The main objective of this project is to dissect the underlying mechanisms of the transcriptional network controlling neuronal differentiation, maintenance and survival. This application specifically focuses on the transcriptional dynamics controlled by the neuronal-specific basic Helix-Loop-Helix (bHLH) transcription factor Nexl/Math-2, which is a member of the NeuroD subfamily. Our studies suggest that Nex 1 is a key regulator of the neuronal differentiation program as its expression is important to the execution of the NGF-induced differentiation pathway. Its overexpression in PC12 cells induces neurite outgrowth and expression of the neuronal marker GAP-43 in an NGF-independent manner. Most importantly, our studies reveal the first evidence that Nex 1 prevents apoptosis of NGF-deprived PC12 cells. Thus, the bHLH Nex 1 may be a key factor linking terminal differentiation to maintenance and survival of differentiated neurons. To test our hypotheses and to elucidate the transcriptional functions of Nex1 during neuronal differentiation and survival, we plan to execute the experiments outlined in the three following aims. Our strategy will employ the rat pheochromocytoma cell line, PC 12, as it is a well-established system to study neuronal differentiation, maintenance, and survival.
Aim I will focus on the Nex 1-modulated transcriptional pathways leading to terminal neuronal differentiation. Initially, we will generate a double stable Nex 1 inducible PC 12 cell line that will permit the manipulation of both the timing and levels of Nex I expression. We will determine the extent of neuronal differentiation by assessing the level of neuronal markers using a combination of western blot and immunocytochemistry. Finally, we will perform a dynamic gene profiling at different stages of Nex1-induced differentiation by differential display and cDNA expression microarray.
Aim II will address the molecular mechanisms by which Nex1 promotes neuronal survival. We will measure the expression of anti-apoptotic and pro-apoptotic genes by RT-PCR, as well as the temporal expression of neuronal markers, using our stable cell lines. Finally, aim III will explore the transcriptional mechanisms controlling Nex1 expression in the NGF-induced differentiation pathway, using a combination of DNase I footprinting, EMSA, and CAT assay analyses. The fact that Nex 1 exhibits neuroprotective properties makes it a promising target to enhance neuronal survival and design novel therapeutical approaches to treat neurodegenerative diseases, age-related neuronal disorders, and CNS injuries.
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