Knockdown of m-calpain increases survival of primary hippocampal neurons following NMDA excitotoxicity.
The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific. In this study, we evaluated the relative role of m-calpain and mu-calpain in a primary hippocampal neuron model of NMDA-mediated excitotoxicity. Baseline mRNA expression for the catalytic subunit of m-calpain (capn2 ) was found to be 50-fold higher than for the mu-calpain catalytic subunit (capn1) based on quantitative real-time PCR. Adeno-associated viral vectors designed to deliver short hairpin RNAs targeting capn1 or capn2 resulted in 60% and 90% knockdown of message respectively. Knockdown of capn2 but not capn1 increased neuronal survival after NMDA exposure at 21 days in vitro. Nuclear translocation of calpain substrates apoptosis inducing factor, p35/p25 and collapsin response mediator protein (CRMP) 2-4 was not detected after NMDA exposure in this model. However, nuclear translocation of CRMP-1 was observed and was prevented by capn2 knockdown. These findings provide insight into potential mechanisms of calpain-mediated neurodegeneration and have important implications for the development of isoform-specific calpain inhibitor therapy.
Bevers
Matthew B
MB
Department of Emergency Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104, USA.
Lawrence
Eric
E
Maronski
Margaret
M
Starr
Neasa
N
Amesquita
Michael
M
Neumar
Robert W
RW
D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects; D006624:Hippocampus / Q000166:cytology*; D016202:N-Methylaspartate / Q000633:toxicity*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects*; D021381:Protein Transport / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D014162:Transfection / Q000379:methods
D018690:Excitatory Amino Acid Agonists; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016202:N-Methylaspartate; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain