significant change to pre-synaptic function as indicated by a similar paired-pulse ratio and miniature excitatory post-synaptic current frequency in σ-1R-KO compared to wild-type (WT) mice. Similarly, the glutamate gated AMPA receptor and NMDA receptors were unaffected with no significant difference in AMPA/NMDA ratio or decay kinetics in σ-1R-KO compared to WT mice. We further examined long-term potentiation in extracellular field recordings in CA1 stratum radiatum following Schaffer collateral stimulation. Interestingly, we found a small but significant reduction in the magnitude of long-term potentiation in mutant compared to WT mice. The results of this investigation suggest that basic cellular physiology is unaffected by σ-1R loss, however the neuronal network is partially compromised. The sigma-1 receptor (σ-1R) is a chaperone protein with roles in neuroprotection and cognition. We determined the consequences to synaptic transmission and plasticity when σ-1R was absent. Utilizing the σ-1R knockout mouse and electrophysiological recordings, we found no change in neuronal excitability and glutamatergic synaptic function. However, we found a significant reduction in long-term potentiation.

NMDA hypofunction as a convergence point for progression and symptoms of schizophrenia.

Schizophrenia is a disabling mental illness that is now recognized as a neurodevelopmental disorder. It is likely that genetic risk factors interact with environmental perturbations to affect normal brain development and that this altered trajectory results in a combination of positive, negative, and cognitive symptoms. Although the exact pathophysiology of schizophrenia is unknown, the N-methyl-D-aspartate receptor (NMDAR), a major glutamate receptor subtype, has received great attention. Proper expression and regulation of NMDARs in the brain is critical for learning and memory processes as well as cortical plasticity and maturation. Evidence from both animal models and human studies implicates a dysfunction of NMDARs both in disease progression and symptoms of schizophrenia. Furthermore, mutations in many of the known genetic risk factors for schizophrenia suggest that NMDAR hypofunction is a convergence point for schizophrenia. In this review, we discuss how disrupted NMDAR function leads to altered neurodevelopment that may contribute to the progression and development of symptoms for schizophrenia, particularly cognitive deficits. We review the shared signaling pathways among the schizophrenia susceptibility genes DISC1, neuregulin1, and dysbindin, focusing on the AKT/GSK3ß pathway, and how their mutations and interactions can lead to NMDAR dysfunction during development. Additionally, we explore what open questions remain and suggest where schizophrenia research needs to move in order to provide mechanistic insight into the cause of NMDAR dysfunction, as well as generate possible new avenues for therapeutic intervention.

Group II metabotropic glutamate receptor agonist LY379268 regulates AMPA receptor trafficking in prefrontal cortical neurons.

Group II metabotropic glutamate receptor (mGluR) agonists have emerged as potential treatment drugs for schizophrenia and other neurological disorders, whereas the mechanisms involved remain elusive. Here we examined the effects of LY379268 (LY37) on the expression and trafficking of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluA1 and GluA2 in prefrontal neurons. We show that LY37 significantly increased the surface and total expression of both GluA1 and GluA2 subunits in cultured prefrontal neurons and in vivo. This effect was mimicked by the selective mGluR2 agonist LY395756 and was blocked by mGluR2/3 antagonist LY341495. Moreover, we found that both GluA1 and GluA2 subunits were colocalized with PSD95 but not synapsin I, suggesting a postsynaptic localization. Consistently, treatment with LY37 significantly increased the amplitude, but not frequency, of miniature excitatory postsynaptic currents. Further, actinomycin-D blocked LY37's effects, suggesting a transcriptional regulation. In addition, application of glycogen synthase kinase-3beta (GSK-3ß) inhibitor completely blocked LY37's effect on GluA2 surface expression, whereas GSK-3ß inhibitor itself induced decreases in the surface and total protein levels of GluA1, but not GluA2 subunits. This suggests that GSK-3ß differentially mediates GluA1 and GluA2 trafficking. Further, LY37 significantly increased the phosphorylation, but not total protein, of extracellular signal-regulated kinase 1/2 (ERK1/2). Neither ERK1/2 inhibitor PD98059 alone nor PD98059 combined with LY37 treatment induced changes in GluA1 or GluA2 surface expression or total protein levels. Our data thus suggest that mGluR2/3 agonist regulates postsynaptic AMPA receptors by affecting the synaptic trafficking of both GluA1 and GluA2 subunits and that the regulation is likely through ERK1/2 signaling in GluA1 and/or both ERK1/2 and GSK-3ß signaling pathways in the GluA2 subunit.

Gestational methylazoxymethanol exposure leads to NMDAR dysfunction in hippocampus during early development and lasting deficits in learning.

The N-methyl-D-aspartate (NMDA) receptor has long been associated with learning and memory processes as well as diseased states, particularly in schizophrenia (SZ). Additionally, SZ is increasingly recognized as a neurodevelopmental disorder with cognitive impairments often preceding the onset of psychosis. However, the cause of these cognitive deficits and what initiates the pathological process is unknown. Growing evidence has implicated the glutamate system and, in particular, N-methyl-D-aspartate receptor (NMDAR) dysfunction in the pathophysiology of SZ. Yet, the vast majority of SZ-related research has focused on NMDAR function in adults leaving the role of NMDARs during development uncharacterized. We used the prenatal methylazoxymethanol acetate (MAM, E17) exposure model to determine the alterations of NMDAR protein levels and function, as well as associated cognitive deficits during development. We found that MAM-exposed animals have significantly altered NMDAR protein levels and function in the juvenile and adolescent hippocampus. Furthermore, these changes are associated with learning and memory deficits in the Morris Water Maze. Thus, in the prenatal MAM-exposure SZ model, NMDAR expression and function is altered during the critical period of hippocampal development. These changes may be involved in disease initiation and cognitive impairment in the early stage of SZ.

Group II metabotropic glutamate receptor agonist ameliorates MK801-induced dysfunction of NMDA receptors via the Akt/GSK-3ß pathway in adult rat prefrontal cortex.

Pharmacological intervention targeting mGluRs has emerged as a potential treatment for schizophrenia, whereas the mechanisms involved remain elusive. We explored the antipsychotic effects of an mGluR2/3 agonist in the MK-801 model of schizophrenia in the rat prefrontal cortex. We found that the mGluR2/3 agonist LY379268 effectively recovered the disrupted expression of NMDA receptors induced by MK-801 administration. This effect was attributable to the direct regulatory action of LY379268 on NMDA receptors via activation of the Akt/GSK-3ß signaling pathway. As occurs with the antipsychotic drug clozapine, acute treatment with LY379268 significantly increased the expression and phosphorylation of NMDA receptors, as well as Akt and GSK-3ß. Physiologically, LY379268 significantly enhanced NMDA-induced current in prefrontal neurons and a GSK-3ß inhibitor occluded this effect. In contrast to the widely proposed mechanism of modulating presynaptic glutamate release, our results strongly argue that mGluR2/3 agonists modulate the function of NMDA receptors through postsynaptic actions and reverse the MK-801-induced NMDA dysfunction via the Akt/GSK-3ß pathway. This study provides novel evidence for postsynaptic mechanisms of mGluR2/3 in regulation of NMDA receptors and presents useful insights into the mechanistic actions of mGluR2/3 agonists as potential antipsychotic agents for treating schizophrenia.

Estradiol potentiation of NR2B-dependent EPSCs is not due to changes in NR2B protein expression or phosphorylation.

The hormone, 17ß-estradiol (E2), influences the structure and function of synapses in the CA1 region of the hippocampus. E2 increases the density of dendritic spines and excitatory synapses on CA1 pyramidal cells, increases CA1 cells' sensitivity to excitatory synaptic input mediated by the NMDA receptor (NMDAR), enhances NMDAR-dependent long-term potentiation, and improves hippocampus-dependent working memory. Smith and McMahon (2006 J Neurosci 26:8517-8522) reported that the larger NMDAR-mediated excitatory postsynaptic currents (EPSCs) recorded after E2 treatment are due primarily to an increased contribution of NR2B-containing NMDARs. We used a combination of electrophysiology, Western blot, and immunofluorescence to investigate two potential mechanisms by which E2 could enhance NR2B-dependent EPSCs: An increase in NMDAR subunit protein levels and/or a change(s) in NR2B phosphorylation. Our studies confirmed the E2-induced increase in NR2B-dependent EPSC amplitude, but we found no evidence that E2 affects protein levels for the NR1, NR2A, or NR2B subunit of the NMDAR, nor that E2 affects phosphorylation of NR2B. Our findings suggest that the effects of E2 on NMDAR-dependent synaptic physiology in the hippocampus likely result from recruitment of NR2B-containing NMDARs to synapses rather than from increased expression of NMDARs or changes in their phosphorylation state.

Multiple ERbeta antisera label in ERbeta knockout and null mouse tissues.

In the process of characterizing a custom-made affinity-purified antiserum for estrogen receptor beta (ERbeta), ck5912, we used a number of common tests for specificity of ck5912 along with that of 8 commercially available ERbeta antisera: Affinity Bioreagents PA1-310B, Invitrogen D7N, Upstate 06-629, Santa Cruz H150, Y19, L20, 1531, and Abcam 9.88. We tested their recognition of recombinant ERbeta (rERbeta) versus rERalpha, ERbeta versus ERalpha transfected into cell lines, as well as labeling in wildtype (WT) versus estrogen receptor beta knockout (betaERKO) and null (ERbeta(ST)(L-/L-)) mouse ovary, hypothalamus, and hippocampus. To our surprise, we found that while most of these antisera passed some tests, giving the initial impression of specificity, western blot analysis showed that all of them recognized apparently identical protein bands in WT, betaERKO and ERbeta(ST)(L-/L-) tissues. We share these results with the goal of helping other researchers avoid pitfalls in interpretation that could come from use of these ERbeta antisera.

Estrogen mobilizes a subset of estrogen receptor-alpha-immunoreactive vesicles in inhibitory presynaptic boutons in hippocampal CA1.

Although the classical mechanism of estrogen action involves activation of nuclear transcription factor receptors, estrogen also has acute effects on neuronal signaling that occur too rapidly to involve gene expression. These rapid effects are likely to be mediated by extranuclear estrogen receptors associated with the plasma membrane and/or cytoplasmic organelles. Here we used a combination of serial-section electron microscopic immunocytochemistry, immunofluorescence, and Western blotting to show that estrogen receptor-alpha is associated with clusters of vesicles in perisomatic inhibitory boutons in hippocampal CA1 and that estrogen treatment mobilizes these vesicle clusters toward synapses. Estrogen receptor-alpha is present in approximately one-third of perisomatic inhibitory boutons, and specifically in those that express cholecystokinin, not parvalbumin. We also found a high degree of extranuclear estrogen receptor-alpha colocalization with neuropeptide Y. Our results suggest a novel mode of estrogen action in which a subset of vesicles within a specific population of inhibitory boutons responds directly to estrogen by moving toward synapses. The mobilization of these vesicles may influence acute effects of estrogen mediated by estrogen receptor-alpha signaling at inhibitory synapses.

The effects of deleting the mouse neurotensin receptor NTR1 on central and peripheral responses to neurotensin.

Mice deficient in the neurotensin (NT)-1 receptor (NTR1) were developed to characterize the NT receptor subtypes that mediate various in vivo responses to NT. F2 generation (C57BL6/Sv129J) NTR1 knockout (-/-) mice were viable, and showed normal growth and overt behavior. The -/- mice lacked detectable NTR1 radioligand binding in brain, whereas NTR2 receptor binding density appeared normal compared with wild-type (+/+) mice. The gene deletion also resulted in the loss of NTR1 expression as determined by reverse transcription-polymerase chain reaction and in situ hybridization. Intracerebroventricular injection of NT (1 microg) to +/+ mice caused a robust hypothermic response (5-6 degrees C) and a significant increase in hot-plate latency. These effects were absent in the -/- mice. Similar results were obtained with i.p. injections of the brain-penetrant NT analog NMe-Arg-Lys-Pro-Trp-Tle-Leu (NT-2, 1 mg/kg i.p.). NT-2 administration also impaired rotarod performance in wild-type mice, but had no effect on motor coordination in knockout mice. In vitro, NT and NT-2 at 30 nM caused predominantly contraction and relaxation in isolated distal colon and proximal ileum, respectively, from +/+ mice, but no responses were observed with tissues from -/- mice. A similar loss of the contractile effects of NT was observed in the isolated stomach fundus from the knockout mice. In vivo, NT-2 administration reduced colonic propulsion substantially in wild-type mice. In contrast, NT-2 had no effect in NTR1 null mice, whereas the hypomotility effect of clonidine was intact. These data indicate that NTR1 mediates several of the central and peripheral effects of NT.