Immunolocalization of calpain I-mediated spectrin degradation to vulnerable neurons in the ischemic gerbil brain

Abstract

Transient ischemia-induced perturbations in calcium homeostasis have been proposed to lead to pathological activation of the cysteine protease calpain I and subsequent delayed neuronal death in the CA1 region of hippocampus. We report here on the design and characterization of antibodies selective for calpain-generated fragments of brain spectrin, and their use for immunoblot and immunohistochemical analyses of calpain activation following cerebral ischemia in the gerbil. Although spectrin was susceptible to degradation in vitro by many mammalian proteases, only calpain degraded spectrin to generate fragments immunoreactive with the antibodies. Following 5 min of global ischemia, immunoreactivity for calpain-degraded spectrin was rapidly (within 30 min) and markedly elevated in the perikarya and dendrites of several populations of forebrain neurons. The rapid calpain activation was completely prevented by the NMDA receptor antagonist MK-801. At later times postischemia, but prior to frank neuronal necrosis, calpain-degraded spectrin was restricted to hippocampal area CA1 pyramidal neurons. Silver impregnation histochemistry confirmed that neuronal damage was confined to area CA1. The results indicate that while nonpathological NMDA receptor stimulation can activate calpain, only those neurons showing sustained calpain activation are destined to die.

Evidence for Activation of Caspase‐3‐Like Protease in Excitotoxin‐ and Hypoxia/Hypoglycemia‐Injured Neurons

“…An increase of the SBDP15O content has been reported in vivo in models for excitotoxicity (Vartanian et a!., 1996), focal ischemia , and global ischemia (Roberts-Lewis et al, 1994) and in cell cultures subjected to excitotoxins ( Siman and Noszek, 1988) and OGD (Wang et a!., 1996a,b). However, those reports preceded the recent discovery that a-spectrin is a substrate for caspases (Nath et a!., 1996a;Cryns et a!., 1996;Vanags et al, 1996).…”

Rapid Calpain I Activation and Cytoskeletal Protein Degradation Following Traumatic Spinal Cord Injury: Attenuation with Riluzole Pretreatment

“…Excessive glutamate receptor activation has also been hypothesized to contribute to spectrin breakdown. Glutamate receptor agonists, as well as ischemic and traumatic brain injury, result in the activation of calpain I and the breakdown of spectrin into calpain Jcleaved fragments (Siman and Noszek, 1988;Siman et al, 1989;Roberts-Lewis et al, 1994;Kampfl et al, 1 996a,b). Finally, several studies have demonstrated that treatments with protease inhibitors that block calpain activity will inhibit MAP2 and spectrmn degradation and will promote neuron survival after excitatory or traumatic CNS injury (Arai et al, 1990;Arlinghaus et al, 1991;Lee et al, 1991;Caner et al, 1993;Rami and Kriegistein, 1993;Bartus et al, 1994).…”

Cellular and Molecular Pathways of Ischemic Neuronal Death

“…This contributes to the dendritic remodeling, interruption of membrane and cytoplasmic transportation, modulation of gene expression, and neuronal degeneration (Wang et al, 1990;Faddis et al, 1997;Wang, 2000). The activation of calpain I has been reported, following the activation of ionotropic glutamate receptors or hypoxic-ischemic insults (Seubert et al, 1988;Ostwald et al, 1993;Roberts-Lewis et al, 1994). Selective inhibitors of calpain I can reduce the NMDA-, kainate-, or AMPA-mediated excitotoxicity to some extent, and attenuate brain damage after hypoxia, focal cerebral ischemia, or transient global cerebral ischemia (Lee et al, 1991;Rami and Krieglstein, 1993;Hong et al, 1994).…”