Excitotoxic Neuronal Cell Death Research Articles

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

Slack K+ channels attenuate NMDA-induced excitotoxic brain damage and neuronal cell death.

The neuronal Na+ -activated K+ channel Slack (aka Slo2.2, KNa 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na+ levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K+ channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca2+ - and Na+ -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca2+ signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K+ following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K+ . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K+ channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K+ signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.

Interactions between Tumor Cells, Neurons, and Microglia in the Glioma Microenvironment.

Despite significant strides made in understanding the pathophysiology of high-grade gliomas over the past two decades, most patients succumb to these neoplasias within two years of diagnosis. Furthermore, there are various co-morbidities associated with glioma and standard of care treatments. Emerging evidence suggests that aberrant glutamate secretion in the glioma microenvironment promotes tumor progression and contributes to the development of co-morbidities, such as cognitive defects, epilepsy, and widespread neurodegeneration. Recent data clearly illustrate that neurons directly synapse onto glioma cells and drive their proliferation and spread via glutamatergic action. Microglia are central nervous system-resident myeloid cells, modulate glioma growth, and possess the capacity to prune synapses and encourage synapse formation. However, current literature has yet to investigate the potential role of microglia in shaping synapse formation between neurons and glioma cells. Herein, we present the literature concerning glutamate’s role in glioma progression, involving hyperexcitability and excitotoxic cell death of peritumoral neurons and stimulation of glioma proliferation and invasion. Furthermore, we discuss instances in which microglia are more likely to sculpt or encourage synapse formation during glioma treatment and propose studies to delineate the role of microglia in synapse formation between neurons and glioma cells. The sex-dependent oncogenic or oncolytic actions of microglia and myeloid cells, in general, are considered in addition to the functional differences between microglia and macrophages in tumor progression. We also put forth tractable methods to safely perturb aberrant glutamatergic action in the tumor microenvironment without significantly increasing the toxicities of the standard of care therapies for glioma therapy.

Dapsone prolong delayed excitotoxic neuronal cell death by interacting with proapoptotic/survival signaling proteins

BackgroundDapsone prevents ischemic injury, inhibits apoptosis and shows functional improvement post-ischemia. However, its effect on proapoptotic or survival proteins in delayed ischemia remains unclear. MethodsMale adult Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by 24 h of ischemic reperfusion (I/R). Dapsone [9.375 or 12.5 mg/kg, intraperitoneally (IP)] was administered at 3, 6 and 12 h of I/R followed by behavioural assessment, brain infarction, histological alteration and cell viability study. Further, dapsone (25 and 50 µM) was added at 3, 6 and 12 h after L-glutamate (100 µM) in primary cortical culture (DIV 14) and cell viability, cytotoxicity, apoptosis was observed. Proteins expression were observed using immunocytochemistry. All experiments were performed after 24 h of I/R (In-Vivo) and 24 h of recovery post glutamate insult (In-Vitro). ResultsReduced brain infarction, improved neurobehavioural functions in addition to reduction in abnormal morphological structures of ischemic brain and improvement in cell viability was observed with treatment of dapsone (12.5 mg/kg) administered upto 6 h. Similarly, dapsone (25, 50 µM) increased cell survival post glutamate insult in cortical culture (In-vitro). Further, dapsone treatment at delayed hours (6 h) reduced apoptotic nuclei and proapoptotic proteins JNK, PTEN, Calpain, Caspase 3 expression along with activation of prosurvival protein BDNF expression post-glutamate insult. ConclusionOur results suggest that dapsone has the potential to limit the neuronal damage post-glutamate insult in delayed hours (6 h) through repressing proapoptotic proteins JNK, PTEN, Calpain, Caspase-3 of cerebral ischemia along with activation of pro-survival protein BDNF.

Extracellular mild acidosis decreases the Ca2+ permeability of the human NMDA receptors

NMDA receptors (NMDARs) are glutamate-gated ion channels involved in excitatory synaptic transmission and in others physiological processes such as synaptic plasticity and development. The overload of Ca2+ ions through NMDARs, caused by an excessive activation of receptors, leads to excitotoxic neuronal cell death. For this reason, the reduction of Ca2+ flux through NMDARs has been a central focus in finding therapeutic strategies to prevent neuronal cell damage.Extracellular H+ are allosteric modulators of NMDARs. Starting from previous studies showing that extracellular mild acidosis reduces NMDA-evoked whole cell currents, we analyzed the effects of this condition on the NMDARs Ca2+ permeability, measured as “fractional calcium current” (Pf, i.e. the percentage of the total current carried by Ca2+ ions), of human NMDARs NR1/NR2A and NR1/NR2B transiently transfected in HeLa cells. Extracellular mild acidosis significantly reduces Pf of both human NR1/NR2A and NR1/NR2B NMDARs, also decreasing single channel conductance in outside out patches for NR1/NR2A receptor. Reduction of Ca2+ flux through NMDARs was also confirmed in cortical neurons in culture. A comparative analysis of both NMDA evoked Ca2+ transients and whole cell currents showed that extracellular H+ differentially modulate the permeation of Na+ and Ca2+ through NMDARs.Our data highlight the synergy of two distinct neuroprotective mechanisms during acidosis: Ca2+ entry through NMDARs is lowered due to the modulation of both open probability and Ca2+ permeability. Furthermore, this study provides the proof of concept that it is possible to reduce Ca2+ overload in neurons modulating the NMDAR Ca2+ permeability.

In vitro evidence for post-insult neuroprotective activity of an evolutionarily conserved motif against excitotoxic neuronal cell death.

In vitro excitotoxic cell death experiments can be considered a screening model of stroke to evaluate the neuroprotective property of specific compounds. Survival of neurons following excitotoxicity is influenced by the neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor). Here, a novel 12 amino-acid peptide [AYKSYVRALPLL (TUF1)] with a high level of evolutionary conservation was assessed for its neuroprotective property in an in vitro model of glutamate-induced N-methyl-D-aspartic acid receptor hyperactivation and excitotoxicity. This peptide shares 100% homology to the conserved motif (SYVRAL) of the neurotrophic factors, which is found in numerous US patents. Following exposure to toxic levels of glutamate (500 µM), cultured primary rat forebrain neurons treated with TUF1 showed a dose-dependent survival rate compared with untreated neurons. The neuroprotective effect was blocked by p75 neurotrophic receptor (p75) inhibitor (MC192), but not by tyrosine kinase receptor inhibitor (K252a) or N-methyl-D-aspartic acid receptor antagonists (MK801 and D-amino-5-phosphonovaleric acid). Serine to alanine substitution that abolishes p75 interaction showed a loss of neuroprotective effect. Collectively, the findings showed that TUF1 can protect cultured primary cortical neurons from excitotoxic cell death through the p75-dependent pathway. Given that TUF1 is derived from TMEM35 (NACHO), which is required for the assembly and expression of nicotinic acetylcholine receptors, mechanism of TUF1 action may involve organization of nicotinic acetylcholine receptor and p75 neurotrophin receptor to modulate neuronal responses, including Ca signaling, to cytotoxic events. Unlike nerve growth factor, which requires a pre-insult exposure, TUF1 has neuroprotective properties even with post-insult administration, making it a potential target for therapeutic development in mitigating neuronal damage due to stroke and brain injury.

Excitatory Dendritic Mitochondrial Calcium Toxicity: Implications for Parkinson's and Other Neurodegenerative Diseases.

Dysregulation of calcium homeostasis has been linked to multiple neurological diseases. In addition to excitotoxic neuronal cell death observed following stroke, a growing number of studies implicate excess excitatory neuronal activity in chronic neurodegenerative diseases. Mitochondria function to rapidly sequester large influxes of cytosolic calcium through the activity of the mitochondrial calcium uniporter (MCU) complex, followed by more gradual release via calcium antiporters, such as NCLX. Increased cytosolic calcium levels almost invariably result in increased mitochondrial calcium uptake. While this response may augment mitochondrial respiration, limiting classic excitotoxic injury in the short term, recent studies employing live calcium imaging and molecular manipulation of calcium transporter activities suggest that mitochondrial calcium overload plays a key role in Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and related dementias [PD with dementia (PDD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD)]. Herein, we review the literature on increased excitatory input, mitochondrial calcium dysregulation, and the transcriptional or post-translational regulation of mitochondrial calcium transport proteins, with an emphasis on the PD-linked kinases LRRK2 and PINK1. The impact on pathological dendrite remodeling and neuroprotective effects of manipulating MCU, NCLX, and LETM1 are reviewed. We propose that shortening and simplification of the dendritic arbor observed in neurodegenerative diseases occur through a process of excitatory mitochondrial toxicity (EMT), which triggers mitophagy and perisynaptic mitochondrial depletion, mechanisms that are distinct from classic excitotoxicity.

Glutamate, T cells and multiple sclerosis.

Glutamate is the major excitatory neurotransmitter in the nervous system, where it induces multiple beneficial and essential effects. Yet, excess glutamate, evident in a kaleidoscope of acute and chronic pathologies, is absolutely catastrophic, since it induces excitotoxicity and massive loss of brain function. Both the beneficial and the detrimental effects of glutamate are mediated by a large family of glutamate receptors (GluRs): the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs), expressed by most/all cells of the nervous system, and also by many non-neural cells in various peripheral organs and tissues. T cells express on their cell surface several types of functional GluRs, and so do few other immune cells. Furthermore, glutamate by itself activates resting normal human T cells, and induces/elevates key T cell functions, among them: T cell adhesion, chemotactic migration, cytokine secretion, gene expression and more. Glutamate has also potent effects on antigen/mitogen/cytokine-activated T cells. Furthermore, T cells can even produce and release glutamate, and affect other cells and themselves via their own glutamate. Multiple sclerosis (MS) and its animal model Experimental Autoimmune Encephalomyelitis (EAE) are mediated by autoimmune T cells. In MS and EAE, there are excess glutamate levels, and multiple abnormalities in glutamate degrading enzymes, glutamate transporters, glutamate receptors and glutamate signaling. Some GluR antagonists block EAE. Enhancer of mGluR4 protects from EAE viaregulatory T cells (Tregs), while mGluR4 deficiency exacerbates EAE. The protective effect of mGluR4 on EAE calls for testing GluR4 enhancers in MS patients. Oral MS therapeutics, namely Fingolimod, dimethyl fumarate and their respective metabolites Fingolimod-phosphate and monomethyl fumarate, can protect neurons against acute glutamatergic excitotoxic damage. Furthermore, Fingolimod reduce glutamate-mediated intracortical excitability in relapsing-remitting MS. Glatiramer acetate -COPAXONE®, an immunomodulator drug for MS, reverses TNF-α-induced alterations of striatal glutamate-mediated excitatory postsynaptic currents in EAE-afflicted mice. With regard to T cells of MS patients: (1) The cell surface expression of a specific GluR: the AMPA GluR3 is elevated in T cells of MS patients during relapse and with active disease, (2) Glutamate and AMPA (a selective agonist for glutamate/AMPA iGluRs) augment chemotactic migration of T cells of MS patients, (3) Glutamate augments proliferation of T cells of MS patients in response to myelin-derived proteins: MBP and MOG, (4) T cells of MS patients respond abnormally to glutamate, (5) Significantly higher proliferation values in response to glutamate were found in MS patients assessed during relapse, and in those with gadolinium (Gd)+ enhancing lesions on MRI. Furthermore, glutamate released from autoreactive T cells induces excitotoxic cell death of neurons. Taken together, the evidences accumulated thus far indicate that abnormal glutamate levels and signaling in the nervous system, direct activation of T cells by glutamate, and glutamate release by T cells, can all contribute to MS. This may be true also to other neurological diseases. It is postulated herein that the detrimental activation of autoimmune T cells by glutamate in MS could lead to: (1) Cytotoxicityin the CNS: T cell-mediated killing of neurons and glia cells, which would subsequently increase the extracellular glutamate levels, and by doing so increase the excitotoxicitymediated by excess glutamate, (2) Release of proinflammatory cytokines, e.g., TNFα and IFNγ that increase neuroinflammation. Finally, if excess glutamate, abnormal neuronal signaling, glutamate-induced activation of T cells, and glutamate release by T cells are indeed all playing a key detrimental role in MS, then optional therapeutic tolls include GluR antagonists, although these may have various side effects. In addition, an especially attractive therapeutic strategy is the novel and entirely different therapeutic approach to minimize excess glutamateand excitotoxicity, titled: 'brain to blood glutamate scavenging', designed to lower excess glutamate levels in the CNS by 'pumping it out' from the brain to the blood. The glutamate scavanging is achieved by lowering glutamate levels in the blood by intravenous injection of the blood enzyme glutamate oxaloacetate transaminase (GOT). The glutamate-scavenging technology, which is still experimental, validated so far for other brain pathologies,but not tested on MS or EAE yet, may be beneficial for MS too, since it could decrease both the deleterious effects of excess glutamate on neural cells, and the activation of autoimmune T cells by glutamate in the brain. The topic of glutamate scavenging, and also its potential benefit for MS, are discussed towards the end of the review, and call for research in this direction.

Synthesis and Biological Evaluation of Novel Multi-target-Directed Benzazepines Against Excitotoxicity.

Excitotoxicty, a key pathogenic event is characteristic of the onset and development of neurodegeneration. The glutamatergic neurotransmission mediated through different glutamate receptor subtypes plays a pivotal role in the onset of excitotoxicity. The role of NMDA receptor (NMDAR), a glutamate receptor subtype, has been well established in the excitotoxicity pathogenesis. NMDAR overactivation triggers excessive calcium influx resulting in excitotoxic neuronal cell death. In the present study, a series of benzazepine derivatives, with the core structure of 3-methyltetrahydro-3H-benzazepin-2-one, were synthesised in our laboratory and their NMDAR antagonist activity was determined against NMDA-induced excitotoxicity using SH-SY5Y cells. In order to assess the multi-target-directed potential of the synthesised compounds, Aβ1-42 aggregation inhibitory activity of the most potent benzazepines was evaluated using thioflavin T (ThT) and Congo red (CR) binding assays as Aβ also imparts toxicity, at least in part, through NMDAR overactivation. Furthermore, neuroprotective, free radical scavenging, anti-oxidant and anti-apoptotic activities of the two potential test compounds (7 and 14) were evaluated using primary rat hippocampal neuronal culture against Aβ1-42-induced toxicity. Finally, in vivo neuroprotective potential of 7 and 14 was assessed using intracerebroventricular (ICV) rat model of Aβ1-42-induced toxicity. All of the synthesised benzazepines have shown significant neuroprotection against NMDA-induced excitotoxicity. The most potent compound (14) showed relatively higher affinity for the glycine binding site as compared with the glutamate binding site of NMDAR in the molecular docking studies. 7 and 14 have been shown experimentally to abrogate Aβ1-42 aggregation efficiently. Additionally, 7 and 14 showed significant neuroprotective, free radical scavenging, anti-oxidant and anti-apoptotic properties in different in vitro and in vivo experimental models. Finally, 7 and 14 attenuated Aβ1-42-induced tau phosphorylation by abrogating activation of tau kinases, i.e. MAPK and GSK-3β. Thus, the results revealed multi-target-directed potential of some of the synthesised novel benzazepines against excitotoxicity.

The Phoneutria nigriventer spider toxin, PnTx4-5-5, promotes neuronal survival by blocking NMDA receptors

Spider toxins are recognized as useful sources of bioactive substances, showing a wide range of pharmacological effects on neurotransmission. Several spider toxins have been identified biochemically and some of them are specific glutamate receptors antagonists. Previous data indicate that PnTx4-5-5, a toxin isolated from the spider Phoneutria nigriventer, inhibits the N-methyl-d-aspartate receptor (NMDAR), with little or no effect on AMPA, kainate or GABA receptors. In agreement with these results, our findings in this study show that PnTx4-5-5 reduces the amplitude of NMDAR-mediated EPSCs in hippocampal slices. It is well established that glutamate-mediated excitotoxic neuronal cell death occurs mainly via NMDAR activation. Thus, we decided to investigate whether PnTx4-5-5 would protect against various cell death insults. For that, we used primary-cultured corticostriatal neurons from wild type (WT) mice, as well as from a mouse model of Huntington's disease, BACHD. Our results showed that PnTx4-5-5 promotes neuroprotection of WT and BACHD neurons under the insult of high levels of glutamate. Moreover, the toxin is also able to protect WT neurons against amyloid β (Aβ) peptide toxicity. These results indicate that the toxin PnTx4-5-5 is a potential neuroprotective drug.

A Cytotoxic, Co-operative Interaction Between Energy Deprivation and Glutamate Release From System xc- Mediates Aglycemic Neuronal Cell Death.

The astrocyte cystine/glutamate antiporter (system xc−) contributes substantially to the excitotoxic neuronal cell death facilitated by glucose deprivation. The purpose of this study was to determine the mechanism by which this occurred. Using pure astrocyte cultures, as well as, mixed cortical cell cultures containing both neurons and astrocytes, we found that neither an enhancement in system xc− expression nor activity underlies the excitotoxic effects of aglycemia. In addition, using three separate bioassays, we demonstrate no change in the ability of glucose-deprived astrocytes—either cultured alone or with neurons—to remove glutamate from the extracellular space. Instead, we demonstrate that glucose-deprived cultures are 2 to 3 times more sensitive to the killing effects of glutamate or N-methyl-D-aspartate when compared with their glucose-containing controls. Hence, our results are consistent with the weak excitotoxic hypothesis such that a bioenergetic deficiency, which is measureable in our mixed but not astrocyte cultures, allows normally innocuous concentrations of glutamate to become excitotoxic. Adding to the burgeoning literature detailing the contribution of astrocytes to neuronal injury, we conclude that under our experimental paradigm, a cytotoxic, co-operative interaction between energy deprivation and glutamate release from astrocyte system xc− mediates aglycemic neuronal cell death.

Fractalkine: multiple strategies to counteract glutamate receptors activation leading to neuroprotection.

Fractalkine: multiple strategies to counteract glutamate receptors activation leading to neuroprotection.

Nitric Oxide Induces Ca2+-independent Activity of the Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)

Both signaling by nitric oxide (NO) and by the Ca(2+)/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca(2+)-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca(2+)/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca(2+). Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death.

NMDA receptor-mediated neuroprotective effect of the Scutellaria baicalensis Georgi extract on the excitotoxic neuronal cell death in primary rat cortical cell cultures.

The objective of the current research work was to evaluate the neuroprotective effect of the ethanol extract of Scutellaria baicalensis (S.B.) on the excitotoxic neuronal cell death in primary rat cortical cell cultures. The inhibitory effects of the extract were qualitatively and quantitatively estimated by phase-contrast microscopy and lactate dehydrogenase (LDH) assays. The extract exhibited a potent and dose-dependent inhibition of the glutamate-induced excitotoxicity in the culture media. Further, using radioligand binding assays, it was observed that the inhibitory effect of the extract was more potent and selective for the N-methyl-D-aspartate (NMDA) receptor-mediated toxicity. The S.B. ethanol extract competed with [3H] MDL 105,519 for the specific binding to the NMDA receptor glycine site with 50% inhibition occurring at 35.1 μg/mL. Further, NMDA receptor inactivation by the S.B. ethanol extract was concluded from the decreasing binding capability of [3H]MK-801 in the presence of the extract. Thus, S.B. extract exhibited neuroprotection against excitotoxic cell death, and this neuroprotection was mediated through the inhibition of NMDA receptor function by interacting with the glycine binding site of the NMDA receptor. Phytochemical analysis of the bioactive extract revealed the presence of six phytochemical constituents including baicalein, baicalin, wogonin, wogonoside, scutellarin, and Oroxylin A.

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter

BackgroundNeural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes.MethodsWild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations.ResultsFollowing T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter.ConclusionGlutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.

Metabotropic glutamate receptor 5 positive allosteric modulators are neuroprotective in a mouse model of Huntington's disease

Background and PurposeHuntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. We have previously demonstrated that the cell signalling of the metabotropic glutamate receptor 5 (mGluR5) is altered in a mouse model of HD. Although mGluR5-dependent protective pathways are more activated in HD neurons, intracellular Ca2+ release is also more pronounced, which could contribute to excitotoxicity. In the present study, we aim to investigate whether mGluR5 positive allosteric modulators (PAMs) could activate protective pathways without triggering high levels of Ca2+ release and be neuroprotective in HD.Experimental ApproachWe performed a neuronal cell death assay to determine which drugs are neuroprotective, Western blot and Ca2+ release experiments to investigate the molecular mechanisms involved in this neuroprotection, and object recognition task to determine whether the tested drugs could ameliorate HD memory deficit.Key ResultsWe find that mGluR5 PAMs can protect striatal neurons from the excitotoxic neuronal cell death promoted by elevated concentrations of glutamate and NMDA. mGluR5 PAMs are capable of activating Akt without triggering increased intracellular Ca2+ concentration ([Ca2+]i); and Akt blockage leads to loss of PAM-mediated neuroprotection. Importantly, PAMs' potential as drugs that may be used to treat neurodegenerative diseases is highlighted by the neuroprotection exerted by mGluR5 PAMs on striatal neurons from a mouse model of HD, BACHD. Moreover, mGluR5 PAMs can activate neuroprotective pathways more robustly in BACHD mice and ameliorate HD memory deficit.Conclusions and ImplicationsmGluR5 PAMs are potential drugs that may be used to treat neurodegenerative diseases, especially HD.

Prostaglandin E2 Type 1 Receptors Contribute to Neuronal Apoptosis after Transient Forebrain Ischemia

Cyclooxygenase-2-derived prostaglandin E2 (PGE2) contributes to excitotoxic and ischemic neuronal cell death by engaging neuronal PGE2 type 1 receptors (EP1R). Our previous studies have shown that EP1R signaling resulted in disturbances of intracellular Ca(2+) homeostasis and suppression of the pro-survival protein kinase AKT. The aim of this study was to investigate whether these pathophysiological mechanism have a role in the neuronal cell death after transient forebrain ischemia. Mice were subjected to ischemia/reperfusion by bilateral common carotid artery occlusion. Hippocampal cornu ammonis area 1 (CA1) neuronal cell death was determined 5 days after reperfusion. Animals treated with the EP1R antagonist SC51089 or EP1R-deficient mice (EP1(-/-)) showed significantly less neuronal injury as compared to vehicle-treated wild-type controls. Benefits of EP1R blockage were still evident 14 days after injury. Better neuronal survival was correlated with reduced neuronal caspase-3 activity and decreased nuclear translocation of the apoptosis-inducing factor . Neuroprotection could be reverted by intracerebroventricular administration of the phosphoinositide 3-kinase inhibitor LY294002 and was not further increased by the calcineurin inhibitor FK506. These data implicate EP1R in postischemic neuronal apoptosis possibly by facilitating AKT inhibition.

Over-expression of X-Linked Inhibitor of Apoptosis Protein Modulates Multiple Aspects of Neuronal Ca2+ Signaling

X-linked inhibitor of apoptosis (XIAP) protects and preserves the function of neurons in both in vitro and in vivo models of excitotoxicity. Since calcium (Ca(2+)) overload is a pivotal event in excitotoxic neuronal cell death, we have determined whether XIAP over-expression influences Ca(2+)-signaling in primary cultures of mouse cortical neurons. Using cortical neuron cultures derived from wild-type (Wt) mice transiently transfected with XIAP or from transgenic mice that over-express XIAP, we show that XIAP opposes the rise in intracellular Ca(2+) concentration by a variety of triggers. Relative to control neurons, XIAP over-expression produced a slight, but significant, elevation of resting Ca(2+) concentrations. By contrast, the rise in intracellular Ca(2+) concentrations produced by N-methyl-D-aspartate receptor stimulation and voltage gated Ca(2+) channel activation were markedly attenuated by XIAP over-expression. The release of Ca(2+) from intracellular stores induced by the sarco/endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin was also inhibited in neurons transiently transfected with XIAP. The pan-caspase inhibitor zVAD did not, however, diminish the rise in intracellular Ca(2+) concentrations elicited by L-glutamate suggesting that XIAP influences Ca(2+) signaling in a caspase-independent manner. Taken together, these findings demonstrate that the ability of XIAP to block excessive rises in intracellular Ca(2+) by a variety of triggers may contribute to the neuroprotective effects of this anti-apoptotic protein.

Conotoxins: Possible Therapeutic Measure for Huntingtons Disease

Huntington’s disease (HD) is a genetic disorder with autosomal dominant inheritance with progressive degeneration of neurons. It is characterized by affective, cognitive, behavioral, and motor dysfunctions. 3 nitropropionic acid is well established animal model which initiates a complex series of neurochemical and signaling changes that lead to pathological events including neuronal excitotoxicity by excessive glutamate release, neuroinflammation and energy impairment. Excitotoxins like NMDA and kainic acid which binds to these receptors to release high levels of glutamate that can cause excitotoxicity by allowing high levels of calcium ions (Ca2+) to enter the cell. Ca2+ influx into cells activates a number of enzymes, including phospholipases, endonucleases, and proteases such as calpain. These enzymes increase apoptosis, oxidative stress and energy impairment thereby causing mitochondrial dysfunctions and go on to damage neuronal structures. The toxic venom used by the cone shells called conotoxin contains up to 50 different peptides that selectively inhibit the function of ion channels and excitatory amino acid receptors such as NMDA involved in the transmission of nerve signals in animals representing an extensive array of ion channel blockers each showing a high degree of selectivity for particular types of channels. Conotoxins selectively target a range of ion channels and NMDA receptors, making them widely useful tool for probing nervous system function. Here we hypothize the protective effect of conotoxins, from genus Conus geographus named w-conotoxin and conantoxin, against excitotoxic neuronal cell death using 3 NP induced Huntington’s model.

Activation of microglial N‐methyl‐D‐aspartate receptors triggers inflammation and neuronal cell death in the developing and mature brain

Activated microglia play a central role in the inflammatory and excitotoxic component of various acute and chronic neurological disorders. However, the mechanisms leading to their activation in the latter context are poorly understood, particularly the involvement of N-methyl-D-aspartate receptors (NMDARs), which are critical for excitotoxicity in neurons. We hypothesized that microglia express functional NMDARs and that their activation would trigger neuronal cell death in the brain by modulating inflammation. We demonstrate that microglia express NMDARs in the murine and human central nervous system and that these receptors are functional in vitro. We show that NMDAR stimulation triggers microglia activation in vitro and secretion of factors that induce cell death of cortical neurons. These damaged neurons are further shown to activate microglial NMDARs and trigger a release of neurotoxic factors from microglia in vitro, indicating that microglia can signal back to neurons and possibly induce, aggravate, and/or maintain neurologic disease. Neuronal cell death was significantly reduced through pharmacological inhibition or genetically induced loss of function of the microglial NMDARs. We generated Nr1 LoxP(+/+) LysM Cre(+/-) mice lacking the NMDAR subunit NR1 in cells of the myeloid lineage. In this model, we further demonstrate that a loss of function of the essential NMDAR subunit NR1 protects from excitotoxic neuronal cell death in vivo and from traumatic brain injury. Our findings link inflammation and excitotoxicity in a potential vicious circle and indicate that an activation of the microglial NMDARs plays a pivotal role in neuronal cell death in the perinatal and adult brain.