Calpain Inhibitors Protect Against Depolarization-induced Neurofilament Protein Loss of Septo-hippocampal 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 septo-hippocampal cultures. One day after depolarization, Western blot analyses revealed losses of all three neurofilament proteins. Increasing the extracellular calcium concentration from 1.8 to 5.8 mM CaCl₂ in the presence of 60 mM KCl produced increased losses of all three neurofilament proteins to ˜80% of control values in the absence of cell death. Calcium-dependent losses of the neurofilament proteins correlated with calcium-dependent increases in calpain 1-mediated breakdown products of alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization and made available to cultures for 24 h, reduced losses of all three neurofilament proteins to ˜14% of control values. The protective effects of calpain inhibitors 1 and 2 were influenced by different levels of extracellular calcium. Qualitative immunohistochemical evaluations confirmed semiquantitative Western blot data on neurofilament loss and protection by calpain inhibitors 1 and 2. We propose that brief depolarization causes loss of neurofilament proteins, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.