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 molecular weight (MW) neurofilament 68 (NF68) break down products (BDPs) detected in cortical homogenates following unilateral cortical impact injury in rats. One dimensional immunoblot of BDPs obtained from in vitro cleavage of enriched neurofilaments (NF) by purified micro-calpain, m-calpain, cathepsin, B, cathepsin D, and CPP32 (caspase-3) were compared to in vivo samples from rats following traumatic brain injury (TBI). Comparison of these blots provided information on the relative contribution of different cysteine or aspartic proteases to NF loss following brain injury. As early as 3 hrs post-injury, cortical impact resulted in the presence of several lower MW NF68 immunopositive bands having patterns similar to those previously reported to be produced by calpain mediated proteolysis of neurofilaments. Only micro-calpain and m-calpain in vitro digestion of enriched neurofilaments contributed to the presence of the low MW 57 kD NF68 break down product (BDP) detected in 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 dimensional peptide maps containing a 1:1 ratio of in vivo and in vitro tissue samples showed complete comigration of lower MW immunopositive spots produced by TBI or in vitro incubation with m-calpain, thus providing additional 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 to the basic pole (+) suggesting that dephosphorylation of the NF68 subunit pool may be associated with NF protein loss following TBI, an observation not previously noted in any model of experimental brain injury.

Regional calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury.

Activity of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein alpha-spectrin into signature spectrin breakdown products (SBDPs) was used to provide the first systematic and simultaneous comparison of changes in activity of these two families of cysteine proteases after traumatic brain injury (TBI) in rats. Distinct regional and temporal patterns of calpain/caspase-3 processing of alpha-spectrin were observed in brain regions ipsilateral to the site of injury after TBI, including large increases of 145 kDa calpain-mediated SBDP in cortex (up to 30-fold), and enduring increases (up to 2 weeks) of 145 kDa SBDP in hippocampus and thalamus. By contrast, 120 kDa caspase-3-mediated SBDP was absent in cortex and showed up to a 2-fold increase in hippocampus and striatum at early (hours) after TBI. Future studies will clarify the pathological significance of large regional differences in activation of calpain and caspase-3 proteases after TBI.

Temporal relationships between de novo protein synthesis, calpain and caspase 3-like protease activation, and DNA fragmentation during apoptosis in septo-hippocampal cultures.

Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 microM), calpain and caspase 3-like proteases processed alpha-spectrin to their signature proteolytic fragments prior to endonuclease-mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of alpha-spectrin by calpains and caspase 3-like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3-like proteases. Calpain inhibitor II and the pan-caspase inhibitor Z-D-DCB each inhibited their respective protease-specific processing of alpha-spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3-like proteases is an early event in staurosporine-induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation.