Data from the initial funding period demonstrated that several neurotrophic factors including basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) can protect cultured hippocampal neurons against excitotoxic and metabolic insults. In each case, the neurotrophic factor prevented glutamate receptor-mediated loss of cellular calcium homeostasis. The proposed studies test the hypothesis that neurotrophic factors regulate the expression and/or activity of specific calcium-regulating proteins, and that mitogen activated protein (MAP) kinases are involved in the signal transduction mechanisms. This work will employ a well-characterized embryonic rat hippocampal cell culture system in which: neuronal survival can be readily quantified; intracellular free calcium levels ([Ca2+]i) can be measured by fluorescence imaging; levels of specific proteins and mRNAs can be quantified; and antisense oligodeoxynucleotides (ODNs) can be used to produce neurons deficient in a specific protein. The first experiments will determine whether bFGF, NGF and/or BDNF affect the expression of specific glutamate receptor mRNAs and proteins. The second set of experiments will test the hypothesis that neurotrophic factors affect the expression and/or activity of the plasma membrane Ca2+ ATPase (PMCA), Na+/Ca2+ exchanger, and the calcium-binding protein calbindin. Activation of MAP kinases by bFGF, NGF and BDNF will be characterized using phosphorylation assays. The hypothesis that MAP kinases mediate the neuroprotective actions of neurotrophic factors will be tested by examining neurons rendered deficient in MAP kinases by exposure to antisense oligodeoxynucleotides In addition, the effects of MAP kinase depletion on [Ca2+]i homeostasis will be determined in calcium imaging studies. Finally, the hypothesis that MAP kinases mediate the actions of neurotrophic factors on the expression of glutamate receptor proteins, PMCA, Na+/Ca2+ exchanger, and calbindin will be tested. This research will provide fundamental information concerning how neurotrophic factors stabilize neuronal calcium homeostasis and protect neurons against excitotoxic insults. Because loss of [Ca2+]i homeostasis and an excitotoxic mechanism of neuronal injury is believed to be involved in the pathogenesis of several major neurological disorders (e.g., stroke Alzheimer's disease Huntington's disease), the findings should prove valuable in developing preventative and therapeutic approaches to such disorders.
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