The Broad, Long Term Objective of this proposal is to understand the role of microtubular proteolysis in ischemic neuron death. Cytoskeletal disruption and loss of microtubule- associated proteins (MAPs) antedate necrosis of selectively vulnerable neurons. The Hypothesis states that these alterations of microtubular elements predict selective vulnerability and are caused by excitotoxic activation of calpain, a calcium-mediated neutral protease. The four-vessel occlusion model of reversible forebrain ischemia will be employed toe test the Hypothesis.
The Specific Aims are: (1) To define the temporal sequence of neuronal calpain activation and cytoskeletal disruption in postischemic neuronal injury, (2) To determine if the regional distribution-of calpain correlates with microtubular proteolysis and selective vulnerability to ischemic injury, and (3) To determine if microtubular proteolysis and ischemic neuronal necrosis may be prevented by calpain inhibitors or antagonists for excitotoxins. Immunocytochemical studies of heat-shock protein and the neuronal cytoskeletal proteins tau, MAP2, MAP 1B, and calpain-cleaved spectrin will be performed and correlated with quantitative western blots to accomplish Specific Aim #1. Experiments designed to accomplish Specific Aim #2 will include immunocytochemical studies of calpain to be correlated with in situ hybridization showing transcription of calpain mRNA A.
Specific Aim #3 will be accomplished by performing quantitative morphometry in ischemic hippocampus taken from animals treated with glutamate receptor antagonists or calpain inhibitors. These experiments will be correlated with immunocytochemical and western blot studies, to determine if inhibition of calpain prevents microtubular proteolysis and ischemic neuronal necrosis. The Health-Relatedness of this project is centered around its inquiry into basic mechanisms of neuronal cell death, observed in ischemic or traumatic brain injury, AIzheimer's disease, and similar conditions.
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