Neuronal nitric oxide synthase and calmodulin-dependent protein kinase IIα undergo neurotoxin-induced proteolysis

Abstract

Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N- methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m- calpain autolytic fragmentation and nonerythroid α-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-α (CaMPK-IIα) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl- Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified μ- and m-calpain produced fragmentation patterns for CaMPK-IIα and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-α are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIα and nNOS. The degradation of CaMPK-IIα, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.