Research reportRoles of p53, c-Myc, Bcl-2, Bax and caspases in glutamate-induced neuronal apoptosis and the possible neuroprotective mechanism of basic fibroblast growth factor
Introduction
Glutamate is the principal excitatory transmitter in the central nervous system. Under certain circumstances it is also toxic to neurons. Excitotoxicity, induced by excessive activation of glutamate receptors, has been postulated to underlie the neuronal death that is associated with some neurodegenerative disorders such as stroke, Huntington's disease and Alzheimer's disease 18, 46. The exact mode of neuronal cell death induced by excitotoxins has remained unresolved, although recent data suggest that both necrotic [9]and apoptotic 20, 38, 44pathways can be activated after over-stimulation of various glutamate receptor subtypes.
Apoptosis is characterized by a series of distinct morphological and biochemical changes. Several apoptosis-related genes have been found 11, 13, 29. One group of apoptosis regulatory genes is the bcl-2 family. Of these genes, bcl-2, bcl-xL are antiapoptotic, whereas bax, bcl-xS, bad, bak and bik are proapoptotic. Dimerization of antiapoptotic factor Bcl-2 with proapoptotic factor Bax seems to be a critical interaction. Cells continue to survive if Bcl-2 predominates over Bax. On the contrary, a higher concentration of Bax, compared with Bcl-2, enhances cell susceptibility to apoptosis 1, 28. p53, a DNA transcription factor originally identified as a tumor suppressor, is also an important cell death regulatory factor. It has been shown to act as a transcriptional activator of bax gene. The activation of p53 may lead to an increase in the Bax/Bcl-2 ratio and therefore contribute to the initiation of apoptosis 15, 23. Some other proapoptotic and antiapoptotic gene products have also been identified in different cell types. For example, c-Myc, the protein encoded by the c-myc oncogene, is a transcription factor. Normally, c-Myc is required for cell cycle entry and promotes cell proliferation, providing that growth factors are available. However, c-Myc induces apoptosis when insulin-like growth factor and platelet-derived growth factor are not available [41]. As to the intracellular death effectors, the most important family in the process of apoptosis is the group of cysteine proteases, called caspases [14]. At least 11 members of the caspases family have been reported.
Up to now, the roles and precise interactions of these apoptosis-regulatory or apoptosis-effector genes remain unclear in neurons. Furthermore, different intracellular mechanisms are likely to be involved in the apoptosis induced by different stimuli or environment conditions. In the present study, we have used rat cortical neuron cultures as models of excitotoxic neuronal damage to study the signalling pathways leading to neuronal cell death and the possible mechanism by which basic fibroblast growth factor (bFGF) protects neurons from excitotoxicity.
Section snippets
Cell culture and glutamate treatment
Cultures of rat primary cortex neurons were prepared from embryonic day 18 Wistar rat embryos as described [27]. Cortices of rat embryos were dissected, placed in ice-cold Hanks' balanced salt solution (HBSS), minced (3–4 mm pieces) and incubated at 37°C for 15 min in Ca2+/Mg2+-free HBSS containing 0.25% trypsin and 0.2 mg/ml DNase I. The cell suspension was centrifuged (300×g for 10 min). The resulting pellets were resuspended in DMEM/F12 (1:1) supplemented with 20% calf serum, 100 units/ml
Effect of glutamate on apoptosis rate
The percentage of cells with subdiploid DNA content (DNA fragmentation) in the all cells was defined as apoptosis rate. There was no significant change in the apoptosis rate in control cells (from 17±3.2% to 21±2.7%) during the 24 h measurement period. After the cortical neurons were treated with 100 or 200 μM glutamate for 24 h, slight increase in the apoptosis rate was found. A significant increase in the apoptosis rate was found when cells were treated with 500 μM glutamate for 12 h or 24 h (
Discussion
The present study showed that cultured cortical neurons exposed to glutamate is accompanied by characteristics of apoptosis such as condensed or fragmented nuclei and the increased apoptosis rate. Our results are in agreement with previous studies, which indicated that excitotoxic neuronal death may be the result of both necrosis and apoptosis 2, 40, depending on the severity of stimulation [8]. The possibility of cell necrosis or other form of death could not be precluded in our experiments
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