Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis

Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previ ously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478*)SVEAL). Here, by us ing a synthetic peptide (NH2-SVEALC) mimicking the neo-N-terminal of SBDP12 0 as antigen, we report the development of chicken antibodies that specific ally recognize the SBDP120 generated by in vitro caspase-3 digestion of bov ine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to hum an neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-sp ectrin nor its other fragments on Western blots. These anti-SBDP120 work eq ually well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical s tudies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN 's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These ant i-SBDP120s might become powerful markers for apoptotic neurons in various n eurological or neurodegenerative conditions in vivo. (C) 2000 Elsevier Scie nce Ltd. All rights reserved.