Overstimulation of ionotropic glutamate receptors leads to excitotoxic neuronal death, which has been implicated in the neurodegeneration of neurological diseases. The present study examined the role of putative low‐affinity kainate receptor subtype (GluR5–7) agonists in excitotoxicity in cultured murine cortical neurons. The concentration‐dependent decrease in cell viability induced by the agonists kainate (1–1,000 μM) and (RS)‐2‐amino‐3‐(hydroxy‐5‐tert‐butylisoxazol‐4‐yl) propanoic acid (ATPA; 1–1,000 μM) was only attenuated by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 10 μM) and 1‐(4‐aminophenyl)‐4‐methyl‐7,8‐methylenedioxy‐5H‐2,3‐benzodiazepine (GYKI 52466; 20 μM). (S)‐5‐iodowillardiine (1–1,000 μM)‐induced toxicity was attenuated by CNQX (20 μM), GYKI 52466 (20 μM) and MK‐801 (10 μM); however, (2S,4R)‐4‐methylglutamate (1–120 μM)‐induced toxicity was not attenuated by the antagonists. None of the agonists possessed selective actions at GluR5–7. Morphological observations (phase‐contrast and fluorescence microscopy) revealed that the agonists induced two distinct patterns of neuronal injury. After 24 hr of treatment, low concentrations of agonists (1–30 μM) produced cellular shrinkage and nuclear granulation consistent with slow, apoptotic‐like neuronal death. Pyknotic labeling with the DNA binding dye Sytox green confirmed these apoptotic characteristics, which significantly decreased with increasing concentrations. After 4 hr, increasing concentrations of agonists (100–1,000 μM) induced cellular swelling, with subsequent extracellular debris; labeling with propidium iodide revealed isolated nuclei consistent with the increased involvement of rapid necrosis. Thus, all putative GluR5–7 agonists produced excitotoxicity across a necrotic‐apoptotic continuum in murine cortical neuron cultures. J. Neurosci. Res. 59:788–796, 2000 © 2000 Wiley‐Liss, Inc.

Abstract: Dysfunctions of the (S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L‐glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor‐mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl‐2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl‐2 gene, the influence of Bcl‐2 on AMPA receptor‐mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1‐100 μM), and cellular injury was analyzed 48 h after insult using phase‐contrast microscopy, a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide viability assay, and DNA staining with 4,6‐diamidino‐2‐phenylindole and Sytox Green. AMPA produced a concentration‐dependent increase in cell death that was significantly attenuated by human Bcl‐2. AMPA (3 μM) increased the number of apoptotic nuclei to 60% of control in wild‐type cultures, and human Bcl‐2 significantly decreased the number of apoptotic nuclei to 30% of AMPA‐treated cultures. Human Bcl‐2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1‐10 nM) and H2O2 (0.1‐30 μM) and where neuronal death was by apoptosis, but not against H2O2‐induced necrosis. Our findings indicate that overexpression of Bcl‐2 in primary cultured neurons protects in an insult‐dependent manner against AMPA receptor‐mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.

The neurotoxic profile of (2S,4R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434), a low-affinity kainate receptor subtype 5 (GluR5) agonist at recombinant human glutamate receptors, was evaluated to investigate the involvement of GluR5 in excitotoxic neuronal death. Murine cortical neurons were exposed to treatments for 24 h and assessed by a cell viability assay and phase-contrast microscopy. LY339434 (1–1000 μM) caused a concentration-dependent decrease in cell viability (EC50=11.4±1.2 μM) that was only attenuated by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801, 10 μM), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 20 μM). Labeling with nucleic acid binding dyes revealed that LY339434 induced few apoptotic-like characteristics. These findings indicate that in cultured murine cortical neurons, LY339434 acts predominantly through N-methyl-d-aspartate (NMDA) receptors rather than GluR5 to effect neuronal death that is rapid and involves predominantly necrosis rather than morphological apoptosis.

Neurotoxic profiles of putative agonists for low-affinity kainate subtypes of L-glutamate receptors (GluR5-7) were determined in cultured cortical neurones. Rank order of neurotoxic potency (microM): (S)-5-iodowillardiine (9) approximately = (2S,4R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434, 11) > (2S,4R)-4-methylglutamate (33) > kainate (100) > (RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA, 360). Using ionotropic glutamate receptor antagonists, neurotoxicity induced by kainate, ATPA and (S)-5-iodowillardiine appeared to involve a GluR5-7 component, unlike LY339434 and (2S,4R)-4-methylglutamate. These putative GluR5-7 agonists exhibited complex excitotoxic profiles highlighting the importance of studying native glutamate receptors.

In neocortical neuronal cultures, (S)-AMPA caused neurotoxicity which was concentration-dependent, receptor-mediated, slow and apoptotic in nature. (S)-AMPA (3–600 μM) failed to produce rapid neuronal swelling, but morphological observations and monitoring of viability at 24–72 h revealed 50% cell death consistent with apoptosis. (S)-AMPA induced cell shrinkage, neurite blebbing and nuclear condensation. Cyclothiazide (50 and 100 μM), which blocks AMPA receptor desensitization potentiated excitotoxicity with 75% of neurones undergoing slow death. The AMPA-selective antagonist GYKI 52466 (10–50 μM), attenuated (S)-AMPA-mediated neurotoxicity. DNA condensation, a hallmark of apoptosis, was found by labelling neurones with the DNA binding dye 4,6-diamidino-2-phenylindole HCl (DAPI). Gel electrophoresis revealed DNA fragmentation, which was increased by cyclothiazide and reduced by GYKI 52466 and cycloheximide. Overstimulation of the AMPA receptor produces a novel form of neuronal death, which is apoptotic, very slow in nature, and which could contribute to various neuropathologies.

Excitotoxicity induced by l-glutamate (Glu), when examined in a pure neuronal cortical culture, involved widespread apoptosis at concentrations of 1–10 μM as part of a continuum of injury, which at its most servere was purely necrotic. Cells, maintained in chemically defined neurobasal/B27 medium, were exposed at d7 for 2 h to Glu (1–500 μM), and cellular injury was analysed 2 and 24 h after insult using morphology (phase-contrast microscopy), a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), terminal transferase-mediated dUTP nick end-labelling (TUNEL) and DNA fragmentation by gel electrophoresis. Glu-mediated neurotoxicity was prevented by MK-801 (5 μM), whilst CNQX (20 μM) attenuated injury by 20%. Exposure to intensive insults (100 and 500 μM Glu) induced necrosis characterized by rapid cell swelling (<2 h) and lack of chromatin condensation, confirmed by DAPI nuclear staining. In contrast, mild insults (<20 μM Glu) failed to produce acute neuronal swelling at <2 h, but 24 h after injury resulted in a large number of apoptotic nuclei as confirmed by TUNEL and electrophoretic evidence of DNA fragmentation, which was attenuated by cycloheximide (0.1 μg/ml). Our findings indicate for the first time that physiological concentrations of Glu produce neuronal injury across a continuum involving apoptosis (<20 μM) and increasingly necrosis(>20 μM), dependent on the severity of the initial insult.

The protooncogene bcl-2 inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE-hbcl-2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE-hbcl-2 transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP+. Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE-hbcl-2 heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.

We have recently shown that dopamine (DA) can trigger apoptosis, an active program of cellular self-destruction, in various neuronal cultures and proposed that inappropriate activation of apoptosis by DA and or its oxidation products may initiate nigral cell loss in Parkinson's disease (PD). Since DA toxicity may be mediated via generation of oxygen-free radical species, we examined whether DA-induced cell death in PC12 cells may be inhibited by antioxidants. We have found that the thiol containing compounds, reduced glutathione (GSH),N-acetyl-cysteine (NAC), and dithiothreitol (DTT) were markedly protective, while vitamins C and E had lesser or no effect. The thiol antioxidants and vitamin C but not vitamin E, prevented dopamine autooxidation and production of dopamine-melanin. Their protective effect has also manifested by inhibiting DA-induced apoptosis; DNA fragmentation was prevented as was shown histochemically by thein situend-labeled DNA technique (TUNEL). Intracellular GSH and other thiols constitute an important natural defense against oxidative stress. We have found that depletion of cellular GSH by the addition of phoron, a substrate of glutathione transferase, and buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl transpeptidase, significantly enhanced DA toxicity. Cotreatment with NAC rescued the cells from the toxic effect of BSO + DA, and phoron + DA, while addition of GSH provided only partial protection from BSO + DA toxicity. Our data indicate that the thiol family of antioxidants, but not vitamins C and E, are highly effective in rescuing cells from DA-induced apoptosis. Further study of the mechanisms underlying the unique protective capacity of thiol antioxidants may lead to the development of new neuroprotective therapeutic strategies for PD.