I have demonstrated that neurotrophins (NTs) protect cultured embryonic rat basal forebrain cholinergic neurons during hypoglycemic stress. The goal of the proposed study is to identify important second messenger and early effector pathways involved in this protection. Many of the experiments will utilize cholinergic neurons purified by immunopanning. I have preliminary evidence that the following contribute importantly to NT-mediated protection of cholinergic neurons during hypoglycemic stress: (1) activation of phosphotidyl inositol (PI) 3-kinase, and (2) activation of NF-kB (perhaps) via the p75 NT receptor (p75NTR). Receptor-blocking antibodies, neurons from receptor-deficient mice, and mutant forms of nerve growth factor (NGF) will be used to test the involvement of the p75 NTR and TrkA NGF receptors. Assays of kinase activity and immunohistochemical staining for activated forms of signalling molecules will be used to clarify the stress-protective pathways activated by NTs under hypoglycemic conditions. Inhibitors and activators of specific kinases and introduction (by transfection) of mutant kinases will be tested to determine whether they block, mimic or occlude NT-mediated protection. To investigate early effector pathways by which NTs might produce their stress-protection, imaging techniques will be used to measure NT effects on cytosolic [Na+] and [Ca2+], mitochondrial [Ca2+] and mitochondrial membrane potential. Alternative energy substrates, respiration measurements and inhibitors of specific mitochondrial complexes will be tested to determine whether NT-mediated protection requires up-regulation of plasma membrane glucose transporters and/or alterations in mitochondrial energy and/or Ca2+ metabolism. I have recently discovered that certain bone morphogenetic proteins (BMPs) robustly enhance NT-mediated protection during hypoglycemic stress, and the proposed experimental plan integrates studies to uncover basic mechanisms underlying this synergistic effect.
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