S. Brecht et al., Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity, MOL BRAIN R, 94(1-2), 2001, pp. 25-34
Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Co
ntributing mechanisms and markers of cell death are the activation of caspa
se-3 and DNA fragmentation. It remains to be resolved to which extent both
cellular reactions overlap and/or indicate different processes of neurodege
neration. In this study, mixed neuronal cultures from newborn mice pubs (0-
24 h) were stimulated with glutamate, and the co-localization of active cas
pase-3 and DNA fragmentation was investigated by immunocytochemistry and th
e TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (ma
rked by MAP-2) displayed activated caspase-3 at different morphological sta
ges of degeneration. TUNEL staining was detected in 5% of cell nuclei inclu
ding GFAP-positive astrocytes. However, co-localization of active caspase-3
with TUNEL was less than 2%. After glutamate stimulation (125 muM), the ma
jority of neurons was dying between 12 and 24 h. The absolute number of act
ive caspase-3 neurons increased only moderately but in relation of survivin
g neurons after 24 h from 8 to 36% (125 muM), to 53% (250 muM) or to 32% (5
00 muM). TUNEL staining also increased after 24 h following glutamate treat
ment to 37% but the co-localization with active caspase-3 remained at the b
asal low level of 2%. In our system, glutamate-mediated excitotoxicity effe
cts the DNA fragmentation and caspase-3 activation. Co-localization of both
parameters, however, is very poor. Active caspase-3 in the absence of TUNE
L indicates a dynamic degenerative process, whereas TUNEL marks the end sta
ge of severe irreversible cell damage regardless to the origin of the cell.
(C) 2001 Elsevier Science BY All rights reserved.