Selective release of calpain produced alphalI-spectrin (alpha-fodrin) breakdown products by acute neuronal cell death.
Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.
Dutta
Satavisha
S
Department of Neuroscience Therapeutics, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA.
Chiu
Yuk Chun
YC
Probert
Albert W
AW
Wang
Kevin K W
KK
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000172:deficiency; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D008387:Marine Toxins / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D035964:Oxocins / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013049:Spectrin / Q000096:biosynthesis / Q000378:metabolism*; D013997:Time Factors; D000095702:Polyether Toxins
D002154:Calpain; D004151:Dipeptides; D005947:Glucose; D008387:Marine Toxins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D035964:Oxocins; D013049:Spectrin; C020394:maitotoxin; D000095702:Polyether Toxins; C058076:calpain inhibitor III