The overall goal of the proposed studies is to reveal the mechanisms that govern the expression of cholinergic-specific genes and therefore regulate acetylcholine (ACh) production and storage. The two major genes that determine the cholinergic phenotype are choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (yAChT). They occupy one genomic locus and they are likely controlled by shared transcriptional mechanisms. ChAT and VAChT expression is coordinately regulated by various extracellular stimuli, in particular by two groups of neurotrophins: the cytokines of the leukemia inhibitory factor/ciliary neurotrophic factor (LIF/CNTF) family, and nerve growth factor (NGF). LIF/CNTF signaling through gpl3O-containing receptors is crucial in vivo for both the development of the cholinergic phenotype and its restoration after injury. Thus its effects on the cholinergic properties of neurons are not limited to embryonal development, but persist into adulthood. The expression of these cytokines and their receptors in the nervous system, as well as cholinergic neurons' responsiveness to them, have therefore been the subject of numerous studies. Several intracellular pathways, by which the signal from gpl3O-containing receptors is transmitted to the cell nucleus, have been described. However, which molecules participate in the activation of the cholinergic locus, and with which specific response elements in the cholinergic promoters they interact, is not known. It is also unknown what other pathways modulate LIF/CNTF-mediated signaling in cholinergic cells. The pathways involved in the activation of cholinergic genes will be investigated in model cell lines and in primary spinal cord neurons. The first specific aim of this proposal is to analyze the CNTF-mediated upregulation of ChAT/VAChT expression, with special emphasis on the hypothesis that the transcription factor Stat3 is a mediator of the CNTF effect on the cholinergic locus. Two strategies will be used to test this hypothesis: inhibiting Stat3 function with either antisense RNA or dominant negative mutants, and analysis of CNTF-responsive elements in two major promoters of the cholinergic locus. An alternative hypothesis, that the CNTF effect on the cholinergic locus is mediated by protein kinase C, will also be tested. The second specific aim will be to investigate the mechanism of the downregulation of cholinergic gene expression by the MEK1IMAPK pathway, and particularly its role in inhibiting the CNTF-mediated effects.
The third aim will focus on the observation that NGF, acting through receptor tyrosine kinase TrkA, can activate cholinergic gene expression and ACh production but, paradoxically, it also down-regulates the CNTF effect, pointing to interference between the NGF and CNTF signaling pathways. The molecular mechanisms of these two opposing functions of NGF will be analyzed with receptor TrkA mutants incapable of using specific subsets of pathways normally activated in NGF/TrkA signaling. Additionally, the importance of subcellular localization of TrkA for its action on cholinergic gene expression will be examined. ? ?
