Voltage-dependent sodium channels are responsible for action potential generation in the axons and cell bodies of peripheral nervous system (PNS) neurons. Through collaborations described in this program project we have determined that the predominant sodium channel in PNS is one that we have named Peripheral Nerve type 1 (PN1). PN1 is the first example of a gene that is expressed exclusively in PNS neurons. PN1 gene expression is rapidly and selectively induced in PC12 cells in response to one-minute treatments of neuronal growth factors and the cytokine, interferon-gamma, leading to membrane excitability. The """"""""triggered"""""""" induction of PN1 mRNA occurs through a novel signal transduction pathway that is being-defined in project 2. The overall goal of this project is to identify the DNA elements and cognate transcription factors that control expression of the PN1 sodium channel gene.
Specific Aim 1 focuses on identifying, through genetic and biochemical approaches, the DNA elements and DNA-binding proteins that restrict the expression of PN1 reporter genes to PC12 cells. The yeast one-hybrid screen, developed originally in the PI's lab to isolate factors required for expression of CNS sodium channel genes, will be exploited for these studies.
Specific Aim 2 focuses on elucidating the DNA elements and transcription factors that mediate the """"""""triggered"""""""" induction of PN1 mRNA in PC12 cells. Reporter genes containing different PN1 gene sequences will be introduced into PC12 cells and then treated briefly with NGF or interferon-gamma. The DNA- binding proteins will be identified using the one-hybrid genetic screen.
Specific Aim 3 focuses on determining whether the genetic elements that regulate expression of PN1 reporter genes in PC12 cells are sufficient to restrict expression of the same reporter genes to PNS neurons in vivo, in transgenic mice. Expression patterns of the reporter genes will be compared to the expression pattern of the endogenous gene and to expression of the type II silencer protein, REST. The results from these studies will provide insights into the basis for processing sensory, and particularly pain, information at the cellular and molecular levels. The knowledge of the DNA elements and proteins that restrict expression of the PN1 gene specifically to the PNS also provides a potential means to manipulate the phenotype of sensory or sympathetic neurons through overexpression of proteins in these neurons.
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