Early calpain-mediated proteolysis following AMPA receptor activation compromises neuronal survival in cultured hippocampal neurons.
In this work, we investigated the involvement of calpains in the neurotoxicity induced by short-term exposure to kainate (KA) in non-desensitizing conditions of AMPA receptor activation (cyclothiazide present, CTZ), in cultured rat hippocampal neurons. The calpain inhibitor MDL28170 had a protective effect in cultures treated with KA plus CTZ (p < 0.01), preventing the decrease in MTT reduction caused by exposure to KA (p < 0.001). Caspase inhibition by ZVAD-fmk was not neuroprotective against the toxic effect of KA. At 1 h after treatment, we could already observe significantly increased calpain activity, which was prevented by MDL 28170 and NBQX. Western blot analysis of calpain substrates, GluR1, neuronal nitric oxide synthase (nNOS) and nonerythroid spectrin (fodrin), showed a time-dependent and MDL 28170-sensitive proteolysis of these proteins. This effect was due to calpains, but not caspases, since ZVAD-fmk was ineffective in preventing proteolytic events. Breakdown products of fodrin (BDPs) were detected as early as 15 min after exposure to KA. Overall, these results show early activation of calpains following activation of AMPA receptors as well as compromise of neuronal survival, likely due to proteolytic events that affect proteins involved in neuronal signaling.
Araújo
Inês M
IM
Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal.
Verdasca
Maria J
MJ
Leal
Ermelindo C
EC
Bahr
Ben A
BA
Ambrósio
António F
AF
Carvalho
Arsélio P
AP
Carvalho
Caetana M
CM
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007608:Kainic Acid / Q000506:poisoning; D008840:Microfilament Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D009498:Neurotoxins / Q000506:poisoning; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000502:physiology*; D013049:Spectrin
D002154:Calpain; D002352:Carrier Proteins; D007608:Kainic Acid; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D018091:Receptors, AMPA; D013049:Spectrin