NSC-34 Motor Neuron-Like Cells Are Unsuitable as Experimental Model for Glutamate-Mediated Excitotoxicity
Blandine Madji HounoumPatrick Vourc’hRomain FelixPhilippe CorciaFranck PatinMaxime GuéguinouMarie Potier‐CartereauChristophe VandierCédric RaoulChristian AndrésSylvie MavelHélène Blasco
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Abstract:
Glutamate-induced excitotoxicity is a major contributor to motor neuron degeneration in the pathogenesis of amyotrophic lateral sclerosis (ALS). The spinal cord × Neuroblastoma hybrid cell line (NSC-34) is often used as a bona fide cellular model to investigate the physiopathological mechanisms of ALS. However, the physiological response of NSC-34 to glutamate remains insufficiently described. In this study, we evaluated the relevance of differentiated NSC-34 (NSC-34D) as an in vitro model for glutamate excitotoxicity studies. NSC-34D showed morphological and physiological properties of motor neuron-like cells and expressed glutamate receptor subunits GluA1-4, GluN1 and GluN2A/D. Despite these diverse characteristics, no specific effect of glutamate was observed on cultured NSC-34D survival and morphology, in contrast to what has been described in primary culture of motor neurons (MN). Moreover, a small non sustained increase in the concentration of intracellular calcium was observed in NSC-34D after exposure to glutamate compared to primary MN. Our findings, together with the inability to obtain cultures containing only differentiated cells, suggest that the motor neuron-like NSC-34 cell line is not a suitable in vitro model to study glutamate-induced excitotoxicity. We suggest that the use of primary cultures of MN is more suitable than NSC-34 cell line to explore the pathogenesis of glutamate-mediated excitotoxicity at the cellular level in ALS and other motor neuron diseases.Keywords:
Excitotoxicity
Excessive or prolonged stimulation of, N ‐methyl‐ D ‐aspartate (NMDA) receptors appears to play an important role in many neurodegenerative processes in brain through a process known as excitotoxicity. This study examined the effects of ethanol on NMDA receptormediated excitotoxicity in primary neuronal cultures obtained from embryonic rat whole brain. Neurotoxicity was quantitated by measuring the amount of lactate dehydrogenase released into the media during a 20‐hr time period following NMDA washout. Exposure of 12‐to 14‐day‐old cultures to NMDA in Mg 2+ ‐free HEPES buffer (pH 7.4) for a 25‐min period resulted in a concentration‐dependent toxicity (EC 50 = 54 μM). Time‐course experiments showed that exposure to NMDA for as little as 5 min was excitotoxic and reached a plateau after a 20‐min exposure period. Preincubation of the cultures with ethanol (25 to 200 mm) resulted in a concentration‐dependent inhibition of NMDA‐mediated toxicity with approximately 38% inhibition produced by 25 mm ethanol and essentially complete inhibition at 200 mm ethanol (IC 50 = 60 mm). Increasing the glycine concentration to 100 μM did not potentiate NMDA neurotoxicity or antagonize the neuroprotective effect of ethanol. NMDA‐Mediated excitotoxicity was reduced by approximately 50% by the glycine antagonist 7‐chlorokynurenate (50 μM). Ethanol (50 mm) reduced NMDA neurotoxicity similar to 7‐chlorokynurenate, and the two together produced greater inhibition than either alone. These results show that intoxicating concentrations of ethanol can potently inhibit NMDA receptor‐mediated excitotoxicity and may have important implications in terms of ethanols interactions with brain trauma, ischemia, and other neuropathologies associated with NMDA receptor‐mediated neurotoxicity.
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Amyotrophic lateral sclerosis is a devastating neurodegenerative disease affecting both upper and lower motor neuron. Despite extensive research the primary cause of the disease has not been indentified and the causative treatment is lacking. The present article describes mechanisms involved in the disease development and progression, including oxidative stress, excitotoxicity, mitochondrial dysfunction, protein aggregation, RNA processing, alterations of cytoskeleton functions and axonal transport, glial cell involvement and programmed cell death. Biomedical Reviews 2011; 22: 7-14.
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Abstract: Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L‐glutamate, kainate (KA), N ‐methyl‐D‐aspartate (NMDA), or RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L‐aspartic acid β‐hydroxamate was added at 500 μ M to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate‐induced cytotoxicity by blocking only one of the glutamate receptor subtpes with either the NMDA receptor antagonist D‐(‐)‐2‐amino‐5‐phosphonopentanoate (APV) or with one of the specific non‐NMDA antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non‐NMDA glutamate agonists with no effect on NMDA‐induced cell death. Likewise, APV prevented NMDA‐induced cell death without affecting the KA‐ or AMPA‐induced neurotoxicity. It is concluded that EAA‐dependent neurotoxicity is induced by NMDA as well as non‐NMDA receptors.
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Immunoreactivity of neuron-specific enolase was measured in cultured embryonic rat motor neurons as an index of neuronal health. Treatment with cerebrospinal fluid from patients with amyotrophic lateral sclerosis and other neuromuscular disorders had no effect on neuron-specific enolase.
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Objective: Compare the concentration of glutamate(Glu) in the cerebrospinal fluid(CSF) of patients with amyotrophic lateral sclerosis( ALS) and those with healthy nervous system to make definite the role of excitotoxicity mediated by Glu in the pathogenesis of ALS. Methods: Subjects were 15 patients with ALS and 20 neurologically normal patients who underwent lumbar anesthesia for surgery. Analyzed their CSF by amino acid measuring machine which utilized the principle of high performance liquid chromatography( HPLC). Results: The concentration of Glu was higher in patients with ALS (48. 81 ±31. 67μmol/L) than in controls (15. 85 ± 6. 70μmol/L) (P0. 01). Conclusions: The excitotoxicity mediated by Glu plays an important role in the pathologic progress of ALS.
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Stroke is one of the three diseases that cause human death in current world, and it is the common, frequently occurring disease in the middle-old ages. NMDA receptors mediate glutamate-induced cell death when intensely or chronically activated, which is an important cause of neuronal cell death after acute injuries. Here, we demonstrated that BQ-869, a potent NMDA receptor antagonist, blocked NMDA receptor in concentration-dependent and dose-dependent manner, attenuated NMDA-induced Ca(2+) influx, inhabited NMDAR-mEPSC in hippocampal pyramidal neurons, improved athletic ability of rats with MACO, decreased infarction size in focal cerebral ischemia rats and reduced stroke mortality. Taken together, our data demonstrate the neuroprotective effect of BQ-869 might be through inhibiting NMDA-mediated excitotoxicity. These findings indicate that BQ-869 is the most potent antagonist of NMDA receptors, and provide new insights with potential therapeutic applications for the treatment of stroke.
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Excitotoxicity is a detrimental condition that accompanies acute brain damage such as stroke. Extensive studies have documented that excitotoxicity is mediated by the NMDA receptors and the excessive Ca2+ influx triggered by prolonged NMDA channel activation. However, the contribution to excitotoxicity by the two main NMDA receptor subtypes in principal neurons, NR2A and NR2B has remained enigmatic. This issue was investigated in cultured cortical neurons prepared from wild-type and gene-targeted mice expressing NR2A or NR2B with altered signalling characteristics. Excitotoxicity experiments including subtype-specific pharmacological tools and electrophysiological measurements were performed. Cultured neurons recapitulate the developmental expression profile of the two main NMDA receptor subunits NR2A and NR2B: at 14DIV neurons express NR2B and very low levels of NR2A. At 21DIV, NR2A subunit expression becomes as prominent as the NR2B subunit. In cultured neurons expressing NR2A only in its C-terminally truncated form, NMDA-mediated excitotoxicity occurred efficiently via the NR2B channels, both at 14 and 21DIV. This probably reflects the inability of truncated NR2A channels to access the “death programme“. Moreover, NMDA-triggered Ca2+-influx at 21DIV was suppressed by the NR2B subunit-specific blocker ifenprodil, indicating that Ca2+-influx occurred only through NR2B channels. Thus, in the absence of fully functional NR2A channels, excitotoxicity can be triggered only by the NR2B subtype. In cultured neurons expressing NR2B only in its C-terminally truncated form, exposure to NMDA had no toxic effect and no Ca2+-influx could be measured. Extensive electrophysiological experiments indicated a strong reduction of synaptic NMDA channels, suggesting that normal NR2B signalling is required for the postsynaptic presence of the NR2A channels. Whereas in young (14DIV) wild-type cultures NMDA-mediated toxicity was mediated solely by the NR2B channels, due to the lack of NR2A expression at this young stage, in older cultures (21DIV) both subtypes were found to be responsible for excitotoxicity. Notably, blocking any of the two subtypes did not reduce toxicity, due to the ability of the other subtype to compensate with no apparent time delay. In summary, this study indicates that both NR2A and NR2B subunits are playing a critical role in excitotoxicity, and any NMDA receptor subtype specific therapeutic intervention in stroke would lead to a failure.
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