Cell-cell interactions play important roles in neuronal development, maintenance and regeneration. These actions are believed to be mediated by both electrical activity and neurotrophic factors. The overall goals of this lab are to determine the molecular mechanisms by which these factors can act. For many cholinergic neurons ciliary neurotrophic factor (CNTF) is a candidate molecule participating in these events. CNTF affects the in vitro development of sensory neurons, autonomic neurons and glia, and rescues target-deprived motor and sensory neurons in vivo. The multiple and dramatic responses of chick ciliary ganglion neurons to CNTF coupled with the accessibility and simplicity of this ganglion make it a valuable model system to study CNTF actions in detail, both in vivo and in vitro. During normal development 50% of these neurons will die, but all can be rescued from this fate in cell culture by a select group of agents including CNTF. Receptor(s) and molecular mechanisms responsible for these effects of CNTF have been proposed, but supporting evidence is lacking, nor is it known what the roles of CNTF may be in vivo. Here the applicants outline plans for determining the molecular mechanisms of action of CNTF and related neurokines on ciliary ganglion neurons by taking three different approaches. First, are studies using biochemical and immunological methods to determine the intracellular and molecular pathways activated by CNTF. These studies will use antibodies for STAT transcription factors and nuclear gel shift assays to identify activated transcription elements and their recognition sites on genes. Second, are microinjection studies using STAT blocking antibodies and response element DNA probes injected into CNTF-sensitive neurons to block specific transduction pathways and determine their role in regulating cell survival and neural phenotype. Third are studies aimed at identifying genes regulated by CNTF on ganglion neurons using reverse transcriptase-polymerase chain reaction coupled with differential display to identify novel genes regulated by CNTF. The value of this research is to advance our understanding of the molecular mechanisms and trophic factor signal pathways in the control of neuronal phenotype. The results may also have implications for the role of trophic factors in neural development, neural disease and neural injury as well.
