Although maintaining the integrity of the central nervous system (CNS) is of extreme importance to human health and function, relatively little is known about the signaling processes involved in the proliferation and differentiation of CNS neurons. Growth factors such as fibroblast derived growth factor (FGF) have been implicated as mitogenic, differentiating agents and neurotrophic agents for neuronal stem cells and neurons in a number of CNS regions. In order to investigate the mechanism by which growth factors influence neuronal development and function, the applicant has previously generated a conditionally immortalized El 7 rat hippocampal cell line (H 1 9- 7) that proliferates in response to EGF and differentiates into a nonproliferating neuronal phenotype in response to bFGF. During the previous grant period, the applicant investigated the signal transduction mechanisms by which FGF activated Raf promotes differentiation in this model system. In particular, she focused on the role of the Ras/Raf/MAPK signaling pathway. The results indicate that there are at least two mechanisms by which differentiation can be induced. The applicant hypothesizes that one of these signaling pathways involves a novel protein kinase C-dependent mechanism for activating MAPK kinase (MEK), and the other signaling pathway involves a new member of the ERK family of MAP kinases activated by Raf. It is further hypothesized that these novel MAP kinase signaling cascades act by inducing expression of p39, a neuronal cyclin that the applicant has shown regulates neurites extension in H19-7 cells. To test these hypotheses, the following specific aims are proposed: 1) Determine whether protein kinase C mediates FGF and Raf activation of MEK; 2) Purify, clone and characterize the novel ERK associated with Raf; and 3) Determine the mechanism by which FGF and Raf regulate the neuronal cyclin p39. These signaling pathways will be tested in both the immortalized hippocampal cell lines and primary neuronal cell cultures. The results of these studies will define key signaling intermediates that influence neuronal differentiation and increase understanding of the processes that control the switch from cell growth to differentiation in the CNS. Failure to complete the proper differentiation program can lead to hyperproliferation and even tumor formation during human development.
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