EFFECTS OF OVEREXPRESSION OF NERVE GROWTH FACTOR

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NGF is a neurotrophic factor known to play a central role in neuron survival, axon growth and gene expression during development and in the adult. NGF can also modulate the physiology of primary sensory neurons involved in pain and temperature sensation. We have developed a new approach to study the role of NGF in neuronal development that utilizes a transgenic mouse model system. Three lines of transgenic mice have been isolated that express increased levels of NGF in the skin, a peripheral target tissue that normally produces NGF. These mice express a transgene containing tissue-specific regulatory sequences from a human epidermal keratin gene (K14) linked to the mouse NGF cDNA. The K14-NGF transgene targets NGF expression to the basal cells of the epidermis and other stratified, squamous epithelium. Expression in the skin begins at approximately E14, the time that endogenous NGF levels decline and neuronal cell death is initiated. We have found that the forced upregulation of NGF in the skin during this critical period has profound effects on the development of the peripheral nervous system. The goal of the proposed research is to elucidate the effect of NGF over- expression on trigeminal and dorsal root ganglia, focusing on changes in cell number, neuronal phenotype, and axon growth. The specific aims of these studies are to: 1) Determine if the amount of NGF affects the number and size of primary sensory neurons in trigeminal and dorsal root ganglia. NGF mRNA will be measured using reverse PCR and in situ hybridization analysis. Relative levels of the NGF polypeptide will be measured using western blot analysis. 2) Determine if increased NGF expression induces changes in primary sensory projections to peripheral and central targets. Immunocytochemistry and western blotting for neurofilaments and NGF will be used to examine the density of innervation of the transgenic skin. In addition, tract tracing will be used to determine if central projections of sensory afferents are affected. 3) Determine if over-expression of NGF differentially affects subpopulations of sensory neurons. In situ hybridization will be used to neurochemically identify primary sensory neurons in the trigeminal and dorsal root ganglia in normal and transgenic mice. Once identified the number and somal size of these subpopulations will be determined. Initial studies will use cRNA and oligonucleotide probes for mRNAs known to be regulated by NGF, including preprotachykinin, calcitonin gene- related peptide, cholecystokinin, somatostatin and tyrosine hydroxylase mRNAs. This model provides a unique means to test the major tenet of the neurotrophic hypothesis in an in vivo system in which changes in the level of NGF are restricted to a single target tissue of peripheral neurons.