Neurotrophins are a growing family of protein factors involved in the development, sculpting and maintenance of the nervous system. Nerve growth factor (NGF) is the first-isolated and most thoroughly characterized member of the neurotrophin family. 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 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 NGF-responsive, PC12 cell culture line. PC12 cells expresses the high affinity NGF receptor, p140trkA, which is a transmembrane protein with tyrosine kinase activity. Upon addition of NGF, the tyrosine kinase activity of p140 trkA 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, an inhibitor of protein kinase activity, blocks selectively all the actions of NGF in PC12 cells including stimulation of p140 trkA tyrosine kinase activity indicating the importance of protein phosphorylation with regard to NGF signal initiation and propagation. Investigative studies conducted in this laboratory have examined 1) the biochemical characteristics of NGF-stimulated phospholipase C-gamma (PLC) phosphorylation and 2) the role of protein kinase C (PKC) in mediating activation of phospholipase A2 (PLA) and subsequent metabolism of arachidonic acid (AA) elicited by NGF . NGF stimulates the phosphorylation of an important signal transducing molecule, PLC, on both tyrosine and serine amino acid residues. The tyrosine phosphorylation is mediated directly by NGF-stimulated, p140 trkA tyrosine kinase activity, whereas, PLC serine phosphorylation appears to occur secondarily. Efforts are ongoing to identify the responsible serine kinase(s). Based upon pharmacologic studies involving inhibitors selective for specific classes of serine kinases it appears that both protein kinase A and PKC may be involved demonstrating the capacity for NGF to activity multiple protein kinase cascades. NGF stimulates the release of esterified AA from PC12 cells as a consequence of PLA activation. The role of PKC in regulating this response has been investigated. Down-regulation of PKC or antagonism of its activity by pharmacologically selective inhibitors blocks NGF-induced release of AA. Conversely, activation of PKC, alone, promotes an increase in AA release. Results from these collaborative studies have been submitted to the Journal of Neurochemistry (1995) for publication.
