Upregulation Of N-methyl-D-aspartate Receptors Research Articles

Upregulated NMDAR-mediated GABAergic transmission underlies autistic-like deficits in Htr3a knockout mice.

Mutations in serotonin pathway genes, especially the serotonergic receptor subunit gene HTR3A, are associated with autism. However, the association of HTR3A deficiency with autism and the underlying mechanisms remain unknown.Methods: The Htr3a knockout (KO) mice were generated using transcription activator-like effector nuclease technology. Various behavior tests, including social interaction, social approach task, olfactory habituation/dishabituation, self-grooming, novel object recognition, contextual fear conditioning, elevated plus maze, open field and seizure susceptibility, were performed to assess the phenotypes. Transcriptome sequencing was carried out to search for molecular network and pathways underlying the phenotypes. Electrophysiological recordings, immunoblotting, immunofluorescence staining, immunoprecipitation, and quantitative real-time PCR were performed to verify the potential mechanisms. The N-methyl-D-aspartate receptor (NMDAR) antagonist memantine was used to treat the KO mice for rescuing the phenotypes.Results: The Htr3a KO mouse model showed three phenotypic domains: autistic-like behaviors (including impaired social behavior, cognitive deficits, and increased repetitive self-grooming), impaired memory, and attenuated susceptibility to pentylenetetrazol-induced seizures. We observed enhanced action potential-driven γ-aminobutyric acid-ergic (GABAergic) transmission in pyramidal neurons and decreased excitatory/inhibitory (E/I) ratio using the patch-clamp recording. Transcriptome sequencing on the hippocampus revealed the converged pathways of the dysregulated molecular networks underlying three phenotypic domains with upregulation of NMDAR. We speculated that Htr3a KO promotes an increase in GABA release through NMDAR upregulation. The electrophysiological recordings on hippocampal parvalbumin-positive (PV+) interneuron revealed increased NMDAR current and NMDAR-dependent excitability. The NMDAR antagonist memantine could rescue GABAergic transmission in the hippocampus and ameliorate autistic-like behaviors of the KO mice.Conclusion: Our data indicated that upregulation of the NMDAR in PV+ interneurons may play a critical role in regulating GABAergic input to pyramidal neurons and maybe involve in the pathogenesis of autism associated with HTR3A deficiency. Therefore, we suggest that the NMDAR system could be considered potential therapeutic target for autism.

Anxiolytic effects of Formononetin in an inflammatory pain mouse model

Chronic pain is commonly accompanied with anxiety disorder, which complicates treatment. In this study, we investigated the analgesic and anxiolytic effects of Formononetin (FMNT), an active component of traditional Chinese medicine red clover (Trifolium pratense L.) that is capable of protecting neurons from N-methyl-D-aspartate (NMDA)-evoked excitotoxic injury, on mice suffering from complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain. The results show that FMNT administration significantly reduces anxiety-like behavior but does not affect the nociceptive threshold in CFA-injected mice. The treatment reverses the upregulation of NMDA, GluA1, and GABAA receptors, as well as PSD95 and CREB in the basolateral amygdala (BLA). The effects of FMNT on NMDA receptors and CREB binding protein (CBP) were further confirmed by the potential structure combination between these compounds, which was analyzed by in silico docking technology. FMNT also inhibits the activation of the NF-κB signaling pathway and microglia in the BLA of mice suffering from chronic inflammatory pain. Therefore, the anxiolytic effects of FMNT are partially due to the attenuation of inflammation and neuronal hyperexcitability through the inhibition of NMDA receptor and CBP in the BLA.

Memantine Differentially Regulates Tau Phosphorylation Induced by Chronic Restraint Stress of Varying Duration in Mice.

Exposure to chronic psychiatric stress has been linked to Alzheimer's disease-related tau hyperphosphorylation and abnormalities in glutamate neurotransmission. However, the pathological relationship between glutamatergic dysfunction and tau phosphorylation in the cerebral cortex under chronic psychiatric stress is not fully understood. The present study investigated the effects of memantine (MEM, 5 and 10 mg/kg), an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, on chronic restraint stress- (CRS-) induced tau phosphorylation in mice. CRS administered for 16 or 28 consecutive days (1 h daily) induced significant tau phosphorylation in the brain. MEM treatment suppressed the elevation of phosphorylated tau (P-tau) levels induced by 16-day CRS in a dose-dependent manner. P-tau reduction was accompanied by the attenuation of the upregulation of GSK3β and CDK5 expression and the downregulation of PP2A activity induced by CRS. Additionally, MEM reduced CRS-induced upregulation of NMDA receptor subunit levels (GluN2A, GluN2B) in the frontal cortex. However, MEM markedly enhanced tau phosphorylation in the frontal cortex and other cerebral cortical regions following 28 days of CRS. The stimulatory effect of MEM on CRS-induced tau phosphorylation was correlated with increased activities of AKT, JNK, and GSK3β, inactivation of PP2A, and downregulation of Pin1 and HSP70. Moreover, MEM did not effectively reverse the NMDA receptor upregulation induced by 28-day CRS and even increased GluN2B subunit levels. In contrast to the duration-dependent effects of MEM on P-tau levels, MEM produced an anxiolytic effect in both regimens as revealed by elevated plus maze testing. However, MEM did not affect the body weight reduction induced by CRS. Thus, MEM exerts distinctive effects on CRS-induced tau phosphorylation, which might be related to the expression of GluN2B. The differential effects of MEM on P-tau levels have crucial implications for its clinical application.

Up-regulation of GluN2A-containing NMDA receptor protects cultured cortical neuron cells from oxidative stress

Neuronal excitotoxicity induced by spreading depolarization occurs during multiple brain diseases. The subsequent extensive releasing of neuronal transmitter glutamate results in over activation of the ionic glutamate receptors and then triggers neuronal cell death. The N-methyl-D-aspartate (NMDA) receptor is one major type of excitatory ionic glutamate receptors in the central nervous system, and it exerts vital functions on the membrane of neurons. Distinct subtypes of the NMDA receptor play different roles and their expression was dynamically regulated according to both physiological and pathological stimulations. During neuronal excitotoxicity the expression of the GluN2A-containing NMDA receptor is specifically up-regulated, and as a result, the ratio of GluN2A- versus GluN2B-containing NMDA receptors is altered. However the physiological significance of this phenomenon is still not clear. In this research, we specifically inhibited the increase of the GluN2A-containing NMDA receptor by a peptide without affecting the basic expression of both GluN2A- and GluN2B-containing NMDA receptors, and found that the oxidative stress of neurons was intensified, with increased endogenous reactive oxygen species (ROS), loss of mitochondrial membrane potential, and elevated expressions of Bcl-2-associated X protein (Bax) and apoptosis-inducing factor (AIF). Furthermore, the phosphorylation of Akt and ERK were also inhibited. These results indicated that the dynamic expression of the GluN2A-containing NMDA receptor played crucial roles in protecting neurons from excitotoxicity.

Thalidomide alleviates postoperative pain and spatial memory deficit in aged rats

Pain is a major risk factor of post-operative cognitive dysfunction (POCD) in aged population. We investigated the effects of thalidomide, an anti-inflammatory and analgesic drug, on POCD in aged rats, and also explored the underlying mechanisms. Laparotomy was performed under anesthesia in aged rats (24–25 months) to establish POCD models. Thalidomide (5–50mg/kg) was intraperitoneally administered immediately after laparotomy. Within 12h after the operation, pain symptoms were assessed by rat grimace scale (RGS). Within postoperative day (POD) 3–14, spatial memory was evaluated using performance errors in a radial arm maze. Protein levels of inflammatory cytokines and N-methyl-D-aspartate (NMDA) receptors were measured on POD 14. POCD rats treated with thalidomide showed decreased RGS and performance errors, compared with saline-treated POCD rats. Single administration of thalidomide significantly reduced production of cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1β) in serum but not in the brain, and attenuated upregulation of NMDA receptor (NR) 2A/B subunits in the hippocampus at POD 14. MK-801 abolished thalidomide-induced attenuation of spatial memory deficits. Our results support that thalidomide could disrupt the development of post-operative memory deficit in aged rats through its long-term regulation of NMDA receptors (NRs) in the hippocampus. Therefore, thalidomide might provide a new means to prevent the development of POCD.

Compound MQA, a Caffeoylquinic Acid Derivative, Protects Against NMDA‐Induced Neurotoxicity and Potential Mechanisms In Vitro

Compound MQA (1,5-O-dicaffeoyl-3-O-[4-malic acid methyl ester]-quinic acid) is a natural derivative of caffeoylquinic acid isolated from Arctium lappa L. roots. However, we know little about the effects of MQA on the central nervous system. This study aims to investigate the neuroprotective effects and underlying mechanisms of MQA against the neurotoxicity of N-methyl-d-aspartate (NMDA). Pretreatment with MQA attenuated the loss of cell viability after SH-SY5Y cells treated with 1mM NMDA for 30min by MTT assay. Hoechst 33342 and Annexin V-PI double staining showed that MQA inhibited NMDA-induced apoptosis. In addition to preventing Ca(2+) influx, the potential mechanisms are associated with increases in the Bcl-2/Bax ratio, attenuation of cytochrome c release, caspase-3, caspase-9 activities, and expressions. Also, MQA inhibited NMDA-induced phosphorylation of ERK1/2, p38, and JNK1/2. Furthermore, deactivation of CREB, AKT, and GSK-3β, upregulation of GluN2B-containing NMDA receptors (NMDARs), and downregulation of GluN2A-containing NMDARs were significantly reversed by MQA treatment. Computational docking simulation indicates that MQA possesses a well affinity for NMDARs. The protective effects of MQA against NMDA-induced cell injury may be mediated by blocking NMDARs. The potential mechanisms are related with mitochondrial apoptosis, ERK-CREB, AKT/GSK-3β, p38, and JNK1/2 pathway.

All trans retinoic acid (ATRA) mediated modulation of N-methyl D-aspartate receptor (NMDAR) and Kruppel like factor 11 (KLF11) expressions in the mitigation of ethanol induced alterations in the brain

BackgroundDamaging effects that chronic ethanol exposure causes to the brain and the neurons are well documented. Ethanol and its toxic metabolites increase the oxidative stress in brain. Chronic exposure to ethanol leads to upregulation of N-methyl D-aspartate receptors (NMDAR) and also activates Kruppel like factor 11 (KLF11) mediated death cascade and thereby neurodegeneration. ObjectiveEthanol depletes vitamin A stores. But supplementation of vitamin A exacerbates ethanol induced toxicity since alcohol and its metabolites are competitive inhibitors of the enzymes involved in the metabolism of vitamin A. Hence, in this study we investigated the impact of co-administration of ethanol and all trans retinoic acid (ATRA), active metabolite of vitamin A, on ethanol induced alterations to the brain. Materials and methodsMale Sprague Dawley rats, adolescent, were grouped as follows and maintained for 90 days. I – Control, II – Ethanol (4 g/kg b.w.), III – ATRA (100 µg/kg b.w.), IV – Ethanol (4 g/kg b.w.), +ATRA (100 µg/kg b.w.). Oxidative stress and the mRNA expression of various receptors for the neurotransmitter involved in glutamergic, serotonergic and gabaergic pathways were studied in the brain homogenate. ResultsEthanol treatment was shown to decrease brain weight and it was increased on ATRA treatment. Increase in oxidative stress due to ethanol treatment was also brought down on ATRA administration. Ethanol induced upregulation of NMDAR and KLF11 was also downregulated on ATRA supplementation. The alterations in the levels of neurotransmitters and the expression of their receptors due to ethanol treatment also were ameliorated on ATRA supplementation. ConclusionOur results show that ATRA supplementation mitigates the ethanol induced alterations in the brain by reducing oxidative stress in the brain with concurrent suppression of NMDAR and KLF11 expression leading to enhanced catabolism of neurotransmitters.

Neuroprotective effects of daphnetin against NMDA receptor-mediated excitotoxicity.

The accumulation of glutamate can excessively activate the N-methyl-d-aspartate (NMDA) receptors and cause excitotoxicity. Daphnetin (Dap), a coumarin derivative, is a protein kinase inhibitor that exhibits antioxidant and neuroprotective properties. However, little is known about the neuroprotective effects of Dap on glutamate-induced excitotoxicity. We evaluated the neuroprotective activities in the primary cultured cortical neurons against NMDA-induced excitotoxicity. Pretreatment with Dap significantly prevented NMDA-induced neuronal cell loss. Dap significantly inhibited the neuronal apoptosis by regulating balance of Bcl-2 and Bax expression. Furthermore, pretreatment of Dap reversed the up-regulation of NR2B-containing NMDA receptors and inhibited the intracellular Ca2+ overload induced by NMDA exposure. In addition, Dap prevented cerebral ischemic injury in mice induced via a 2 h middle cerebral artery occlusion and a 24 h reperfusion in vivo. The findings suggest that Dap prevents the excitotoxicity through inhibiting the NR2B-containing NMDA receptors and the subsequent calcium overload in cultured cortical neurons.

Role of glutamate transporter 1 in the attenuation of alcohol intake.

Role of glutamate transporter 1 in the attenuation of alcohol intake.

HIF‐1α increases sympathoexcitation via upregulation of NMDA receptors in the PVN during heart failure (1130.16)

Increased sympathetic outflow is a prominent feature of chronic heart failure (HF) associated with higher mortality. Multiple studies from our lab have reported that higher glutamatergic tone (increase in N‐methyl‐D‐aspartate (NMDA)‐type 1 (NR1) receptors expression) within the paraventricular nucleus (PVN) increase the sympathetic drive in HF. Further, HF (coronary artery ligation‐induced) augmented the expression of hypoxia‐inducible‐factor (HIF)‐1α both at mRNA (9 fold) and protein (2.5 fold) levels in the PVN which, led us to hypothesized that increased levels of HIF‐1α may contribute to exaggerated sympathetic drive via increasing NR1 expression in the PVN of rats with HF. Neuronal cell line (NG108‐15) exposed to hypoxia (2% O₂, 5% CO₂, 93% N₂) showed 2‐fold increase in HIF‐1α (12h) expression with concomitant increases in NR1 at transcription and protein levels after 4h (6.5 fold) and 12h (40%) respectively. Live cell imaging also showed marked increase in intracellular calcium in response to glutamate under hypoxic conditions. Mechanistically, hypoxia promotes the direct binding of HIF‐1α (3.2 fold) at NR1 promoter in neurons exposed to hypoxia as demonstrated by chromatin immunoprecipitation analysis. Furthermore, silencing of HIF‐1α by using HIF‐1α ‐targeted small interfering RNA in neurons led to 2.9 fold decrease in the expression of NR1. Validating our in vitro data, HIF‐1α knockdown by HIF‐1α‐siRNA in the PVN of rats with HF ameliorated the increased sympathoexcitation to NMDA (57±5% vs. 27±1% of basal value). Taken together, our study suggests a contribution of HIF‐1α in the regulation of NR1 expression within the PVN in HF, and provides a novel mechanism for the modulation of sympathoexcitation in HF. Supported by NIH HL62222.Grant Funding Source: NIH HL62222.

Postoperative pain impairs subsequent performance on a spatial memory task via effects on N-methyl-d-aspartate receptor in aged rats

AimsPain may be associated with postoperative cognitive dysfunction (POCD); however, this relationship remains underinvestigated. Therefore, we examined the impact of postoperative pain on cognitive functions in aged animals. Main methodsRats were allocated to the following groups: control (C), 1.2 % isoflurane for 2hours alone (I), I with laparotomy (IL), IL with analgesia using local ropivacaine (IL+R), and IL with analgesia using systemic morphine (IL+M). Pain was assessed by rat grimace scale (RGS). Spatial memory was evaluated using a radial maze from postoperative days (POD) 3 to 14. NMDA receptor (NR) 2 subunits in hippocampus were measured by ELISA. Finally, effects of memantine, a low-affinity uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, on postoperative cognitive performance were tested. Key findingsPostoperative RGS was increased in Group IL, but not in other groups. The number of memory errors in Group I were comparable to that in Group C, whereas errors in Group IL were increased. Importantly, in Group IL+R and IL+M, cognitive impairment was not found. The memory errors were positively correlated with the levels of NMDA receptor 2 subunits in hippocampus. Prophylactic treatment with memantine could prevent the development of memory deficits observed in Group IL without an analgesic effect. SignificancePostoperative pain contributes to the development of memory deficits after anesthesia and surgery via up-regulation of hippocampal NMDA receptors. Our findings suggest that postoperative pain management may be important for the prevention of POCD in elderly patients.

Neuroprotective effects of vitexin by inhibition of NMDA receptors in primary cultures of mouse cerebral cortical neurons

The accumulation of glutamate can excessively activate the N-methyl-D-aspartate (NMDA) receptors and cause excitotoxicity. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside, Vit) is a c-glycosylated flavone which was found in the several herbs, exhibiting potent hypotensive, anti-inflammatory, and neuroprotective properties. However, little is known about the neuroprotective effects of Vit on glutamate-induced excitotoxicity. In present study, primary cultured cortical neurons were treated with NMDA to induce the excitotoxicity. Pretreatment with Vit significantly prevented NMDA-induced neuronal cell loss and reduced the number of apoptotic neurons. Vit significantly inhibited the neuronal apoptosis induced by NMDA exposure by regulating balance of Bcl-2 and Bax expression and the cleavages of poly (ADP-ribose) polymerase and pro-caspase 3. Furthermore, pretreatment of Vit reversed the up-regulation of NR2B-containing NMDA receptors and the intracellular Ca(2+) overload induced by NMDA exposure. The neuroprotective effects of Vit are related to inhibiting the activities of NR2B-containing NMDA receptors and reducing the calcium influx in cultured cortical neurons.

The neuroprotective effect of praeruptorin C against NMDA-induced apoptosis through down-regulating of GluN2B-containing NMDA receptors

Praeruptorin C (Pra-C), one of the principal bioactive components derived from the root of Peucedanum praeruptorum Dunn, has been widely used as an antioxidant and a calcium antagonist to treat diseases. The present study investigated the protective effect of Pra-C on cultured cortical neuron injury induced by glutamate. After challenge with 200μM N-methyl-d-aspartate (NMDA) for 30min, loss of cell viability and excessive apoptotic cell death were observed in cultured cortical neurons. Pra-C conferred protective effects against loss of cellular viability in a concentration-dependent manner. Pra-C also significantly inhibited neuronal apoptosis induced by NMDA exposure by reversing intracellular Ca2+ overload and balancing Bcl-2 and Bax expression. Furthermore, Pra-C significantly reversed the upregulation of GluN2B-containing NMDA receptors by exposure to NMDA but did not affect the expression of GluN2A-containing NMDA receptors. These findings suggest that Pra-C partially protects cortical neurons by inhibiting the expression of GluN2B-containing NMDA receptors and regulating the Bcl-2 family.

Cytisine confers neuronal protection against excitotoxic injury by down-regulating GluN2B-containing NMDA receptors

Cytisine (CYT), one of the principal bioactive components derived from the seeds of Cytisus laborinum L, has been widely used for central nervous system (CNS) diseases treatment. The present study investigated the protective effect of CYT on cultured cortical neural injury induced by N-methyl-d-aspartate (NMDA). Our data showed that CYT conferred protective effect against loss of cellular viability induced by brief exposure to 200μM NMDA in a concentration-dependent manner. CYT significantly inhibited the neuronal apoptosis induced by NMDA exposure by reversing intracellular Ca2+ overload and balancing Bcl-2 and Bax expression levels. Furthermore, CYT significantly reversed the up-regulation of GluN2B-containing NMDA receptors by exposure to NMDA, but it did not affect the level of GluN2A-containing NMDA receptors. These findings suggest that CYT protects cortical neurons, at least partially, by inhibiting the level of GluN2B-containing NMDA receptors and regulating Bcl-2 family.

Neuroprotective Effects of Flax Lignan Against NMDA‐Induced Neurotoxicity In Vitro

Flax Lignan (FLL), a chemical widespread within the plant and animal kingdoms, has antioxidant, antiinfectious, and antitumor activities. However, little is known about the effects of FLL on the central nervous system (CNS). The neuroprotective actions of FLL against N-methyl-d-aspartate (NMDA) are investigated in primary cultured cortical neurons by MTT assay. The expression levels of proteins related to apoptosis and GluN2-containing receptor were detected by Western blot analysis. Intracellular Ca(2+) was measured under a confocal laser scanning microscope. After challenged with 100μM NMDA for 30min, loss of cell viability and excessive apoptotic cell death were observed in cultured cortical neurons. FLL protected the neurons against the NMDA-induced cell loss in a concentration-dependent manner. FLL also significantly inhibited the neuronal apoptosis induced by NMDA exposure through reversing intracellular concentration of Ca(2+) overload and balancing of Bcl-2 and Bax expression. Furthermore, FLL significantly reversed the upregulation of GluN2B-containing NMDA receptors by exposure to NMDA, but did not affect the expression of GluN2A-containing NMDA receptor. These findings suggest that FLL protects cortical neurons by inhibiting the expression of GluN2B-containing NMDA receptor and regulating the Bcl-2 family.

Upregulation of NMDA receptors in Spinal Cord Dorsal Horn Contributes to Diabetic Neuropathic Pain

Diabetic neuropathic pain is a common complication in patients with diabetes. Spinal N‐methyl‐D‐aspartate (NMDA) receptors play critical role in neuropathic pain. In this study, we determined that role of enhanced NMDA receptors function in diabetic neurophathic pain. Diabetic neuropathy was induced by a single injection of streptozotocin (STZ, 50 mg/kg, intraperitoneal). Blood glucose level and paw withdraw threshold (PWT) were measured 3 weeks after STZ treatment to verify diabetic neuropathy. Evoked NMDA excitatory postsynaptic currents (NMDAR‐EPSCs) were recorded in lamina II neurons by using whole‐cell voltage‐clamp patch in rats treated with STZ and vehicle. The blood glucose level was significantly higher in STZ‐treated rats than in vehicle‐treated rats. The PWT in STZ‐treated rats was significantly lower than that in vehicle group. Immunohistochemical staining revealed that the number of NR1 and NR2B‐positive neurons were significantly higher in STZ‐treated rats than in vehicle‐treated rats. Furthermore, the amplitude of evoked NMDA‐EPSCs in spinal neurons was significantly higher in STZ‐treated rats than vehicle‐treated rats. Application of 50 μM AP5 completely blocked the evoked NMDAR‐EPSCs. Taken together, these data suggest that upregulation of NMDA receptors in the spinal cord dorsal horn contributes to the diabetic neuropathic pain

Dysregulated Src upregulation of NMDA receptor activity: a common link in chronic pain and schizophrenia

Upregulation of N-methyl-D-aspartate (NMDA) receptor function by the nonreceptor protein tyrosine kinase Src has been implicated in physiological plasticity at glutamatergic synapses. Here, we highlight recent findings suggesting that aberrant Src upregulation of NMDA receptors may also be key in pathophysiological conditions. Within the nociceptive processing network in the dorsal horn of the spinal cord, pathologically increased Src upregulation of NMDA receptors is critical for pain hypersensitivity in models of chronic inflammatory and neuropathic pain. On the other hand, in the hippocampus and prefrontal cortex, the physiological upregulation of NMDA receptors by Src is blocked by neuregulin 1-ErbB4 signaling, a pathway that is genetically implicated in the positive symptoms of schizophrenia. Thus, either over-upregulation or under-upregulation of NMDA receptors by Src may lead to pathological conditions in the central nervous system. Therefore, normalizing Src upregulation of NMDA receptors may be a novel therapeutic approach for central nervous system disorders, without the deleterious consequences of directly blocking NMDA receptors.

Regulation of NMDA receptors by the tyrosine kinase Fyn

The phosphorylation and trafficking of N-methyl-d-aspartate (NMDA) receptors are tightly regulated by the Src family tyrosine kinase Fyn, through dynamic interactions with various scaffolding proteins in the NMDA receptor complex. Fyn acts as a point of convergence for many signaling pathways that upregulate GluN2B-containing NMDA receptors. In the following review, we focus on Fyn signaling downstream of different G-protein-coupled receptors: the dopamine D1 receptor, and receptors cognate to the pituitary adenylate cyclase-activating polypeptide. The net result of activation of each of these signaling pathways is upregulation of GluN2B-containing NMDA receptors. The NMDA receptor is a major target of ethanol in the brain, and accumulating evidence suggests that Fyn mediates the effects of ethanol by regulating the phosphorylation of GluN2B NMDA receptor subunits. Furthermore, Fyn has been shown to regulate alcohol withdrawal and acute tolerance to ethanol through a GluN2B-dependent mechanism. In addition to its effects on NMDA receptor function, Fyn also modifies the threshold for synaptic plasticity at CA1 synapses, an effect that probably contributes to the effects of Fyn on spatial and contextual fear learning.

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1enu/+ mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1enu/+ mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.

Pilocapine alters NMDA receptor expression and function in hippocampal neurons: NADPH oxidase and ERK1/2 mechanisms

The molecular basis for epileptogenesis remains poorly defined, but repeated or prolonged seizures can cause altered hippocampal N-methyl d-aspartate receptor (NMDAR) stoichiometry, loss of hippocampal neurons, and aberrant mossy fiber sprouting. Using the muscarinic receptor 1 (m1R) agonist, pilocarpine (PILO), in hippocampal cell cultures we explored the early sequence of molecular events that occur within 24h of the initial insult and result in altered neuronal function during epileptogenesis. Our findings show that PILO-induced, m1R-mediated, inositol 1,4,5-trisphosphate (IP3) synthesis constitutes an early, crucial biochemical event required for NMDAR hyperactivation and subsequent NADPH oxidase (NOX) activation and NMDAR-independent ERK1/2 phoshorylation. Together, but not separately, NOX activation and ERK1/2 phosphorylation induce alterations in NMDAR stoichiometry through the upregulation of NR1 and NR2B subunits. Lastly, we demonstrated that PILO-mediated oxidative stress alters NMDAR function through the redox modulation of cysteine residues. The in vitro results related to thiol oxidation, NOX activation, ERK1/2 phosphorylation and NMDAR upregulation were confirmed in vivo, 24h after treatment of adult rats with PILO. These results obtained in PILO-treated primary hippocampal neurons – and confirmed in vivo at the same time-point after PILO – provide a better understanding of the early cellular responses during epileptogenesis and identify potential therapeutic targets to prevent development of chronic epilepsy.