Studies on the mechanisms of excitatory amino acid (EAA) neurotoxicity conducted in the initial period (1900-1992) of the program project have established that during abusive EAA receptor stimulation the neuronal homeostatic mechanisms are overwhelmed and a pathological homeostatic destabilization (HD) of free cytosolic Ca ([Ca2+](i) and protein kinase C (PKC) translocation ensues. This ISD of [Ca2+](i) and PKC triggers the subsequent overactivation of Ca2+-dependent enzymes (proteases, ornithine, decarboxylase, NO synthase) that ultimately may bring about delayed neuronal death. The long term goal of our research is to utilize homeostatic destabilization inhibitors (HDI) for the treatment of secondary neuronal damage due to abusive EAA receptor stimulation following brain ischemia, trauma, and Alzheimer's disease. An ideal HDI should: 1) be targeted specifically to the brain structures that are in danger, without blocking EAA mediated synaptic transmission in unaffected brain areas that perform compensatory vital functions; 2) allow the pharmacological rectification of only the pathological consequences of the pathological process leading to neuronal death; (3) allow drug intervention after termination of the EAA receptor abusive insult. To design ideal HDI we should be able to fully understand the signal amplification steps that specifically differentiate signal transduction following physiological glutamate receptor use from that following pathological receptor abuse. To this end the project will be focused on the following: 1) to determine in vitro (primary cultured neurons, cerebellar and cortical) the molecular mechanisms of [Ca2+](i) following EAAs receptor abuse. A number of mechanisms operates to maintain the neuronal homeostasis of [Ca2+](i) - these mechanisms include a) plasma membrane Ca2+ channels (VOC or ROC), b) plasma membrane Na+ exchanger, c) Ca2+ ATPase, which pumps Ca2+ across the neuronal membrane, d) transport proteins on the membrane of the endoplasmic reticulum and the mitochondria, e) Ca2+ binding proteins. In this specific aim we plan to single out the principal mechanism or the constellation of mechanisms that contribute to the HD of [Ca](i) 2) to study in vivo how EAA receptor abuse relates to protracted PKC activation and translocation and to neuronal damage in the area that surrounds a focal ischemic brain lesion. We began to study biochemical, histochemical and neurological changes occurring in rats subjected to focal brain ischemia induced in the cerebral cortex with photochemical Rose Bengal method (35). If EAA abuse receptor stimulation is involved, blockade of NMDA, non-NMDA glutamate receptors and gangliosides should curtail the cascade of pathophysiological events in the neurons of the area penumbra and reduce behavioral deficits caused by the photochemical stroke.
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