The Mechanisms of Neuronal Phenotype Control by Neuronal Growth Factors

Simon Halegoua
Professor
PhD, SUNY Stony Brook

Christine Parker - Graduate Student
Yafang Shao - Graduate Student
Julie Rosenbaum - Research Technician

The elaboration of neuronal phenotype induced in cultured cells by neuronal growth factors involves dramatic changes in major cellular processes, including the induction of neurite growth, simulation of neurotransmitter synthesis, cessation of cell division and establishment of the action potential. These changes are evident from as early as seconds to as long as days in the continual presence of the growth factor. Evidence has accumulated for a
model of growth factor action in which a variety of cellular control mechanisms are coordiantely recruited to bear on the process of growth factor induced neuronal differentiation. An ongoing combination of transcriptional, post-transcriptional, and post-translational events coordinate the differentiation process under the reversible and continual control of the neuronal growth factor.

We have been dissecting the signal transduction pathways for Nerve Growth Factor (NGF) actions in a neuronal cell line, PC12, using the following approaches.

(1) We have defined a proto-oncoprotein signaling pathway which underlies major NGF actions such as the induction of neurite growth. This has been determined by microinjection of proto-oncogene protein-blocking antibodies into living cells treated with these growth factors, and by the creation of new PC12 cell lines transfected with inducible oncogenes.

(2) The proto-oncoprotein pathway stimulates second messenger pathways and protein kinase activities responsible for growth factor-regulated activities. These events are being studied by a variety of biochemical techniques such as phosphopeptide mapping of activated enzymes like tyrosine hydroxylase (TH), the enzyme controlling neurotransmitter biosynthesis. Protein kinases mediating site-specific phosphorylations of TH are being identified, purified and characterized.

(3) The NGF signaling pathways leading to both short and long term gene expression changes have been examined. We have used a variety of molecular approaches to assess gene inductions including Northern blot analysis, nuclear run-on transcription analysis, analysis using fusion response element-reporter vectors for transient and permanent transfection. These approaches have led to the identification of branchpoints in the proto-oncoprotein signaling pathway. These branched pathways independently mediate gene inductions leading to the differential establishment of neuronal phenotypic characteristics, such as the action potential, neurite growth, or the expression of neuronal-specific markers. We are further defining the molecular components of these pathways which lead to gene transcriptional regulation.

(4) In collaboration with Dr. Gail Mandel's lab, we have found that the establishment of eletrical excitability in PC12 cells involves the induction of two voltage-dependent sodium channel genes encoding type II and PN1. In contrast to other major neuronal traits such as neurite growth and neurotransmitter synthesis, the expression of PN1 sodium channel (and excitability) is induced in a triggered manner, after only a one minute pulse of NGF treatment. We are defining the novel mechanism of triggered gene induction, using mutational and biochemical analysis of the NGF receptor and its signaling components, and have extended these studies to cytokine-induced triggering via a JAK-STAT pathway. We have identified through differential display, and are currently analyzing, a host of RNAs which are also induced in a triggered manner by growth factors and cytokines.



Relevant Publications:

Kremer, N.E., D'Arcangelo, G., Thomas, S.M., DeMarco, M., Brugge, J.S. & Halegoua, S. (1991) Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of Src and Ras actions. J. Cell Biol. 115: 809-819.

Thomas, S.M., DeMarco, M., D'Arcangelo, G., Halegoua, S. & Brugge, J.S. (1992) Ras is essential to nerve growth factor and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell 68: 1031-1040.

Keegan, K. & Halegoua, S. (1993) Signal transduction pathways in neuronal differentiation. Curr. Opin. in Neurobiol. 3: 14-19.

D'Arcangelo, G. & Halegoua, S. (1993) A branched signaling pathway for nerve growth factor is revealed by Src- Ras-, and Raf-mediated gene inductions. Mol. Cell Biol. 13: 3146-3155.

D'Arcangelo, G., Paradiso, K., Shepherd, D., Brehm, P., Halegoua, S., and Mandel, G. (1993) J. Cell Biol. 122: 915-921.

Rusanescu, G., Qi, H., Thomas, S.M., Brugge, J.S. and Halegoua, S. (1995) Calcium influx induces neurite growth through a Src-Ras signaling cassette. Neuron, 5:1415-1425.

Toledo-Aral, J.J., Brehm, P., Halegoua, S., and Mandel, G. (1995) A single pulse of Nerve Growth Factor triggers long-term neuronal excitability through sodium channel gene induction. Neuron 14: 607-611.

D'Arcangelo, G., Habas, R., Wang, S., Halegoua, S. and Salton, S.R.J. (1996) Activation of co-dependent transcription factors is required for transcriptional induction of the vgf gene by NGF and Mol. Cell Biol. 9: 4621-4631.