IMMUNOBLOT ANALYSES OF THE RELATIVE CONTRIBUTIONS OF CYSTEINE AND ASPARTIC PROTEASES TO NEUROFILAMENT BREAKDOWN PRODUCTS FOLLOWING EXPERIMENTAL BRAIN INJURY IN RATS

Analyses using either one or two-dimensional gel electrophoresis were performed to identify the contribution of several proteases to lower m olecular weight (MW) neurofilament 68 (NF68) break down products (BDPs ) detected in cortical homogenates following unilateral cortical impac t injury in rats. One dimensional immunoblot of BDPs obtained from in vitro cleavage of enriched neurofilaments (NF) by purified mu-calpain, m-calpain, cathepsin, B, cathepsin D, and CPP32 (caspase-3) were comp ared to in vivo samples from rats following traumatic brain injury (TB I). Comparison of these blots provided information on the relative con tribution of different cysteine or aspartic proteases to NF loss follo wing brain injury. As early as 3 hrs post-injury, cortical impact resu lted in the presence of several lower MW NF68 immunopositive bands hav ing patterns similar to those previously reported to be produced by ca lpain mediated proteolysis of neurofilaments. Only mu-calpain and m-ca lpain in vitro digestion of enriched neurofilaments contributed to the presence of the low MW 57 kD NF68 break down product (BDP) detected i n post-TBI samples. Cathepsin B, cathepsin D, and caspase-3 failed to produce either the 53 kD or 57 kD NF BDPs. Further, 1 and 2 dimensiona l peptide maps containing a 1:1 ratio of in vivo and in vitro tissue s amples showed complete comigration of lower MW immunopositive spots pr oduced by TBI or in vitro incubation with m-calpain, thus providing ad ditional evidence for the potential role of calpain activation to the production of NF68 BDPs following TBI. More importantly, 2-dimensional gel electrophoresis detected that immunopositive NF68 spots shifted t o the basic pole (+) suggesting that dephosphorylation of the NF68 sub unit pool may be associated with NF protein loss following TBI, an obs ervation not previously noted in any model of experimental brain injur y.