Abstract
Activation of calpain results in the breakdown of α II
spectrin (αfodrin), 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 cellconditioned media
from SHSY5Y 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 timedependent
manner. MDL28170, a selective calpain inhibitor, was
the only protease inhibitor tested that significantly reduced
MTXinduced SBDP150 release. The cellconditioned
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 cellconditioned
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.