Glutamate Challenge Research Articles

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl1-NH-Aryl2 scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.

FGF-21, a novel metabolic regulator, has a robust neuroprotective role and is markedly elevated in neurons by mood stabilizers

Fibroblast growth factor-21 (FGF-21) is a new member of the FGF super-family and an important endogenous regulator of glucose and lipid metabolism. It has been proposed as a therapeutic target for diabetes and obesity. Its function in the central nervous system (CNS) remains unknown. Previous studies from our laboratory demonstrated that aging primary neurons are more vulnerable to glutamate-induced excitotoxicity, and that co-treatment with the mood stabilizers lithium and valproic acid (VPA) induces synergistic neuroprotective effects. This study sought to identify molecule(s) involved in these synergistic effects. We found that FGF-21 mRNA was selectively and dramatically elevated by co-treatment with lithium and VPA in primary rat brain neurons. FGF-21 protein levels were also robustly increased in neuronal lysates and culture medium following lithium-VPA co-treatment. Combining glycogen synthase kinase-3 (GSK-3) inhibitors with VPA or histone deacetylase (HDAC) inhibitors with lithium synergistically increased FGF-21 mRNA levels, supporting that synergistic effects of lithium and VPA are mediated via GSK-3 and HDAC inhibition, respectively. Exogenous FGF-21 protein completely protected aging neurons from glutamate challenge. This neuroprotection was associated with enhanced Akt-1 activation and GSK-3 inhibition. Lithium-VPA co-treatment dramatically prolonged lithium-induced Akt-1 activation and augmented GSK-3 inhibition. Akt-1 knockdown markedly decreased FGF-21 mRNA levels, and reduced the neuroprotection induced by FGF-21 or lithium-VPA co-treatment. In addition, FGF-21 knockdown reduced lithium-VPA co-treatment-induced Akt-1 activation and neuroprotection against excitotoxicity. Together, our novel results suggest that FGF-21 is a key mediator of the effects of these mood stabilizers, and a potential new therapeutic target for CNS disorders.

Hypoxia-Ischemia or Excitotoxin-Induced Tissue Plasminogen Activator- Dependent Gelatinase Activation in Mice Neonate Brain Microvessels

Hypoxia-ischemia (HI) and excitotoxicity are validated causes of neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor-mediated pathways. Brain microvascular endothelial cells from neonates in culture, contain and release more t-PA and gelatinases upon glutamate challenge than adult cells. We have studied t-PA to gelatinase (MMP-2 and MMP-9) activity links in HI and excitotoxicity lesion models in 5 day–old pups in wild type and in t-PA or its inhibitor (PAI-1) genes inactivated mice. Gelatinolytic activities were detected in SDS-PAGE zymograms and by in situ fluorescent DQ-gelatin microscopic zymographies. HI was achieved by unilateral carotid ligature followed by a 40 min hypoxia (8%O2). Excitotoxic lesions were produced by intra parenchymal cortical (i.c.) injections of 10 µg ibotenate (Ibo). Gel zymograms in WT cortex revealed progressive extinction of MMP-2 and MMP-9 activities near day 15 or day 8 respectively. MMP-2 expression was the same in all strains while MMP-9 activity was barely detectable in t-PA−/− and enhanced in PAI-1−/− mice. HI or Ibo produced activation of MMP-2 activities 6 hours post-insult, in cortices of WT mice but not in t-PA−/− mice. In PAI-1−/− mice, HI or vehicle i.c. injection increased MMP-2 and MMP-9 activities. In situ zymograms using DQ-gelatin revealed vessel associated gelatinolytic activity in lesioned areas in PAI-1−/− and in WT mice. In WT brain slices incubated ex vivo, glutamate (200 µM) induced DQ-gelatin activation in vessels. The effect was not detected in t-PA−/−mice, but was restored by concomitant exposure to recombinant t-PA (20 µg/mL). In summary, neonatal brain lesion paradigms and ex vivo excitotoxic glutamate evoked t-PA-dependent gelatinases activation in vessels. Both MMP-2 and MMP-9 activities appeared t-PA-dependent. The data suggest that vascular directed protease inhibition may have neuroprotection potential against neonatal brain injuries.

Histamine up-regulates astrocytic glutamate transporter 1 and protects neurons against ischemic injury

Astrocytic glutamate transporter 1 (GLT-1) is responsible for the majority of extracellular glutamate clearance and is essential for preventing excitotoxicity in the brain. Up-regulation of GLT-1 shows benefit effect on ischemia-induced neuronal damage. In present study, we examined the effect of histamine, a neurotransmitter or neuromodulator, on GLT-1 expression and function. In acute hippocampal slices, histamine selectively increased GLT-1 expression independent of neuronal activities. Similar up-regulation of GLT-1 was also observed after histamine treatment in pure cultured astrocytes, which was abolished by H1 receptor antagonist or PKC inhibitor. Cell surface biotinylation and whole-cell patch recordings of glutamate transporter current confirmed the up-regulation of functional GLT-1 following histamine exposure. Histamine treatment decreased the extracellular glutamate content and alleviated neuronal cell death induced by exogenous glutamate challenge. Moreover, we found a significant neuroprotective effect of histamine in brain slices after oxygen-glucose deprivation (OGD). In addition, histidine, the precursor of histamine, also showed neuroprotection against ischemic injury, which was accompanied by reversion of declined expression of GLT-1 in adult rats subjected to middle cerebral artery occlusion (MCAO). These neuroprotective effects of histamine/histidine were blocked by GLT-1 specific inhibitor dihydrokainate or H1 receptor antagonist. In summary, our results suggest that histamine up-regulates GLT-1 expression and function via astrocytic H1 receptors, thus resulting in neuroprotection against excitotoxicity and ischemic injury.

High t-PA release by neonate brain microvascular endothelial cells under glutamate exposure affects neuronal fate

Glutamate excitotoxicity is a consolidated hypothesis in neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor mediated effects. In brain microvascular endothelial cell (nBMEC) cultures from neonates, t-PA content and release upon glutamate are higher than in adult (aBMECs) cultures. Owing to the variety of t-PA substrates and receptor targets, the study was aimed at determining the putative roles of endothelial t-PA in the neonatal brain parenchyma under glutamate challenge. Basal t-PA release was 4.4 fold higher in nBMECs vs aBMECs and glutamate was 20 fold more potent to allow Evans blue vascular permeability in neonate microvessels indicating that, under noxious glutamate (50μM) exposure, high amounts of endothelial t-PA stores may be mobilized and may access the nervous parenchyma. Culture media from nBMECS or aBMECs challenged by excitotoxic glutamate were applied to neuron cultures at DIV 11. While media from adult cells did not evoke more LDH release in neuronal cultures that under glutamate alone, media from nBMECs enhanced 2.2 fold LDH release. This effect was not observed with media from t-PA−/− nBMECs and was inhibited by hr-PAI-1. In Cortical slices from 10day-old mice, hrt-PA associated with glutamate evoked neuronal necrosis in deeper (more mature) layers, an effect reversed by NMDA receptor GluN1 amino-terminal domain antibody capable of inhibiting t-PA potentiation of the receptor. In superficial layers (less mature), hrt-PA alone inhibited apoptosis, an effect reversed by the EGF receptor antagonist AG1478. Applied to immature neurons in culture (DIV5), media from nBMEC rescued 85.1% of neurons from cell death induced by serum deprivation. In cortical slices, the anti-apoptotic effect of t-PA fitted with age dependent localization of less mature neurons. These data suggest that in the immature brain, propensity of vessels to release high amounts of t-PA may not only impact vascular integrity but may also influence neuronal fate, via regulation of apoptosis in immature cells and, as in adult by potentiating glutamate toxicity in mature neurons. The data point out putative implication of microvessels in glutamate neurotoxicity in the development, and justify research towards vessel oriented neuroprotection strategies in neonates.

Deficient mitochondrial Ca 2+ buffering in the Cln8mnd mouse model of neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses (NCLs) are a group of genetic childhood-onset progressive brain diseases characterized by a decline in mental and motor capacities, epilepsy, visual loss and premature death. Using patch clamp, fluorescence imaging and caged Ca 2+ photolysis, we evaluated the mechanisms of neuronal Ca 2+ clearance in Cln8 mnd mice, a model of the human NCL caused by mutations in the CLN8 gene. In Cln8 mnd hippocampal slices, Ca 2+ clearance efficiency in interneurons and, to some extent, principal neurons declined with age. In cultured Cln8 mnd hippocampal neurons, clearance of large Ca 2+ loads was inefficient due to impaired mitochondrial Ca 2+ uptake. In contrast, neither Ca 2+ uptake by sarco/endoplasmic reticulum Ca 2+ ATPase, nor Ca 2+ extrusion through plasma membrane was affected by the Cln8 mutation. Excitotoxic glutamate challenge caused Ca 2+ deregulation more readily in Cln8 mnd than in wt neurons. We propose that neurodegeneration in human CLN8 disorders is primarily caused by reduced mitochondrial Ca 2+ buffering capacity.

Excitotoxic neuroprotection and vulnerability with CaMKII inhibition

Aberrant calcium signaling is a common feature of ischemia and multiple neurodegenerative diseases. While activation of calcium–calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key event in calcium signaling, its role in excitotoxicity is controversial. Our findings demonstrate neuroprotection in neuronal cultures treated with the small molecule (KN-93) and peptide (tat-AIP and tat-CN21) inhibitors of CaMKII immediately prior to excitotoxic glutamate/glycine insult. Unlike KN-93 which blocks CaMKII activation, but not constitutively active forms of CaMKII, tat-CN21 and tat-AIP significantly reduced excitotoxicity in cultured neurons when applied post-insult. We observed that the neuroprotective effects of tat-CN21 are greatest when applied before the toxic glutamate challenge and diminish with time, with the neuroprotection associated with CaMKII inhibition diminishing back to control 3 h post glutamate insult. Mechanistically, tat-CN21 inhibition of CaMKII resulted in an increase in CaMKII activity and the percentage of soluble αCaMKII observed in neuronal lysates 24 h following glutamate stimulation. To address the impact of prolonged CaMKII inhibition prior to excitotoxic insult, neuronal cultures were treated with CaMKII inhibitors overnight and then subjected to a sub-maximal excitotoxic insult. In this model, CaMKII inhibition prior to insult exacerbated neuronal death, suggesting that a loss of CaMKII enhances neuronal vulnerability to glutamate. Although changes in αCaMKII or NR2B protein levels are not responsible for this enhanced glutamate vulnerability, this process is blocked by the protein translation inhibitor cycloheximide. In total, the neuroprotection afforded by CaMKII inhibition can be seen as neuroprotective immediately surrounding the excitotoxic insult, whereas sustained CaMKII inhibition produced by excitotoxicity leads to neuronal death by enhancing neuronal vulnerability to glutamate.

Pharmacological enhancement of glutamate transport reduces excitotoxicity in vitro

Glutamate transporters are responsible for removing glutamate from the extracellular space and have the potential to protect neurons from excitotoxicity. In the present study, the effects of ceftriaxone and (2R, 4R)-APDC (APDC) on the protein expression of GLAST and GLT-1, the rate of glutamate uptake, and neuroprotection were evaluated in a cell culture model of glutamate excitotoxicity. Mixed neuron/astrocyte cultures were prepared from 1 day old rat pups. Protein levels of GLAST and GLT-1 glutamate transporters were quantified using In-Cell Western techniques after acute or 5-day treatment with either ceftriaxone or APDC. Glutamate uptake was measured using Michaelis-Menten kinetics to evaluate the effects of 5-day treatment with ceftriaxone or APDC. Neuronal cell death in response to a 10-minute 1 mM glutamate challenge was measured following 5-day treatment with either ceftriaxone or APDC. Five-day treatment with 100 μM ceftriaxone significantly increased both GLAST and GLT-1 protein levels 31.3% and 47.5% above control, respectively, increased the Vmax 29.3%, increased the Km of glutamate uptake 117.9%, and reduced neuronal death 22.0% after a 1 mM glutamate challenge. Five-day treatment with 1 mM APDC significantly increased GLAST protein levels 27.6%, increased the Vmax 92.4%, increased the Km of glutamate transport 118.9%, and decreased neuronal death 36.8% after a 1 mM glutamate challenge. Chronic treatment with ceftriaxone or APDC provided neuroprotection from glutamate excitotoxicity while increasing GLAST and GLT-1 protein levels and increasing glutamate uptake. These compounds may have therapeutic potential in chronic excitotoxic neurodegenerative diseases.

First evidence that drugs of abuse produce behavioral sensitization and cross sensitization in planarians

Behavioral sensitization in mammals, including humans, is sensitive to factors such as administration route, testing environment, and pharmacokinetic confounds, unrelated to the drugs themselves that are difficult to eliminate. Simpler animals less susceptible to these confounding influences may be advantageous substitutes for studying sensitization. We tested this hypothesis by determining whether planarians display sensitization and cross sensitization to cocaine and glutamate. Planarian hyperactivity was quantified as the number of C-like hyperkinesias during a 1-min drug exposure. Planarians exposed initially to cocaine (or glutamate) on day 1 were challenged with cocaine (or glutamate) after 2 or 6 days of abstinence. Acute cocaine or glutamate produced concentration-related hyperactivity. Cocaine or glutamate challenge after 2 and 6 days of abstinence enhanced the hyperactivity, indicating the substances produced planarian behavioral sensitization. Cross-sensitization experiments showed that cocaine produced greater hyperactivity in planarians earlier exposed to glutamate than in glutamate-naive planarians, and vice versa. Behavioral responses were pharmacologically selective because neither scopolamine nor caffeine produced planarian behavioral sensitization despite causing hyperactivity after initial administration, and acute gamma-aminobutyric acid did not cause hyperactivity. Demonstration of pharmacologically selective behavioral sensitization in planarians suggests that these flatworms represent a sensitive in-vivo model to study cocaine behavioral sensitization and to screen potential abuse-deterrent therapeutics.

Alexander Disease Mutant Glial Fibrillary Acidic Protein Compromises Glutamate Transport in Astrocytes

Alexander disease (AxD) is a leukodystrophy caused by heterozygous mutations in the gene for glial fibrillary acidic protein, an intermediate filament protein expressed by astrocytes. The mutation causes prominent protein aggregates inside astrocytes; there is also loss of myelin and oligodendrocytes and neuronal degeneration. We show that immunohistochemical staining for glutamate transporter 1, the major brain glutamate transporter expressed primarily in astrocytes suggests decreased levels in the hippocampi of infantile AxD patients. A knock-in mouse model of AxD also shows significant reduction of glutamate transporter 1 in the hippocampus. To explore this phenomenon at the cellular level, wild-type and R239C mutant glial fibrillary acidic proteins (the most common mutation) were overexpressed in astrocytes in culture. Western blotting and whole-cell patch clamp recordings demonstrated that the R239C astrocytes exhibited markedly reduced glutamate transporter 1 protein levels; this resulted in attenuated or abolished glutamate-induced inward transporter current. Neurons cocultured with the R239C astrocytes exhibited increased death after glutamate challenge. These results indicate that aberrant astrocytes have decreased glutamate uptake, which may play an important role in the pathogenesis of neuronal and oligodendrocyte injury and death in AxD.

Nanomolar vitamin E α‐tocotrienol inhibits glutamate‐induced activation of phospholipase A2 and causes neuroprotection

Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.

Prolonged Aβ treatment leads to impairment in the ability of primary cortical neurons to maintain K+ and Ca2+ homeostasis

BackgroundAlzheimer's disease (AD) is a progressive neurodegenerative disease, characterised by the formation of insoluble amyloidogenic plaques and neurofibrillary tangles. Beta amyloid (Aβ) peptide is one of the main constituents in Aβ plaques, and is thought to be a primary causative agent in AD. Neurons are likely to be exposed to chronic, sublethal doses of Aβ over an extended time during the pathogenesis of AD, however most studies published to date using in vitro models have focussed on acute studies. To experimentally model the progressive pathogenesis of AD, we exposed primary cortical neurons daily to 1 μM of Aβ1-40 over 7 days and compared their survival with age-similar untreated cells. We also investigated whether chronic Aβ exposure affects neuronal susceptibility to the subsequent acute excitotoxicity induced by 10 μM glutamate and assessed how Ca2+ and K+ homeostasis were affected by either treatment.ResultsWe show that continuous exposure to 1 μM Aβ1-40 for seven days decreased survival of cultured cortical neurons by 20%. This decrease in survival correlated with increased K+ efflux from the cells. One day treatment with 1 μM Aβ followed by glutamate led to a substantially higher K+ efflux than in the age-similar untreated control. This difference further increased with the duration of the treatment. K+ efflux also remained higher in Aβ treated cells 20 min after glutamate application leading to 2.8-fold higher total K+ effluxed from the cells compared to controls. Ca2+ uptake was significantly higher only after prolonged Aβ treatment with 2.5-fold increase in total Ca2+ uptake over 20 min post glutamate application after six days of Aβ treatment or longer (P < 0.05).ConclusionsOur data suggest that long term exposure to Aβ is detrimental because it reduces the ability of cortical neurons to maintain K+ and Ca2+ homeostasis in response to glutamate challenge, a response that might underlie the early symptoms of AD. The observed inability to maintain K+ homeostasis might furthermore be useful in future studies as an early indicator of pathological changes in response to Aβ.

TNF‐α‐mediates neuroprotection against glutamate‐induced excitotoxicity via NF‐κB‐dependent up‐regulation of KCa2.2 channels

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) induces neuroprotection against excitotoxic damage in primary cortical neurons via sustained nuclear factor-kappa B (NF-kappaB) activation. The transcription factor NF-kappaB can regulate the expression of small conductance calcium-activated potassium (K(Ca)) channels. These channels reduce neuronal excitability and as such may yield neuroprotection against neuronal overstimulation. In the present study we investigated whether TNF-alpha-mediated neuroprotective signaling is inducing changes in the expression of small conductance K(Ca) channels. Interestingly, the expression of K(Ca)2.2 channel was up-regulated by TNF-alpha treatment in a time-dependent manner whereas the expression of K(Ca)2.1 and K(Ca)2.3 channels was not altered. The increase in K(Ca)2.2 channel expression after TNF-alpha treatment was shown to be dependent on TNF-R2 and NF-kappaB activation. Furthermore, activation of small conductance K(Ca) channels by 6,7-dichloro-1H-indole-2,3-dione 3-oxime or cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine-induced neuroprotection against a glutamate challenge. Treatment with the small conductance K(Ca) channel blocker apamin or K(Ca)2.2 channel siRNA reverted the neuroprotective effect elicited by TNF-alpha. We conclude that treatment of primary cortical neurons with TNF-alpha leads to increased K(Ca)2.2 channel expression which renders neurons more resistant to excitotoxic cell death.

Thyroid hormone increases astrocytic glutamate uptake and protects astrocytes and neurons against glutamate toxicity

Thyroid hormone (T(3)) regulates the growth and differentiation of rat cerebellar astrocytes. Previously, we have demonstrated that these effects are due, at least in part, to the increased expression of extracellular matrix molecules and growth factors, such as fibroblast growth factor-2. T(3) also modulates neuronal development in an astrocyte-mediated manner. In the mammalian central nervous system, excitatory neurotransmission is mediated mainly by glutamate. However, excessive stimulation of glutamate receptors can lead to excitotoxicity and cell death. Astrocytic glutamate transporters, GLT-1 and GLAST, play an essential role in the clearance of the neuronal-released glutamate from the extracellular space and are essential for maintaining physiological extracellular glutamate levels in the brain. In the present study, we showed that T(3) significantly increased glutamate uptake by cerebellar astrocytes compared with control cultures. Inhibitors of glutamate uptake, such as L-PDC and DL-TBOA, abolished glutamate uptake on control or T(3)-treated astrocytes. T(3) treatment of astrocytes increased both mRNA levels and protein expression of GLAST and GLT-1, although no significant changes on the distribution of these transporters were observed. The gliotoxic effect of glutamate on cultured cerebellar astrocytes was abolished by T(3) treatment of astrocytes. In addition, the neuronal viability against glutamate challenge was enhanced on T(3)-treated astrocytes, showing a putative neuroprotective effect of T(3). In conclusion, our results showed that T(3) regulates extracellular glutamate levels by modulating the astrocytic glutamate transporters. This represents an important mechanism mediated by T(3) on the improvement of astrocytic microenvironment in order to promote neuronal development and neuroprotection.

Mitochondrial Dysfunction and Dendritic Beading during Neuronal Toxicity

Mitochondrial dysfunction (depolarization and structural collapse), cytosolic ATP depletion, and neuritic beading are early hallmarks of neuronal toxicity induced in a variety of pathological conditions. We show that, following global exposure to glutamate, mitochondrial changes are spatially and temporally coincident with dendritic bead formation. During oxygen-glucose deprivation, mitochondrial depolarization precedes mitochondrial collapse, which in turn is followed by dendritic beading. These events travel as a wave of activity from distal dendrites toward the neuronal cell body. Despite the spatiotemporal relationship between dysfunctional mitochondria and dendritic beads, mitochondrial depolarization and cytoplasmic ATP depletion do not trigger these events. However, mitochondrial dysfunction increases neuronal vulnerability to these morphological changes during normal physiological activity. Our findings support a mechanism whereby, during glutamate excitotoxicity, Ca(2+) influx leads to mitochondrial depolarization, whereas Na(+) influx leads to an unsustainable increase in ATP demand (Na(+),K(+)-ATPase activity). This leads to a drop in ATP levels, an accumulation of intracellular Na(+) ions, and the subsequent influx of water, leading to microtubule depolymerization, mitochondrial collapse, and dendritic beading. Following the removal of a glutamate challenge, dendritic recovery is dependent upon the integrity of the mitochondrial membrane potential, but not on a resumption of ATP synthesis or Na(+),K(+)-ATPase activity. Thus, dendritic recovery is not a passive reversal of the events that induce dendritic beading. These findings suggest that the degree of calcium influx and mitochondrial depolarization inflicted by a neurotoxic challenge, determines the ability of the neuron to recover its normal morphology.

Na+/Ca2+ exchange and regulation of cytoplasmic concentration of calcium in rat cerebellar neurons treated with glutamate

In the present work, the forward and/or reversed Na+/Ca2+ exchange in cerebellar granular cells was suppressed by substitution of Na+o by Li+ before, during, and after exposure to glutamate for varied time and also using the inhibitor KB-R7943 of the reversed exchange. After glutamate challenge for 1 min, Na+o/Li+ substitution did not influence the recovery of low [Ca2+]i in a calcium-free medium. A 1-h incubation with 100 microM glutamate induced in the neurons a biphasic and irreversible [Ca2+]i rise (delayed calcium deregulation (DCD)), enhancement of [Na+]i, and decrease in the mitochondrial potential. If Na+o had been substituted by Li+ before the application of glutamate, i.e. the exchange reversal was suppressed during the exposure to glutamate, the number of cells with DCD was nearly fourfold lowered. However, addition of the Na+/K+-ATPase inhibitor ouabain (0.5 mM) not preventing the exchange reversal also decreased DCD in the presence of glutamate. Both exposures decreased the glutamate-caused loss of intracellular ATP. Glucose deprivation partially abolished protective effects of the Na+o/Li+ substitution and ouabain. KB-R7943 (10 microM) increased 7.4-fold the number of cells with the [Ca2+]i decreased to the basal level after the exposure to glutamate. Thus, reversal of the Na+/Ca2+ exchange reinforced the glutamate-caused perturbations of calcium homeostasis in the neurons and slowed the recovery of the decreased [Ca2+]i in the post-glutamate period. However, for development of DCD, in addition to the exchange reversal, other factors are required, in particular a decrease in the intracellular concentration of ATP.

Synergistic Neuroprotection by Bis(7)-tacrine via Concurrent Blockade of N-Methyl-d-aspartate Receptors and Neuronal Nitric-Oxide Synthase

The excessive activation of the N-methyl-D-aspartate receptor (NMDAR)/nitric oxide (NO) pathway has been proposed to be involved in the neuropathology of various neurodegenerative disorders. In this study, NO was found to mediate glutamate-induced excitotoxicity in primary cultured neurons. Compared with the NO synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), and the NMDAR antagonist memantine, bis(7)-tacrine was found to be more potent in reducing NO-mediated excitotoxicity and the release of NO caused by glutamate. Moreover, like L-NMMA but not like 5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and memantine, bis(7)-tacrine showed greater neuroprotection and inhibition on NO release when neurons were pretreated for a prolonged time between 0 and 24 h and remained quite potent even when neurons were post-treated 1 h after the glutamate challenge. Bis(7)-tacrine was additionally found to be as moderately potent as memantine in competing with [(3)H]MK-801, inhibiting NMDA-evoked currents and reducing glutamate-triggered calcium influx, which eventually reduced neuronal NOS activity. More importantly, at neuroprotective concentrations, bis(7)-tacrine substantially reversed the overactivation of neuronal NOS caused by glutamate without interfering with the basal activity of NOS. Furthermore, in vitro pattern analysis demonstrated that bis(7)-tacrine competitively inhibited both purified neuronal and inducible NOS with IC(50) values at 2.9 and 9.3 microM but not endothelial NOS. This result was further supported by molecular docking simulations that showed hydrophobic interactions between bis(7)-tacrine and three NOS isozymes. Taken together, these results strongly suggest that the substantial neuroprotection against glutamate by bis(7)-tacrine might be mediated synergistically through the moderate blockade of NMDAR and selective inhibition of neuronal NOS.

Meso‐dihydroguaiaretic acid and licarin A of Machilus thunbergii protect against glutamate‐induced toxicity in primary cultures of a rat cortical cells

1 We previously reported that four lignans isolated from the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae) protected primary cultures of rat cortical neurons from neurotoxicity induced by glutamate. 2 Among the lignans, meso-dihydroguaiarectic acid (MDGA) and licarin A significantly attenuated glutamate-induced neurotoxicity when added prior to or right after the excitotoxic glutamate challenge. 3 The neuroprotective activities of two lignans appeared to be more effective in protecting neurons against neurotoxicity induced by NMDA than that induced by kainic acid. 4 MDGA and licarin A diminished the calcium influx that routinely accompanies with the glutamate-induced neurotoxicity, and inhibited the subsequent overproduction of cellular nitric oxide and peroxide to the level of control cells. They also preserved cellular activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and glutathione reductase reduced in the glutamate-injured neuronal cells. 5 Thus, our results suggest that MDGA and licarin A significantly protect primary cultured neuronal cells against glutamate-induced oxidative stress, via antioxidative activities.

Neuroprotective Properties of the Natural Vitamin E α-Tocotrienol

The current work is based on our previous finding that in neuronal cells, nmol/L concentrations of alpha-tocotrienol (TCT), but not alpha-tocopherol (TCP), blocked glutamate-induced death by suppressing early activation of c-Src kinase and 12-lipoxygenase. The single neuron microinjection technique was used to compare the neuroprotective effects of TCT with that of the more widely known TCP. Stroke-dependent brain tissue damage was studied in 12-Lox-deficient mice and spontaneously hypertensive rats orally supplemented with TCT. Subattomole quantity of TCT, but not TCP, protected neurons from glutamate challenge. Pharmacological as well as genetic approaches revealed that 12-Lox is rapidly tyrosine phosphorylated in the glutamate-challenged neuron and that this phosphorylation is catalyzed by c-Src. 12-Lox-deficient mice were more resistant to stroke-induced brain injury than their wild-type controls. Oral supplementation of TCT to spontaneously hypertensive rats led to increased TCT levels in the brain. TCT-supplemented rats showed more protection against stroke-induced injury compared with matched controls. Such protection was associated with lower c-Src activation and 12-Lox phosphorylation at the stroke site. The natural vitamin E, TCT, acts on key molecular checkpoints to protect against glutamate- and stroke-induced neurodegeneration.

Novel Dimeric Acetylcholinesterase Inhibitor Bis(7)-tacrine, but Not Donepezil, Prevents Glutamate-induced Neuronal Apoptosis by Blocking N-Methyl-d-aspartate Receptors

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.