NGF is the first-isolated and most thoroughly characterized member of a family of neurotrophic factors collectively referred to as neurotrophins. Among the most significant biologic effects of NGF are promotion of the survival and neuronal differentiation of distinct elements of the nervous system. These include peripheral sympathetic and sensory neurons as well as central cholinergic neurons of the basal forebrain, a region prominently involved, pathologically, in the neural degeneration associated with Alzheimer's Disease. Understanding the mechanisms of NGF action could lead to treatments for a variety of neurodegenerative conditions. The principle model used to investigate NGF signaling is the PC12 cell culture line. Upon addition of NGF, tyrosine kinase activity of p140 trkA, the high affinity NGF receptor, is stimulated and summarily, previously round, replicating cells cease to divide and become, phenotypically, sympathetic neurons replete with electrically excitable neuritic processes. The alkaloid, K-252a, a kinase inhibitor, blocks selectively all the actions of NGF in PC12 cells including stimulation of p140 trkA tyrosine kinase activity indicating the importance of protein phosphorylation in NGF signaling. Investigative studies conducted in this laboratory have examined 1) the biochemical characteristics of NGF-stimulated phospholipase C (PLC)-gamma phosphorylation and 2) the possibility that non-neurotrophic peptides capable of promoting protein phosphorylation in PC12 cells will, when added in combination, induce an NGF-like neuronal differentiation. NGF stimulates the phosphorylation of an important signal transducing molecule, PLC-gamma, on both tyrosine and serine. The tyrosine phosphorylation is mediated directly by NGF-stimulated kinase activity of p140 trkA while serine phosphorylation appears to occur secondarily. Efforts are ongoing to identify the responsible kinase. A number of known serine/threonine kinases activated by NGF including B-raf, MAP kinase, cAMP-dependent protein kinase, and protein kinase N do not seem to be involved, suggesting a novel, NGF-stimulated serine kinase is responsible. Preliminary findings indicate that the mitogen, EGF, which promotes tyrosine phosphorylation in PC12 cells, and the neuropeptide VIP, an activator of adenylate cyclase when added in combination promote a neuronal differentiation in PC12 cells comparable to that observed for NGF. Investigations are being conducted to determine whether, in fact, biochemically, the responses are equivalent.
