CALPAIN INHIBITORS PROTECT AGAINST DEPOLARIZATION-INDUCED NEUROFILAMENT PROTEIN LOSS OF SEPTOHIPPOCAMPAL NEURONS IN CULTURE

We examined the effect of a 6 min depolarization with 60 mM KCl and 1. 8, 2.8 or 5.8 mM extracellular CaCl2 on neurofilament proteins of high (NF-H), medium (NF-M) and low (NF-L) molecular weight in primary sept ohippocampal cultures. One day after depolarization, Western blot anal yses revealed losses of all three neurofilament proteins. Increasing t he extracellular calcium concentration from 1.8 to 5.8 mM CaCl2 in the presence of 60 mM KCl produced increased losses of all three neurofil ament proteins to similar to 80% of control values in the absence of c ell death. Calcium-dependent losses of the neurofilament proteins corr elated with calcium-dependent increases in calpain 1-mediated breakdow n products of alpha-spectrin. Calpain inhibitors 1 and 2, applied imme diately after depolarization and made available to cultures for 24 h, reduced losses of all three neurofilament proteins to similar to 14% o f control values. The protective effects of calpain inhibitors 1 and 2 were influenced by different levels of extracellular calcium. Qualita tive immunohistochemical evaluations confirmed semiquantitative Wester n blot data on neurofilament loss and protection by calpain inhibitors 1 and 2. We propose that brief depolarization causes loss of neurofil ament proteins, possibly due to calpain activation. Thus, calpain inhi bitors could represent a viable strategy for preserving the cytoskelet al structure of injured neurons.