Biochemical Studies Of Neurons And Other Cell Types

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This section investigates vasoactive intestinal peptide (VIP) as a regulator of brain development and neuroprotection. VIP can influence many processes that are important to development including the survival of nerve cells, neurite extension, excitatory synaptogenesis and embryonic growth and development. Many of the neurotrophic and growth-stimulating actions of VIP are mediated indirectly through secreted, glia-derived substances. Much of the effort in this section resides in identifying the substances that VIP releases and studies their mechanism of action. After identifying the VIP-related, neuroprotective substances, the continuing goal is to develop therapeutic agents that prevent neurodegenerative disease. Previous studies have indicated that one of the protective proteins released by VIP is a femtomolar-acting protein: activity dependent neurotrophic factor (ADNF). A continuing priority of this section is to characterize ADNF at the protein and genetic level and to develop therapeutic agents based on peptides derived from ADNF. Recent evidence indicates that ADNF is a complex of proteins that are characterized by three peaks of biological activity that greatly differ in potency in preventing apoptotic death in cell cultures derived from the central nervous system. During the last year, the fundamental nature of the ADNF-related protective activity has become apparent: multiple proteases are involved. Through the use of specific inhibitors of protease action, all the survival-promoting activity can be inhibited. Furthermore, the protease activity of one of the ADNF components has been demonstrated in a cell-free system utilizing a fluorescent peptide substrate. The protease activity detected with this methodology corresponded very closely to the potency of the survival-promoting activity. These data strongly support the hypothesis that ADNF is a multi-protease complex that can increase the survival of developing neurons exposed to toxic agents that produce apoptotic death. Thus, the proof of principal is emphasized with studies on the protease activity of component II of the ADNF complex; however, all of the neurotrophic activities of the ADNF components may reside in their proteolytic actions. Previous studies have shown that short peptides derived from ADNF and a pharmacologically related protein (activity dependent neuroprotective protein, ADNP) exhibit neuroprotection at femtomolar concentrations. The protective action of these peptides is observed in cultures treated with clinically relevant toxins including glutamate, beta amyloid peptide, iron, hydrogen peroxide and gp120, the HIV envelope protein. Recently, eleven new peptides derived from ADNF have been discovered. All of these peptides are novel, although a number of these structures show homology to known proteases. This is an interesting corollary in that the biological activity ADNF complex is now clearly linked to protease activity. All eleven new peptides were tested for intrinsic survival-promoting activity utilizing a cell culture model of tetrodotoxin-induced neuronal cell death. These experiments indicated that four of the eleven peptides showed potent survival-promoting activity. Complex dose/response relationships were confirmed by both neuronal cell counts and the carboxyfluorescene diacetate methodology used to assess neuronal survival. These new peptides provide further basis for the exploration of these structures as lead compounds for therapeutic intervention in the treatment of neurodegenerative disease. VIP is an important regulator of embryonic growth and development during the early postimplantation period of development. VIP receptors are localized to the neural tube at this stage of development and we have performed studies of VIP-mediated regulation of gene expression and neurotrophin release with the use of explanted neural tubes. With this model system we have shown that nerve growth factor (NGF) is among the factors regulated by VIP in neural tube preparation explants from the early postimplantation mouse embryo. Although a small amount of NGF was found in the fully processed form, VIP stimulation releases NGF primarily in the form of a prohormone from the embryonic mouse neural tube preparation. VIP induced significantly more immunoreactive NGF in both conditioned medium and within the neural tube preparation itself, than was found in untreated neural tube preparations and preparations treated with equal concentrations of the highly homologous neuropeptide, PACAP. The VIP concentrations used were within its' biologically active range in CNS tissues and these concentrations released immunoreactive NGF at concentrations which elicit biological actions. However, the concentrations of immunoreactive NGF in the conditioned medium represent NGF diluted into the incubation medium and suggest that, within the local environment of neural tube tissues, VIP stimulates the release of NGF prohormone at concentrations many fold greater. This model system has revealed a functional relationship between VIP and NGF and support the hypothesis that VIP mediates developmental events through its action as a secretagogue. This study is the first report linking the actions of VIP to releasable NGF prohormone in neural tissue and implies that drugs mimicking the neurotrophic action of VIP in the central nervous system could elicit the protective and repair modalities of NGF.