We investigate vasoactive intestinal peptide (VIP) as a regulator of brain development and neuroprotection. VIP can influence many important processes that are central to development, including the survival of nerve cells, neurite extension, excitatory synaptogenesis, and embryonic growth. Many of the neurotrophic and growth-stimulating actions of VIP are mediated indirectly through secreted, glia-derived substances. Much of our effort is devoted to identifying substances whose release is triggered by VIP and its mechanism of action. After identifying the VIP-related, neuroprotective substances, the continuing goal is to develop therapeutic agents that prevent neurodegenerative disease.

Activity-Dependent Neurotrophic Factor
Hill, Li, Zhao, Hauser, McCune, Spong, Brenneman; in collaboration with Gozes, Jaffee, Yergey
Previous studies have indicated that one of the protective proteins released by VIP is a femtomolar-acting protein: activity-dependent neurotrophic factor (ADNF). One of our continuing priorities is to characterize ADNF at the protein and genetic levels and to develop therapeutic agents based on peptides derived from ADNF. Recent evidence indicates that ADNF is a complex of proteins characterized by three peaks of biological activity that greatly differ in potency to prevent 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 by using a fluorescent peptide substrate. The protease activity detected with this methodology corresponded very closely to the potency of the survival-promoting activity. The 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, studies on the protease activity of component II of the ADNF complex emphasize the proof of principle; however, all the neurotrophic activities of the ADNF components may reside in their proteolytic actions.

Neuroprotective Peptides
Spong, Goodwin, Hauser, Poggi, Brenneman; in collaboration with Gozes
Previous studies have shown that short peptides derived from ADNF and a pharmacologically related protein (activity-dependent neuropro-tective 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, 11 new peptides derived from ADNF have been discovered, although a number of the 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 11 new peptides were tested for intrinsic survival-promoting activity by utilizing a cell culture model of tetrodotoxin-induced neuronal cell death. The experiments indicated that four of the 11 peptides showed potent survival-promoting activity. Both neuronal cell counts and the carboxyfluorescene diacetate methodology used to assess neuronal survival confirmed complex dose/response relationships. The new peptides provide further basis for the exploration of these structures as lead compounds for therapeutic intervention in the treatment of neurodegenerative disease.

VIP and the Neural Tube
Hill, McCune, Sahir, Brenneman
VIP is an important regulator of embryonic growth and development during the early post-implantation period of development, with VIP receptors localized to the neural tube. We have used explanted neural tubes to perform studies of VIP-mediated regulation of gene expression and neurotrophin release. With this model system, we have shown that nerve growth factor (NGF) is one of the factors regulated by VIP in neural tube preparation explants from the early post-implantation 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 in preparations treated with equal concentrations of the highly homologous neuropeptide, PACAP.

The VIP concentrations used were within the peptide’s biologically active range in central nervous system tissues, and immunoreactive NGF was released at concentrations that 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 much greater concentrations.

The model system has revealed a functional relationship between VIP and NGF, and the findings support the hypothesis that VIP mediates development through its action as a secretagog. The study links, for the first time, the actions of VIP with releasable NGF prohormone in neural tissue. The results of this work imply that drugs mimicking the neurotrophic action of VIP in the central nervous system could elicit protective and repair modalities of NGF and thereby are of therapeutic value.