Nicholas J. Pantazis - US grants

Nerve growth factor, NGF, is a protein which is involved in the development and maintenance of the sensory and sympathetic nervous systems. Most of our information concerning the biochemical and biological properties of this important protein is derived from studies on NGF found in the mouse salivary gland, and high concentration source for NGF. Although NGF is believed to play a role in many species, including humans, the salivary gland is unlikely to be the source of NGF since NGF is not found in the glands of most animals. No universal source of NGF has been identified and the possibility exists that numerous tissues in vivo synthesize NGF in low concentrations. Since the mouse salivary gland is unique in having such high concentrations of NGF, are the properties of NGF produced in this gland applicable to NGF produced elsewhere? To answer this question, NGF synthesized by several non-salivary gland sources will be studied. Numerous types of cells synthesize NGF in vitro and cell culture systems will be used to study non-salivary gland NGF. Our recent results indicate that fibroblast cells produce two new forms of NGF. The biochemical and biological properties of these new fibroblast NGF molecules will be studied using biochemical techniques (column chromatography, immunoaffinity columns, polyacrylamide gel electrophoresis) coupled with specific NGF radioimmunoassays. Several types of cells in culture (fibroblast, glial, muscle) synthesize NGF and this project will determine if they all produce similar molecules. Cells from different species will be studied in order to establish similarities or differences among NGF molecules seen in various animals. Human NGF will be examined in normal fibroblasts and in fibroblasts taken from patients with neurofibromatosis, a disease in which changes in NGF have been reported. This project will therefore compare NGF from different cell types, different animals, and in normal versus diseased situations. The properties of NGF precursor molecules will be examined using a cell-free translation system directed with fibroblast cell RNA. NGF precursor molecules may be more readily studied in this system since fibroblasts lack a protein believed to be involved in cleaving an NGF precursor molecule. Immunocytochemical studies on the mouse salivary gland will establish the cellular location for each of the subunits of the NGF molecule. Results from this study will provide additional insight into the possible functional role of this molecule.

DESCRIPTION: (Adapted from the Investigator's Abstract) One of the most detrimental effects of ethanol exposure during nervous system development is depletion of neuronal cells. Ethanol may deplete neurons by several means, one of them is ethanol-induced cell death, which is the focus of this proposal. Ethanol exposure of primary neuronal cultures of cerebellar granule cells (CGC) produces cell death and this cell loss is similar to that observed in animal studies. A valuable property of this model is that both ethanol-sensitive and ethanol-resistant cultures can be obtained in two ways. First, as CGC mature in culture they become ethanol-resistant, called time-dependent ethanol resistance. Second, treating ethanol-sensitive CGC with either NMDA or growth factors makes cells ethanol-resistant, called neuroprotection-dependent ethanol resistance. Of considerable value, ethanol-sensitive and ethanol-resistant cells can be compared to identify molecular differences which may be linked to ethanol-induced cell death. Specific Aim 1 will determine whether ethanol causes both necrotic and apoptotic cell death in CGC cultures. Our previous studies determined that the nitric oxide-cGMP (NO-cGMP) pathway is essential for NMDA-mediated neuroprotection against ethanol-induced cell death in CGC cultures. Specific Aim 2 will study whether the pathway plays an essential role in both time-dependent and neuroprotection-dependent ethanol resistance. CGC cultures will be established from mutant mice which lack the NO-cGMP pathway, in order to evaluate the function of the pathway in ethanol neurotoxicity and resistance. This specific aim may establish that the NO-cGMP pathway is activated by diverse signals (NMDA, growth factors, and time) and it plays an essential role in ethanol resistance. Since Ca2+ has been linked to cell death, Specific Aim 3 will examine the role of Ca2+ in ethanol-induced cell death in CGC cultures. Both ethanol-sensitive and ethanol-resistant cultures will be compared to determine whether there are differences in the maintenance of intracellular Ca2+, which may be linked to ethanol-induced cell death. In summary, this proposal will use ethanol-sensitive and ethanol-resistant cultures to obtain information about the nature of ethanol-induced cell death (necrotic versus apoptotic), the functional role of the NO-cGMP pathway in protecting neurons against this cell death, and the role of Ca2+ in this cell death. These closely-linked specific aims will greatly enhance our knowledge about molecular mechanisms involved in one of ethanol's most detrimental effects, neuronal cell death.

DESCRIPTION (provided by applicant): Fetal alcohol syndrome (FAS) is an important cause of mental retardation. Exposure of the developing brain to alcohol can induce the death of neurons, which contributes strongly to learning deficits and neurological problems associated with FAS. Understanding the factors that influence neuronal vulnerability to alcohol induced loss in the developing brain is of considerable significance. We hypothesize that as neurons mature, they acquire a neuroprotective, signaling pathway that protects them against alcohol toxicity. We have discovered a neuroprotective pathway, which is mediated by nitric oxide (NO). This proposal will elucidate the function and molecular mechanisms of the NO-signaling pathway in protecting the developing brain against alcohol-induced neuronal loss. Our experiments will utilize a mouse strain carrying a null mutation for neuronal nitric oxide synthase (nNOS-/-), thus these animals lack NO-mediated neuroprotection. Specific Aim 1 includes morphological experiments examining the importance of NO-mediated neuroprotection on neuronal survival. Stereological methods will measure alcohol-induced neuronal losses in the hippocampus and cerebellum of mice that express or lack the protective pathway. Alcohol exposure will occur at a variety of ages in order to determine the impact of NO-mediated neuroprotection at different stages of brain development. Specific Aim 2 utilizes behavioral testing (eyeblink conditioning, Morris Water Maze) to determine whether the enhanced alcohol-induced neuronal losses in nNOS -/- mice are linked to greater functional deficits. Alcohol-induced deficits on these tests will be compared in nNOS / and nNOS+/+ mice and linked with neuronal losses. Specific Aim 3 includes molecular studies to determine the mechanism by which NO-signaling provides neuroprotection against alcohol toxicity. Experiments will determine whether nNOS is developmentally regulated in vivo and link alcohol vulnerability to expression levels and activity of nNOS. "Rescue" experiments in which the nNOS gene will be transfected into pathway-deficient neurons in culture derived from nNOS -/- mice will determine whether neuroprotection against alcohol toxicity can be restored. Immunohistochemistry and quantitative RT-PCR will explore the role of cAMP-responsive element binding protein (CREB) as a downstream effector molecule for the NO-signaling and elucidate CREB's role in regulating apoptotic proteins, which may underlie this neuroprotection.