Lesiuk, Howard,Hewitt, Kimberley E.2009-03-232009-03-2319991999Source: Dissertation Abstracts International, Volume: 61-01, Section: B, page: 0119.9780612465237http://hdl.handle.net/10393/8852http://dx.doi.org/10.20381/ruor-16025The ubiquitous, Ca2+-dependent, neutral proteases mu-calpain and m-calpain are heterodimeric regulatory enzymes consisting of an 80 kDa catalytic subunit and a 30 kDa regulatory subunit. In the central nervous system (CNS), calpain-mediated proteolysis of structural proteins and metabolic enzymes is a common feature of Ca2+-dependent neuronal death in both in vitro and in vivo models of excitotoxicity and stroke. A number of inhibitor studies have lead to the conclusion that calpain activation is a necessary and contributory event in the death of neurons under ischemic or ischemic-like conditions. Although the mechanism of calpain activation has been extensively studied in vitro, a precise understanding of the activation and regulation of the enzyme within cells remains elusive. In this thesis we have examined the effects of lethal and non-lethal Ca2+ influx on the activation of mu-calpain, and the mechanism by which that activation occurs. We have demonstrated that calpain activation in cultured neurons proceeds by a mechanism clearly distinct from the "traditional" route observed in erythrocytes and other non-CNS cells. Transiently exposing primary rat cortical neurons to lethal NMDA caused protracted calpain activation, measured as increased endogenous spectrin hydrolysis, that was independent of translocation and autolysis of the protease. Calpain was largely membrane associated in cortical neurons and consequently neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. One intracellular mediator of NMDA effects is protein kinase C (PKC). The rapid inactivation of PKC is a characteristic and necessary event in the excitotoxic death of neurons both in vivo and in vitro. We demonstrate that the NMDA-induced inactivation of PKC in cortical neurons contributes to calpain activation. The neurotrophin BDNF, which protects neurons from excitotoxicity by preventing the NMDA-induced inactivation of PKC, also blocked calpain activation. The use of PKC inhibitors showed this effect of BDNF on calpain activity to be mediated by the NMDA selective inactivation of PKC. Furthermore, sublethal NMDA receptor activation and PKC inhibition, treatments unable to activate calpain when applied separately, were in combination able to promote calpain-mediated spectrin hydrolysis. (Abstract shortened by UMI.)245 p.Biology, Neuroscience.Thesis