Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity.
Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Contributing mechanisms and markers of cell death are the activation of caspase-3 and DNA fragmentation. It remains to be resolved to which extent both cellular reactions overlap and/or indicate different processes of neurodegeneration. In this study, mixed neuronal cultures from newborn mice pubs (0-24 h) were stimulated with glutamate, and the co-localization of active caspase-3 and DNA fragmentation was investigated by immunocytochemistry and the TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (marked by MAP-2) displayed activated caspase-3 at different morphological stages of degeneration. TUNEL staining was detected in 5% of cell nuclei including GFAP-positive astrocytes. However, co-localization of active caspase-3 with TUNEL was less than 2%. After glutamate stimulation (125 microM), the majority of neurons was dying between 12 and 24 h. The absolute number of active caspase-3 neurons increased only moderately but in relation of surviving neurons after 24 h from 8 to 36% (125 microM), to 53% (250 microM) or to 32% (500 microM). TUNEL staining also increased after 24 h following glutamate treatment to 37% but the co-localization with active caspase-3 remained at the basal low level of 2%. In our system, glutamate-mediated excitotoxicity effects the DNA fragmentation and caspase-3 activation. Co-localization of both parameters, however, is very poor. Active caspase-3 in the absence of TUNEL indicates a dynamic degenerative process, whereas TUNEL marks the end stage of severe irreversible cell damage regardless to the origin of the cell.
Brecht
S
S
Institut für Pharmakologie, Christian-Albrechts-Universität, Hospitalstrasse 4, 24105, Kiel, Germany. sbrecht@pharmakologie.uni-kiel.de
Gelderblom
M
M
Srinivasan
A
A
Mielke
K
K
Dityateva
G
G
Herdegen
T
T
D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000378:metabolism*; D018920:Coculture Techniques; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005189:False Positive Reactions; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D018698:Glutamic Acid / Q000633:toxicity*; D006624:Hippocampus / Q000166:cytology; D020287:In Situ Nick-End Labeling / Q000592:standards; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000032:analysis; D009457:Neuroglia / Q000737:chemistry / Q000166:cytology; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology*; D009498:Neurotoxins / Q000633:toxicity
D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; D009498:Neurotoxins; D018698:Glutamic Acid; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases