title: Mechanisms of calpain mediated proteolysis of voltage gated sodium channel α-subunits following in vitro dynamic stretch injury.
abstract: Although enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) α-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of α-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh α-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Catherine R
authors.initials: CR
authors.lastname: von Reyn
authors.forename: Rosalind E
authors.initials: RE
authors.lastname: Mott
authors.forename: Robert
authors.initials: R
authors.lastname: Siman
authors.forename: Douglas H
authors.initials: DH
authors.lastname: Smith
authors.forename: David F
authors.initials: DF
authors.lastname: Meaney
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001930:Brain Injuries / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D007150:Immunohistochemistry; D009474:Neurons; D059748:Proteolysis; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*
chemical_list: D016194:Receptors, N-Methyl-D-Aspartate; D015222:Sodium Channels; D002154:Calpain