Degradation of beta II-Spectrin Protein by Calpain-2 and Caspase-3 Under Neurotoxic and Traumatic Brain Injury Conditions

A major consequence of traumatic brain injury (TBI) is the rapid proteolytic degradation of structural cytoskeletal proteins. This process is largely reflected by the interruption of axonal transport as a result of extensive axonal injury leading to neuronal cell injury. Previous work from our group has described the extensive degradation of the axonally enriched cytoskeletal alpha II-spectrin protein which results in molecular signature breakdown products (BDPs) indicative of injury mechanisms and to specific protease activation both in vitro and in vivo. In the current study, we investigated the integrity of beta II-spectrin protein and its proteolytic profile both in primary rat cerebrocortical cell culture under apoptotic, necrotic, and excitotoxic challenge and extended to in vivo rat model of experimental TBI (controlled cortical impact model). Interestingly, our results revealed that the intact 260-kDa beta II-spectrin is degraded into major fragments (beta II-spectrin breakdown products (beta sBDPs)) of 110, 108, 85, and 80 kDa in rat brain (hippocampus and cortex) 48 h post-injury. These beta sBDP profiles were further characterized and compared to an in vitro beta II-spectrin fragmentation pattern of naive rat cortex lysate digested by calpain-2 and caspase-3. Results revealed that beta II-spectrin was degraded into major fragments of 110/85 kDa by calpain-2 activation and 108/80 kDa by caspase-3 activation. These data strongly support the hypothesis that in vivo activation of multiple protease system induces structural protein proteolysis involving beta II-spectrin proteolysis via a specific calpain and/or caspase-mediated pathway resulting in a signature, protease-specific beta sBDPs that are dependent upon the type of neural injury mechanism. This work extends on previous published work that discusses the interplay spectrin family (alpha II-spectrin and beta II-spectrin) and their susceptibility to protease proteolysis and their implication to neuronal cell death mechanisms.