Generation of spectrin breakdown products in peripheral nerves by addition of m-calpain

Abstract

Identification of spectrin breakdown products (SBP) in tissues of the central nervous system (CNS) has been used to monitor calpain activity in models of neurodegeneration. We investigated the use of this technique in the peripheral nervous system (PNS) in order to use it as a marker of calpain- mediated proteolysis during axonal degeneration. Using in vitro methods for activation of calpains, we compared brains and sciatic nerves from rats for the presence of calpain-specific SBP. The 150-kDa SBP identified on western blots was demonstrated in brain and nerve homogenates subjected to membrane disruption in the presence of calcium. Incubation of tissues with recombinant m-calpain generated SBP in a dose-dependent fashion, and calpastatin inhibited the generation of SBF by either paradigm. In contrast to brain, sciatic nerves showed the presence of SBP even in noninjured tissues, suggesting a basal level of calpain activity in peripheral nerves. Time- course experiments showed that the generation of SBP in sciatic nerves correlated with the breakdown of axonal neurofilaments. SBP peaked within minutes after addition of m-calpain and disappeared in the homogenates before 1 h, indicating that identification of SBP is a transient phenomenon. These data provide a potential new way for studying axonal degeneration in both experimental and human neuropathies.