Possible involvement of mitochondrial uncoupling protein-2 in cytotoxicity mediated by acquired N-methyl-d-aspartate receptor channels

We have previously shown the possible involvement of mitochondrial membrane potential disruption in the mechanisms underlying the neurotoxicity seen after activation of N-methyl-d-aspartate (NMDA) receptors (NMDAR) in primary cultured rat hippocampal neurons. In this study, we attempted to demonstrate a pivotal role of mitochondrial uncoupling protein-2 (UCP2) as a determinant of the NMDA neurotoxicity by using acquired NMDAR channels artificially orchestrated in HEK293 cells. In cells with overexpression of UCP2, immunoreactive UCP2 was exclusively detected at intracellular locations stained with the mitochondrial marker MitoTracker. In cells with acquired NMDAR channels, exposure to either NMDA or the calcium ionophore A23187 similarly led to a significant increase in cytosolic Ca(2+) levels determined by Fluo-3 imaging irrespective of the overexpression of UCP2. By contrast, NMDA, but not A23187, was significantly more effective in increasing mitochondrial Ca(2+) levels determined by Rhod-2 fluorescence imaging in cells transfected with NMDAR subunit and UCP2 expression vectors than in those without UCP2 overexpression. Overexpression of UCP2 significantly increased the number of cells stained with propidium iodide in cultures with acquired NMDAR channels, but failed to significantly affect that in cells exposed to A23187. Immunocytochemical and immunoprecipitation analyses similarly revealed the possible interaction between GluN1 subunit and UCP2 in HEK293 cells with acquired NMDAR channels and UCP2 overexpression. These results suggest that UCP2 could play a role as a determinant of the neurotoxicity mediated by NMDAR through a mechanism related to the unidentified interaction with the essential GluN1 subunit toward modulation of mitochondrial Ca(2+) levels in neurons.     

Benzyl alcohol inhibits <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptor-mediated neurotoxicity and calcium accumulation in cultured rat cortical neurons

The purpose of this study is to examine whether benzyl alcohol affects N-methyl-D-aspartate (NMDA) receptor in cortical cells. Benzyl alcohol (0.5–2 mM) inhibited NMDA-induced cytotoxicity. The protective effect of benzyl alcohol on NMDA-induced toxicity disappeared by washing cells with buffer to remove benzyl alcohol. Benzyl alcohol reduced NMDA receptor-mediated calcium accumulation, indicating that benzyl alcohol inhibits NMDA receptor activity.     

Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons

There is increasing evidence that a functional interaction exists between interleukin-1β (IL-1β) and N-methyl-d-aspartate (NMDA) receptors. The present study attempted to elucidate the effect of IL-1β on the NMDA-induced outward currents in mechanically dissociated hippocampal neurons using a perforated patch recording technique. IL-1β (30-100 ng/ml) inhibited the mean amplitude of the NMDA-induced outward currents that were mediated by charybdotoxin (ChTX)-sensitive Ca²⁺-activated K⁺ (K_Ca) channels. IL-1β (100 ng/ml) also significantly increased the mean ratio of the NMDA-induced inward current amplitudes measured at the end to the beginning of a 20-s application of NMDA. In hippocampal neurons from acute slice preparations, IL-1β significantly inhibited ChTX-sensitive K_Ca currents induced by a depolarizing voltage-step. IL-1 receptor antagonist antagonized effects of IL-1β. These results strongly suggest that IL-1β increases the neuronal excitability by inhibition of ChTX-sensitive K_Ca channels activated by Ca²⁺ influx through both NMDA receptors and voltage-gated Ca²⁺ channels.     

Alleviation by GABA<Subscript>B</Subscript> Receptors of Neurotoxicity Mediated by Mitochondrial Permeability Transition Pore in Cultured Murine Cortical Neurons Exposed to <Emphasis Type="Italic">N</Emphasis>-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate

Mitochondrial permeability transition pore (PTP) is supposed to at least in part participate in molecular mechanisms underlying the neurotoxicity seen after overactivation of N-methyl-d-aspartate (NMDA) receptor (NMDAR) in neurons. In this study, we have evaluated whether activation of GABA_B receptor (GABA_BR), which is linked to membrane G protein-coupled inwardly-rectifying K⁺ ion channels (GIRKs), leads to protection of the NMDA-induced neurotoxicity in a manner relevant to mitochondrial membrane depolarization in cultured embryonic mouse cortical neurons. The cationic fluorescent dye 3,3′-dipropylthiacarbocyanine was used for determination of mitochondrial membrane potential. The PTP opener salicylic acid induced a fluorescence increase with a vitality decrease in a manner sensitive to the PTP inhibitor ciclosporin, while ciclosporin alone was effective in significantly preventing both fluorescence increase and viability decrease by NMDA as seen with an NMDAR antagonist. The NMDA-induced fluorescence increase and viability decrease were similarly prevented by pretreatment with the GABA_BR agonist baclofen, but not by the GABA_AR agonist muscimol, in a fashion sensitive to a GABA_BR antagonist. Moreover, the GIRK inhibitor tertiapin canceled the inhibition by baclofen of the NMDA-induced fluorescence increase. These results suggest that GABA_BR rather than GABA_AR is protective against the NMDA-induced neurotoxicity mediated by mitochondrial PTP through a mechanism relevant to opening of membrane GIRKs in neurons.     

Glutamate-induced metabolic changes influence the cytoplasmic redox state of hippocampal neurons

Brain cell metabolism is intimately associated with intracellular oxidation–reduction (redox) balance. Glutamatergic transmission is accompanied with changes in substrate preference in neurons. Therefore, we studied cytoplasmatic redox changes in hippocampal neurons in culture exposed to glutamate. Neurons were transfected with HyPer, a genetically encoded redox biosensor for hydrogen peroxide which allows real-time imaging of the redox state. The rate of fluorescence decay, corresponding to the reduction of the biosensor was found to be augmented by low doses of glutamate (10 μM) as well as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca²⁺ abolished the glutamate-induced effect observed on HyPer fluorescence. Additional experiments indicated that mitochondrial function and hence energetic substrate availability commands the redox state of neurons and is required for the glutamate effect observed on the biosensor signal. Furthermore, our results implicated astrocytic metabolism in the changes of neuronal redox state observed with glutamate.     

Lycopene protects against trimethyltin-induced neurotoxicity in primary cultured rat hippocampal neurons by inhibiting the mitochondrial apoptotic pathway

Lycopene is a potent free radicals scavenger with demonstrated protective efficacy in several experimental models of oxidative damage. Trimethyltin (TMT) is an organotin compound with neurotoxic effects on the hippocampus and other limbic structures and is used to model neurodegenerative diseases targeting these brain areas. Oxidative stress is widely accepted as a central pathogenic mechanism of TMT-mediated neurotoxicity. The present study investigated whether the plant carotene lycopene protects against TMT-induced neurotoxicity in primary cultured rat hippocampal neurons. Lycopene pretreatment improved cell viability in TMT-treated hippocampal neurons and inhibited neuronal apoptosis. Microfluorometric imaging revealed that lycopene inhibited the accumulation of mitochondria-derived reactive oxygen species (ROS) during TMT exposure. Moreover, lycopene ameliorated TMT-induced activation of the mitochondrial permeability transition pore (mPTP) and the concomitant depolarization of the mitochondrial membrane potential (ΔΨ_m). Consequently, cytochrome c release from the mitochondria and ensuing caspase-3 activation were markedly reduced. These findings reveal that lycopene protects against TMT-induced neurotoxicity by inhibiting the mitochondrial apoptotic pathway. The anti-apoptotic effect of lycopene on hippocampal neurons highlights the therapeutic potential of plant-derived antioxidants against neurodegenerative diseases.     

Identification of an N-Methyl-d-aspartate Receptor in Isolated Nervous System Mitochondria

NMDA ionotropic glutamate receptors gate the cytoplasmic influx of calcium, which may, depending on the intensity of the stimulus, subserve either normal synaptic communication or cell death. We demonstrate that when isolated mitochondria are exposed to calcium and NMDA agonists, there is a significant increase in mitochondrial calcium levels. The agonist/antagonist response studies on purified mitochondria suggest the presence of a receptor on mitochondria with features similar to plasma membrane NMDA receptors. Immunogold electron microscopy of hippocampal tissue sections revealed extensive localization of NR2a subunit immunoreactivity on mitochondria. Transient transfection of neuronal GT1-7 cells with an NR1-NR2a NMDA receptor subunit cassette specifically targeting mitochondria resulted in a significant increase in mitochondrial calcium and neuroprotection against glutamate-induced cell death. Mitochondria prepared from GT1-7 cells in which the NR1 subunit of NMDA receptors was silenced demonstrated a decrease in calcium uptake. Our findings are the first to demonstrate that mitochondria express a calcium transport protein that shares characteristics with the NMDA receptor and may play a neuroprotective role.     

[+]-Huperzine A treatment protects against N-methyl-d-aspartate-induced seizure/status epilepticus in rats

The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood–brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [−]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [−]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20 μg/kg NMDA (intracerebroventricular) 20–30 min later. For post-exposure, rats were treated with [+]-Hup A (3 mg/kg, intramuscularly) 1 min after NMDA (20 μg/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3 mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [−]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.     

Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture

The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca(2+) level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca(2+) rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca(2+) rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pre-treatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBX-induced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that both in astrocytes and neurons gap junctions provide protection against NMDA-induced cytotoxicity.     

NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

Accumulating evidence indicates the involvement of N-methyl-d-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.     

Taurine attenuates amyloid β 1–42-induced mitochondrial dysfunction by activating of SIRT1 in SK-N-SH cells

Amyloid β (Aβ) plays a critical role in the pathogenesis of Alzheimer disease (AD). Studies indicate that Aβ causes reactive oxygen species (ROS) generation, mitochondrial dysfunction and neurons loss in vivo and in vitro. Taurine, a naturally occurring β-amino acid in the brain, has been demonstrated to have neuroprotective properties. In the present study, the effects of taurine on cell viability and mitochondrial function in Aβ1–42-treated SK-N-SH cells were investigated. Pretreatment of taurine significantly attenuated Aβ1–42-induced neuronal death. Similarly, taurine suppressed the mPTP opening and reversed mitochondrial function in the presence of Aβ1–42. Additionally, taurine attenuated the intracellular Ca²⁺ and ROS generation induced by Aβ1–42. Moreover, the expression of Sirtuin 1 (SIRT1) was obviously recovered by taurine in Aβ1–42-treated SK-N-SH cells. Our results suggest that taurine prevents Aβ1–42-induced mitochondrial dysfunction by activation of SIRT1. This study implies that taurine is a prospective additive for AD patients.     

The role of antagonism of NMDA receptor-mediated neurotransmission and inhibition of the dopamine reuptake in the neuroendocrine effects of phencyclidine

Phencyclidine (PCP) activates the hypothalamo-pituitary-adrenal (HPA) axis and decreases plasma prolactin levels in the rat. PCP is a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, but it also inhibits the reuptake of dopamine, serotonin and norepinephrine. The purpose of the present study was to utilize the PCP analogue N-[1-(2-thienyl)cyclohexyl]piperidine; (TCP), the potent dopamine reuptake inhibitor N-[1-(2-benzo(b)thiophenyl) cyclohexyl]piperidine; (BTCP) and the nonselective monoamine reuptake inhibitor cocaine as pharmacologic probes in order to determine the roles of noncompetitive NMDA receptor blockade and inhibition of dopamine reuptake in the neuroendocrine effects of PCP. PCP, TCP and cocaine increased plasma levels of adrenocorticotropin and corticosterone, but BTCP had no effect. In contrast, PCP, BTCP and cocaine decreased plasma prolactin, but TCP produced no such effect. The data suggest that mechanisms besides inhibition of dopamine reuptake are involved in the effects of PCP on the HPA axis, and the PCP-induced decrease in plasma prolactin is not a consequence of inhibition of NMDA receptor-mediated neurotransmission.     

Automated column-switching high-performance liquid chromatography system for quantifying N-methyl-D- and -L-aspartate

The occurrence and biological significance of the D-amino acids, N-methyl-D-aspartate (NMDA) and N-methyl-L-aspartate (NMLA), have been recently studied in a variety of living organisms. In this study, we established a highly sensitive and reliable fluorometric HPLC system for determining levels of N-methyl-aspartate (NMA). The system comprises fluorescent derivatization of NMA with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and two chromatographic steps: one that separates NMA from other primary amino acids in reverse-phase mode and another that enantioseparates NMDA and NMLA in a normal-phase mode. These two steps are linked by an automated column-switching system. A simple pretreatment step with o-phthalaldehyde to remove primary amino acids that can interfere with sensitivity is also described. The detection limit for NMDA is as low as 5fmol and the correlation between peak heights and concentrations between 5fmol and 1pmol is satisfactory (r=0.999). Following sample preparation and separation using the column-switching HPLC system, more than 80% of NMDA was recovered from rat liver homogenates spiked with NMDA. This method was employed to determine the levels of NMDA in tissues from bivalves and the results obtained were consistent with the values reported previously.     

Neuroprotective effects of interleukin-6 on NMDA-induced rat retinal damage

This study shows that interleukin-6 (IL-6) combined with soluble interleukin-6 receptors (sIL-6R) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Eyes pretreated with a combined injection of IL-6 and sIL-6R had NMDA administered into the vitreous cavity. Morphometric analysis and retrograde labeling analysis found that pretreatment with either IL-6 or sIL-6R alone did not bring about any neuroprotective effect. However, pretreatment with a combined administration of IL-6 and sIL-6R induced a significant neuroprotective effect against NMDA-induced retinal damage. Apoptotic changes in the retina were assessed by the TUNEL method. The results indicated that pretreatment with IL-6 combined with sIL-6R prevents NMDA-induced apoptosis. Western blotting studies demonstrated upregulation of gp130 expression in the NMDA-injected retina. Present studies suggest that IL-6 combined with sIL-6R provides a neuroprotective effect on NMDA-induced retinal damage.     

Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (–)-epigallocatechin-3-gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer''s disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca²⁺ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca²⁺ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.     

Study of neuroprotection of donepezil, a therapy for Alzheimer's disease

Donepezil is a potent acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease. Although acetylcholinesterase inhibitors are thought to be symptomatic treatment of Alzheimer's disease, it is not clear whether they are effective against progressive degeneration of neuronal cells. In this study, we investigated the neuroprotective effects of donepezil against ischemic damage, N-methyl-d-aspartate (NMDA) excitotoxicity, and amyloid-beta (Abeta) toxicity using rat brain primary cultured neurons. Lactate dehydrogenase (LDH) released into the culture medium was measured as a marker of neuronal cell damage. As an ischemic damage model, we used oxygen-glucose deprivation in rat cerebral cortex primary cultured neurons. Pretreatment with donepezil (0.1, 1 and 10muM) significantly decreased LDH release in a concentration-dependent manner. However, other acetylcholinesterase inhibitors (galantamine, tacrine and rivastigmine) did not significantly decrease LDH release. In a NMDA excitotoxicity model, pretreatment with donepezil (0.1, 1 and 10muM) decreased the LDH release in a concentration-dependent manner. In binding assay for glutamate receptors, donepezil at 100muM only slightly inhibited binding to the glycine and polyamine sites on NMDA receptor complex. We further examined the effect of donepezil on Abeta (1-40)- and Abeta (1-42)-induced toxicity in primary cultures of rat septal neurons. Pretreatment with donepezil (0.1, 1 and 10muM) significantly decreased LDH release induced by Abetas in a concentration-dependent manner. However, other acetylcholinesterase inhibitors (galantamine and tacrine) and NMDA receptor antagonists (memantine and dizocilpine (MK801)) did not significantly decrease LDH release. These results demonstrate that donepezil has protective effects against ischemic damage, glutamate excitotoxicity and Abeta toxicity to rat primary cultured neurons and these effects are not dependent on acetylcholinesterase inhibition and antagonism of NMDA receptors. Thus, donepezil is expected to have a protective effect against progressive degeneration of brain neuronal cells in ischemic cerebrovascular disease and Alzheimer's disease.     

Differential mechanisms of glutamate receptor regulation of SynGAP in cortical neurones

One prime candidate linking N-methyl-D-aspartate (NMDA) receptors to the regulation of the MAP kinase cascade is SynGAP, a negative regulator of Ras. In order to assess how a physiological stimulus can alter SynGAP activity, an appropriate whole cell system must be used and SynGAP must be specifically extracted from membranes whilst preserving the catalytic activity of the protein. Here, we have achieved this and studied the effect of NMDA/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptor stimulations on SynGAP activity in cortical neurones. Furthermore, we have examined the role of extracellular Ca2+, CaM kinase II and the PSD-95-NR2B subunit interaction in SynGAP activity regulation and propose a novel convergence of signalling between AMPA, kainate and NMDA receptors.