NMDA-induced Toxicity Research Articles

Cytosolic PSD-95 interactor alters functional organization of neural circuits and AMPA receptor signaling independent of PSD-95 binding.

Cytosolic PSD-95 interactor (cypin) regulates many aspects of neuronal development and function, ranging from dendritogenesis to synaptic protein localization. While it is known that removal of postsynaptic density protein-95 (PSD-95) from the postsynaptic density decreases synaptic N-methyl-D-aspartate (NMDA) receptors and that cypin overexpression protects neurons from NMDA-induced toxicity, little is known about cypin’s role in AMPA receptor clustering and function. Experimental work shows that cypin overexpression decreases PSD-95 levels in synaptosomes and the PSD, decreases PSD-95 clusters/μm2, and increases mEPSC frequency. Analysis of microelectrode array (MEA) data demonstrates that cypin or cypinΔPDZ overexpression increases sensitivity to CNQX (cyanquixaline) and AMPA receptor-mediated decreases in spike waveform properties. Network-level analysis of MEA data reveals that cypinΔPDZ overexpression causes networks to be resilient to CNQX-induced changes in local efficiency. Incorporating these findings into a computational model of a neural circuit demonstrates a role for AMPA receptors in cypin-promoted changes to networks and shows that cypin increases firing rate while changing network functional organization, suggesting cypin overexpression facilitates information relay but modifies how information is encoded among brain regions. Our data show that cypin promotes changes to AMPA receptor signaling independent of PSD-95 binding, shaping neural circuits and output to regions beyond the hippocampus.

Development of a Neuroprotective Erythropoietin Modified with a Novel Carrier for the Blood-Brain Barrier.

Extremely high doses of erythropoietin (EPO) has been used for neuroprotection in ischemia-reperfusion brain injury to deliver sufficient amounts of EPO across the blood-brain barrier (BBB); however, harmful outcomes were observed afterward. We aimed to test the ability of HBHAc (heparin-binding haemagglutinin adhesion c), an intracellular delivery peptide for macromolecules, as an EPO carrier across the BBB. The cellular internalization and transcytosis ability of HBHAc-modified EPO (EPO-HBHAc) were evaluated in bEnd.3 cells and in the bEnd.3/CTX TNA2 co-culture BBB model, respectively. Subsequently, the NMDA-induced-toxicity model and ischemia-reperfusion rat model were used to understand the neuronal protective activity of EPO-HBHAc. The biodistribution of EPO-HBHAc was demonstrated in rats by the quantification of EPO-HBHAc in the brain, plasma, and organs by ELISA. Our results demonstrate that EPO-HBHAc exhibited significantly higher cellular internalization in dose- and time-dependent manners and better transcytosis ability than EPO. In addition, the transported EPO-HBHAc in the co-culture transwell system maintained the neuronal protective activity when primary rat cortical neurons underwent NMDA-induced toxicity. The calculated cerebral infarction area of rats treated with EPO-HBHAc was significantly reduced compared to that of rats treated with EPO (29.9 ± 7.0% vs 48.9 ± 7.9%) 24h after occlusion in 3VO rat experiments. Moreover, the EPO amount in both CSF and damaged cortex from the EPO-HBHAc group was 4.0-fold and 3.0-fold higher than the EPO group, respectively. These results suggest that HBHAc would be a favorable tool for EPO brain delivery and would further extend the clinical applications of EPO in neuroprotection.

BACE1 Mediates HIV-Associated and Excitotoxic Neuronal Damage Through an APP-Dependent Mechanism.

HIV-associated neurocognitive disorders (HANDs) share common symptoms with Alzheimer's disease (AD), which is characterized by amyloid-β (Aβ) plaques. Plaques are formed by aggregation of Aβ oligomers, which may be the toxic species in AD pathogenesis, and oligomers are generated by cleavage of amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 inhibitors reverse neuronal loss and cognitive decline in animal models of AD. Although studies have also found evidence of altered APP processing in HIV+ patients, it is unknown whether increased BACE1 expression or Aβ oligomer production is a common neuropathological feature of HAND. Moreover, it is unknown whether BACE1 or APP is involved in the excitotoxic, NMDAR-dependent component of HIV-associated neurotoxicity in vitro Herein, we hypothesize that HIV-associated neurotoxicity is mediated by NMDAR-dependent elevation of BACE1 and subsequent altered processing of APP. Supporting this, we observed elevated levels of BACE1 and Aβ oligomers in CNS of male and female HIV+ patients. In a model of HIV-associated neurotoxicity in which rat neurons are treated with supernatants from HIV-infected human monocyte-derived macrophages, we observed NMDAR-dependent elevation of BACE1 protein. NMDA treatment also increased BACE1 and both pharmacological BACE1 inhibition and genetic loss of APP were partially neuroprotective. Moreover, in APP knock-out (APP-/-) mouse neurons, NMDA-induced toxicity was BACE1 independent, indicating that cytotoxicity of BACE1 is dependent upon APP cleavage. Our findings suggest that increased BACE1 and the resultant Aβ oligomer production may contribute to HIV-associated neuropathogenesis and inhibition of BACE1 could have therapeutic potential in HANDs.SIGNIFICANCE STATEMENT HIV-associated neurocognitive disorders (HANDs) represent a range of cognitive impairments affecting ∼50% of HIV+ individuals. The specific causes of HAND are unknown, but evidence suggests that HIV-infected macrophage infiltration into the brain may cause neuronal damage. Herein, we show that neurons treated with conditioned media from HIV-infected macrophages have increased expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protein implicated in Alzheimer's disease pathogenesis. Moreover, inhibition of BACE1 prevented neuronal loss after conditioned media exposure, but had no effect on HIV-associated neurotoxicity in neurons lacking its cleavage target amyloid precursor protein. We also observed increased BACE1 expression in HIV+ patient brain tissue, confirming the potential relevance of BACE1 as a therapeutic target in HANDs.

A Small Molecule Mimetic of the Humanin Peptide as a Candidate for Modulating NMDA-Induced Neurotoxicity.

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aβ and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is reported to be primarily through its agonist binding properties to the gp130 receptor. However, the peptidic nature of HN presents challenges in its development as a therapeutic for AD. We report here for the first time the elucidation of the binding site of Humanin (HN) peptide to the gp130 receptor extracellular domain through modeling and the synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and provide protection against NMDA-induced neurotoxicity in primary hippocampal neurons. A brain permeable small molecule mimetic was identified through exploratory medicinal chemistry using microfluidic flow chemistry to facilitate the synthesis of new analogues for screening and SAR optimization.

Humanin rescues cultured rat cortical neurons from NMDA-induced toxicity through the alleviation of mitochondrial dysfunction.

N-methyl-D-aspartate (NDMA) receptor-mediated excitotoxicity has been implicated in a variety of pathological situations such as Alzheimer’s disease (AD) and Parkinson’s disease. However, no effective treatments for the same have been developed so far. Humanin (HN) is a 24-amino acid peptide originally cloned from the brain of patients with AD and it prevents stress-induced cell death in many cells/tissues. In our previous study, HN was found to effectively rescue rat cortical neurons. It is still not clear whether HN protects the neurons through the attenuation of mitochondrial dysfunction. In this study, excitatory toxicity was induced by NMDA, which binds the NMDA receptor in primarily cultured rat cortical neurons. We found that NMDA (100 μmol/L) dramatically induced the decrease of cell viability and caused mitochondrial dysfunction. Pretreatment of the neurons with HN (1 μmol/L) led to significant increases of mitochondrial succinate dehydrogenase (SDH) activity and membrane potential. In addition, HN pretreatment significantly reduced the excessive production of both reactive oxygen species (ROS) and nitric oxide (NO). Thus, HN could attenuate the excitotoxicity caused by the overactivation of the NMDA receptor through the alleviation of mitochondrial dysfunction.

In vitro and invivo effects of a novel dimeric inhibitor of PSD-95 on excitotoxicity and functional recovery after experimental traumatic brain injury.

PSD-95 inhibitors have been shown to be neuroprotective in stroke, but have only to a very limited extent been evaluated in the treatment of traumatic brain injury (TBI) that has pathophysiological mechanisms in common with stroke. The aims of the current study were to assess the effects of a novel dimeric inhibitor of PSD-95, UCCB01-147, on histopathology and long-term cognitive outcome after controlled cortical impact (CCI) in rats. As excitotoxic cell death is thought to be a prominent part of the pathophysiology of TBI, we also investigated the neuroprotective effects of UCCB01-147 and related compounds on NMDA-induced cell death in cultured cortical neurons. Anesthetized rats were given a CCI or sham injury, and were randomized to receive an injection of either UCCB01-147 (10mg/kg), the non-competitive NMDAR-receptor antagonist MK-801 (1mg/kg) or saline immediately after injury. At 2 and 4weeks post-trauma, spatial learning and memory were assessed in a water maze, and at 3months, brains were removed for estimation of lesion volumes. Overall, neither treatment with UCCB01-147 nor MK-801 resulted in significant improvements of cognition and histopathology after CCI. Although MK-801 provided robust neuroprotection against NMDA-induced toxicity in cultured cortical neurons, UCCB01-147 failed to reduce cell death and became neurotoxic at high doses. The data suggest potential differential effects of PSD-95 inhibition in stroke and TBI that should be investigated further in future studies taking important experimental factors such as timing of treatment, dosage, and anesthesia into consideration.

Neuroprotection Mediated through GluN2C-Containing N-methyl-D-aspartate (NMDA) Receptors Following Ischemia.

Post-ischemic activation of NMDA receptors (NMDARs) has been linked to NMDAR subunit-specific signaling that mediates pro-survival or pro-death activity. Although extensive studies have been performed to characterize the role of GluN2A and GluN2B following ischemia, there is less understanding regarding the regulation of GluN2C. Here, we show that GluN2C expression is increased in acute hippocampal slices in response to ischemia. Strikingly, GluN2C knockout mice, following global cerebral ischemia, exhibit greater neuronal death in the CA1 area of the hippocampus and reduced spatial working memory compared to wild-type mice. Moreover, we find that GluN2C-expressing hippocampal neurons show marked resistance to NMDA-induced toxicity and reduced calcium influx. Using both in vivo and in vitro experimental models of ischemia, we demonstrate a neuroprotective role of GluN2C, suggesting a mechanism by which GluN2C is upregulated to promote neuronal survival following ischemia. These results may provide insights into development of NMDAR subunit-specific therapeutic strategies to protect neurons from excitotoxicity.

Altered relation between lipopolysaccharide-induced inflammatory response and excitotoxicity in rat organotypic hippocampal slice cultures during ethanol withdrawal.

Ethanol (EtOH) causes neurotoxicity by several mechanisms including excitotoxicity and neuroinflammation, but little is known about the interaction between these mechanisms. Because neuroinflammation is known to enhance excitotoxicity, we hypothesized that neuroinflammation contributes to the enhanced excitotoxicity, which is associated with EtOH withdrawal (EWD). The aim of this study was to evaluate the lipopolysaccharide (LPS)-induced inflammatory response of cultured hippocampal tissue during EWD and its effects on the enhanced N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, which occurs at this time. Using a neonatal organotypic hippocampal slice culture (OHSC) model, we assessed the effects of NMDA and LPS (separately or combined) during EWD after 10days of EtOH exposure. Neurotoxicity was assessed using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (quantified by enzyme-linked immunosorbent assay) and nitric oxide (NO; quantified by the Griess reaction) into culture media. Furthermore, we explored the potential role of the microglial cell type using immortalized BV2 microglia treated with EtOH for 10days and challenged with LPS during EWD. As predicted, NMDA-induced toxicity was potentiated by LPS under control conditions. However, during EWD, the reverse was observed and LPS inhibited peak NMDA-induced toxicity. Additionally, LPS-induced release of TNF-alpha and NO during EWD was reduced compared to control conditions. In BV2 microglia, following EtOH exposure, LPS-induced release of NO was reduced, whereas TNF-alpha release was potentiated. During EWD following chronic EtOH exposure, OHSC exhibited a desensitized inflammatory response to LPS and the effects of LPS on NMDA toxicity were reversed. This might be explained by a change in microglia to an anti-inflammatory and neuroprotective phenotype. In support, studies on BV2 microglia indicate that EtOH exposure and EWD do alter the response of these cells to LPS, but this cannot fully explain the changes observed in the OHSC. The data suggest that neuroinflammation and excitotoxicity do interact during EWD. However, the interaction is not as simple as we originally proposed. This in turn illustrates the need to assess the extent, importance, and relation of these mechanisms in models of EtOH exposure producing neurotoxicity.

Humanin rescues cultured rat cortical neurons from NMDA-induced toxicity not by NMDA receptor.

Excitatory neurotoxicity has been implicated in many pathological situations and there is no effective treatment available. Humanin is a 24-aa peptide cloned from the brain of patients with Alzheimer's disease (AD). In the present study, excitatory toxicity was induced by N-methyl-D-aspartate (NMDA) in primarily cultured rat cortical neurons. MTT assessment, lactate dehydrogenase (LDH) release, and calcein staining were employed to evaluate the protective activity of humanin on NMDA induced toxicity. The results suggested that NMDA (100 μmol/L, 2.5 hr) triggered neuronal morphological changes, lactate dehydrogenase (LDH) release (166% of the control), reduction of cell viability (about 50% of the control), and the decrease of living cell density (about 50% of the control). When pretreated with humanin, the toxicity was suppressed. The living cells' density of humanin treated group was similar to that of control. The cell viability was attenuated dose-dependently (IC50 = 0.132 nmol/L). The LDH release was also neutralized in a dose-dependent manner. In addition, the intracellular Ca2+ overloading triggered by NMDA reverted quickly and humanin could not inhibit it. These findings indicate that humanin can rescue cortical neurons from NMDA-induced toxicity in rat but not through interfering with NMDA receptor directly.

BDNF prevents NMDA‐induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved

NMDA receptor-mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain-derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A2 ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs. In corticostriatal slices from wild-type mice and age-matched symptomatic R6/2 mice (a model of HD), NMDA application (75μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10ng/mL) potentiated NMDA effects in wild-type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A2 AR blockade. The protective effect of BDNF against NMDA-induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A2 AR ligands in HD.

Curcumin Protects against NMDA-Induced Toxicity: A Possible Role for NR2A Subunit

Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.

The Mitochondrial Inner Membrane GTPase, Optic Atrophy 1 (Opa1), Restores Mitochondrial Morphology and Promotes Neuronal Survival following Excitotoxicity

Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.

N‐Methyl‐d‐aspartate receptor subunit‐ and neuronal‐type dependence of excitotoxic signaling through post‐synaptic density 95

NMDA receptors (NMDARs) mediate excitatory synaptic transmission during repetitive or prolonged glutamate release, playing a critical role in synaptic plasticity or cell death, respectively. Evidence indicates that a major pathway of NMDAR signaling to cell death in cortical and hippocampal neurons requires the scaffolding protein post-synaptic density 95 (PSD-95) and activation of neuronal nitric oxide synthase. However, it is not known if this PSD-95-dependent pathway contributes to excitotoxicity in other brain regions. It is also unclear whether the neuroprotective effects of Tat-NR2B9c, a membrane-permeant peptide that disrupts PSD-95/NMDAR binding, correlate with uncoupling NR2B- and/or NR2A-type NMDARs from PSD-95. In this study, we used cultured hippocampal and striatal neurons to test the potency of Tat-NR2B9c on uncoupling NR2 subunits from PSD-95 and protecting against NMDA-induced excitotoxicity. We found that the concentration of Tat-NR2B9c required to dissociate 50% of PSD-95 was fourfold lower for NR2B than NR2A in cultured hippocampal and striatal neurons, and that this concentration correlated tightly with protection against NMDA-induced toxicity in hippocampal neurons without altering NMDAR current. In contrast, NMDAR signaling to cell death in cultured striatal neurons occurred independently of the NR2B/PSD-95 interaction or neuronal nitric oxide synthase activation. These results will facilitate development of neuronal type-specific protective therapies.

Neuroprotective Effect of PACAP Against NMDA-Induced Retinal Damage in the Mouse

Retinal excitotoxicity is one of the major causes of retinal ganglion cell (RGC) death in glaucoma. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide with potent neuroprotective activity; however, whether it exerts such an effect in the retina and the mechanism by which RGCs are protected is still not well understood. In this study, we examined the effect of exogenous and endogenous PACAP on RGC death induced by N-methyl-D: -aspartate acid (NMDA). The vitreous body of anesthetized adult male mice (C57/BL6J) was injected with NMDA (40 nmol in a 2 μL saline solution). The number of RGCs decreased from days 1 to 7 after NMDA injection, and the number of dUTP end-labeling (TUNEL)-positive cells, an indicator of cell death, peaked at day 3. However, when PACAP38 (10(-8), 10(-10), 10(-12), 10(-14), or 10(-16)M) was co-administered with NMDA, the 10(-10)M dose resulted in significantly increased RGC survival at day 7, and a decrease in the number of TUNEL-positive RGCs at day 3. We next investigated the neuroprotective effect of endogenous PACAP using PACAP heterozygote(+/-) mice. Under normal circumstances, there was no significant difference in the number of RGCs in the PACAP(+/-) mice compared with their wild-type counterparts. However, the number of RGCs significantly decreased in the PACAP(+/-) mice 7 days after NMDA injection, relative to their wild-type counterparts. The number of TUNEL-positive RGCs peaked at day 1 in the PACAP(+/-) mice. These effects in the PACAP(+/-) mice were reversed by intravitreous injection of 10(-10)M PACAP38. This suggests that exogenous PACAP is able to counteract NMDA-induced toxicity, and that endogenous PACAP exerts a neuroprotective effect in the retina.

Inhibition of sigma-1 receptor reduces N-methyl- d-aspartate induced neuronal injury in methamphetamine-exposed and -naive hippocampi

Acute and prolonged methamphetamine (METH) exposure has been reported to moderate the function of N-methyl- d-aspartate type glutamate receptors (NMDAr) in the hippocampus. These effects have been found to be associated with enhanced NMDAr-dependent release of Ca 2+ from IP 3-sensitive intracellular stores. The present studies were designed to extend these findings and examine the role of the endoplasmic membrane (ER) bound orphan receptor, the sigma-1 receptor, in NMDA-induced neuronal injury and METH withdrawal-potentiated NMDA-induced neuronal injury. Organotypic hippocampal slice cultures were exposed to METH (0 or 100 μM) for 6 days and withdrawn for 7 days, then exposed to NMDA (0 or 5 μM) for 24 h. Additional cultures were also exposed to this regimen and were co-incubated with BD1047 (100 μM), a specific inhibitor of ER-bound sigma-1 receptors, for the 24 h NMDA exposure. Cytotoxicity was assessed by analysis of propidium iodide uptake. These studies demonstrated that protracted METH exposure and withdrawal significantly potentiated the neuronal injury produced by NMDA exposure. Further, co-exposure to BD1047 with NMDA markedly attenuated neuronal injury in METH-naïve and METH-withdrawn organotypic cultures. As a whole, these data demonstrate that prolonged METH exposure, even at non-toxic concentrations, significantly alters glutamate receptor signaling. Inhibition of sigma-1 receptor-dependent Ca 2+ release from the ER entirely prevented NMDA-induced toxicity in METH-naïve cultures and markedly reduced METH-potentiated toxicity. These findings demonstrate the importance of Ca 2+-induced intracellular Ca 2+ release in excitotoxic insult and suggest that blockade of glutamatergic overactivity may represent a therapeutic target in the treatment of METH withdrawal.

The DREAM Protein Negatively Regulates the NMDA Receptor through Interaction with the NR1 Subunit

Glutamate-induced excitotoxicity has been implicated in the etiology of stroke, epilepsy, and neurodegenerative diseases. NMDA receptors (NMDARs) play a pivotal role in excitotoxic injury; however, clinical trials testing NMDAR antagonists as neuroprotectants have been discouraging. The development of novel neuroprotectant molecules is being vigorously pursued. Here, we report that downstream regulatory element antagonist modulator (DREAM) significantly inhibits surface expression of NMDARs and NMDAR-mediated current. Overexpression of DREAM showed neuroprotection against excitotoxic neuronal injury, whereas knockdown of DREAM enhanced NMDA-induced toxicity. DREAM could directly bind to the C0 domain of the NR1 subunit. Although DREAM contains multiple binding sites for the NR1 subunit, residues 21-40 of the N terminus are the main binding site for the NR1 subunit. Thus, 21-40 residues might relieve the autoinhibition conferred by residues 1-50 and derepress the DREAM core domain by a competitive mechanism. Intriguingly, the cell-permeable TAT-21-40 peptide, constructed according to the critical binding site of DREAM to the NR1 subunit, inhibits NMDAR-mediated currents in primary cultured hippocampal neurons and has a neuroprotective effect on in vitro neuronal excitotoxic injury and in vivo ischemic brain damage. Moreover, both pretreatment and posttreatment of TAT-21-40 is effective against excitotoxicity. In summary, this work reveals a novel, negative regulator of NMDARs and provides an attractive candidate for the treatment of excitotoxicity-related disease.

The use of Organotypic Hippocampal Slice Cultures to Evaluate Protection by Non-competitive NMDA Receptor Antagonists against Excitotoxicity

There is great interest in testing neuroprotectants which inhibit the neurodegeneration that results from excessive activation of N-methyl-D-aspartate (NMDA) receptors. As an alternative to in vivo testing in animal models, we demonstrate here the use of a complex in vitro model to compare the efficacy and toxicity of NMDA receptor inhibitors. Organotypic hippocampal slice cultures were used to compare the effectiveness of the Alzheimer's disease drug, memantine, the Parkinson's disease drug, procyclidine, and the novel neuroprotectant, gacyclidine (GK11), against NMDA-induced toxicity. All three drugs are non-competitive NMDA receptor open-channel blockers that inhibit excitotoxic injury, and their neuroprotective capacities have been extensively investigated in vivo in animal models. They have also been evaluated as potential countermeasure agents against organophosphate poisoning. Quantitative densitometric image analysis of propidium iodide uptake in hippocampal regions CA1, CA3 and DG, showed that, after exposure to 10microM NMDA for 24 hours, GK11 was the most potent of the three drugs, with an IC50 of about 50nM and complete protection at 250nM. When applied at high doses, GK11 was still the more potent neuroprotectant, and also the least cytotoxic. These findings are consistent with those from in vivo tests in rodents. We conclude that the slice culture model provides valuable pre-clinical data, and that applying the model to the screening of neuroprotectants might significantly limit the use of in vivo tests in animals.

Influence of CGS 21680, a selective adenosine A 2A receptor agonist, on NMDA receptor function and expression in the brain of Huntington's disease mice

The effect of chronic treatment with the selective adenosine A 2A receptor agonist CGS 21680 on N-Methyl- d-Aspartate (NMDA) receptor function and expression has been studied in the striatum and cortex of R6/2 mice, a genetic mouse model of Huntington's disease (HD). Starting from 8 weeks of age, R6/2 and wild type (WT) mice were treated daily with CGS 21680 (0.5 mg/kg i.p.) for 3 weeks and the expression levels of NMDA receptor subunits were then evaluated. In addition, to study CGS 21680-induced changes in NMDA receptor function, NMDA-induced toxicity in corticostriatal slices from both R6/2 and WT mice was investigated. We found that CGS 21680 increased NR2A subunit expression and the NR2A/NR2B ratio in the cortex of R6/2 mice, having no effect in WT mice. In the striatum, CGS 21680 reduced NR1 expression in both R6/2 and WT mice while the effect on NR2A and NR2/NR2B expression was genotype-dependent, reducing and increasing their expression in WT and R6/2 mice, respectively. On the contrary, NMDA-induced toxicity in corticostriatal slices was not modified by the treatment in WT or HD mice. These results demonstrate that in vivo activation of A 2A receptors modulates the subunit composition of NMDA receptors in the brain of HD mice.

Remodeling of striatal NMDA receptors by chronic A2A receptor blockade in Huntington's disease mice

Excitotoxicity plays a major role in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disorder. Adenosine A2A receptors (A2ARs) modulate excitotoxicity and have been suggested to play a pathogenetic role in HD. The main aim of this study was to evaluate the effect of A2AR blockade on the expression and functions of NMDA receptors in the striatum of HD mice (R6/2). We found that 3 weeks' treatment with SCH 58261 (0.01 mg/kg/day i.p. from the 8th week of age) modified NR1 and NR2A/NR2B expression in the striatum of R6/2 (Western blotting) while had no effect on NMDA-induced toxicity in corticostriatal slices (electrophysiological experiments). In conclusion, in vivo A2AR blockade induced a remodeling of NMDA receptors in the striatum of HD mice. Even though the functional relevance of the above effect remains to be fully elucidated, these results add further evidence to the modulatory role of A2ARs in HD.

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum

Adenosine A(2A), cannabinoid CB(1) and metabotropic glutamate 5 (mGlu(5)) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB(1)R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A(2A)Rs. Such a permissive effect of A(2A)Rs towards CB(1)Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A(2A)Rs. The selective mGlu(5)R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A(2A)R blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A(2A)Rs regulates the synaptic effects of CB(1)Rs and that A(2A)Rs may control CB(1) effects also indirectly, namely through mGlu(5)Rs.