Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Czeta.
Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 micro m NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.
Koponen
Susanna
S
Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Finland.
Kurkinen
Kaisa
K
Akerman
Karl E O
KE
Mochly-Rosen
Daria
D
Chan
Pak H
PH
Koistinaho
Jari
J
D000818:Animals; D001253:Astrocytes / Q000166:cytology; D002545:Brain Ischemia / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D016202:N-Methylaspartate / Q000633:toxicity*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology; D009498:Neurotoxins / Q000633:toxicity; D010455:Peptides / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000099221:Protein Kinase C zeta
D002118:Calcium; D004791:Enzyme Inhibitors; D016202:N-Methylaspartate; D018696:Neuroprotective Agents; D009498:Neurotoxins; D010455:Peptides; D011493:Protein Kinase C; D012333:RNA, Messenger; D000099221:Protein Kinase C zeta