Colchicine induces apoptosis in organotypic hippocampal slice cultures.
The microtubule-disrupting agent colchicine is known to be particular toxic for certain types of neurons, including the granule cells of the dentate gyrus. In this study we investigated whether colchicine could induce such neuron-specific degeneration in developing (1 week in vitro) and mature (3 weeks in vitro) organotypic hippocampal slice cultures and whether the induced cell death was apoptotic and/or necrotic. When applied to 1-week-old cultures for 48 h, colchicine induced primarily apoptotic, but also a minor degree of necrotic cell death in the dentate granule cells, as investigated by cellular uptake of the fluorescent dye propidium iodide (PI), immunostaining for active caspase 3 and c-Jun/AP-1 (N) and fragmentation of nuclei as seen in Hoechst 33342 staining. All four markers appeared after 12 h of colchicine exposure. Two of them, active caspase 3 and c-Jun/AP-1 (N) displayed a similar time course and reached a maximum after 24 h of exposure, 24 h ahead of both PI uptake and Hoechst 33342 staining, which together displayed similar time profiles and a close correlation. In 3-week-old cultures, colchicine did not induce apoptotic or necrotic cell death. Attempts to interfere with the colchicine-induced apoptosis in 1-week-old cultures showed that colchicine-induced PI uptake and formation of apoptotic nuclei were temporarily prevented by coapplication of the protein synthesis inhibitor cycloheximide. Application of the pancaspase inhibitor z-VAD-fmk almost completely abolished the formation of active caspase 3 protein and apoptotic nuclei induced by colchicine, but the formation of necrotic nuclei increased correspondingly and the PI uptake was unaffected. We conclude that colchicine induces caspase 3-dependent apoptotic cell death of dentate granule cells in hippocampal brain slice cultures, but the apoptotic cell death is highly dependent on the developmental stage of the cultures.
Kristensen
Bjarne W
BW
Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Winsløwparken 21, DK-5000 C, Odense, Denmark. bkristensen@health.sdu.dk
Noer
Helle
H
Gramsbergen
Jan Bert
JB
Zimmer
Jens
J
Noraberg
Jens
J
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D003078:Colchicine / Q000633:toxicity*; D003513:Cycloheximide / Q000494:pharmacology; D018891:Dentate Gyrus / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D009336:Necrosis; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D009498:Neurotoxins / Q000633:toxicity*; D011419:Propidium / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D018696:Neuroprotective Agents; D009498:Neurotoxins; D011500:Protein Synthesis Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011419:Propidium; D003513:Cycloheximide; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C017807:bisbenzimide ethoxide trihydrochloride; D003078:Colchicine