The Section on Developmental and Molecular Pharmacology investigates vasoactive intestinal peptide (VIP) as a regulator of brain development and neuroprotection. VIP can influence many important processes that are important 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 focuses on identifying the substances that VIP releases and on studying their mechanism of action. After identifying the VIP-related, neuroprotective substances, our continuing goal is to develop therapeutic agents based on these proteins that may prevent neurodegenerative disease.

Figure 25

Excised neural tube and an E9.5 mouse embryo: experimental systems for investigation of neuropeptide –related growth regulation and central nervous system development.

Activity-Dependent Neurotrophic Factor
Hill, Li, Hauser, McCune, Spong, Brenneman in collaboration with Gozes,a Jaffe,b Yergeyc
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 levels and to develop therapeutic agents based on peptides derived from ADNF. Although initially thought to be a single 14 kDa protein, ADNF is now known to exist as a complex of at least three components. MALDI (Matrix Assisted Laser Desorption/Ionization) and SELDI (Surface Enhanced Laser Desorption/Ionization) mass spectrometric/time of flight analyses have yielded additional information on the ADNF complex. With these techniques, we determined that the molecular weight of the ADNF complex was 14,590 Daltons. Furthermore, both the MALDI and SELDI spectra of ADNF exhibited an unusually broad peak molecular range (in excess of 1,000 Daltons), an observation consistent with the conclusion that ADNF is an array of proteins or glycoproteins that are stably associated to form a functional unit. Further support for this molecular model is the separation of three component peaks of ADNF by N-linked carbohydrate (N-CHO) capillary electrophoresis. The three peaks isolated from the N-CHO column exhibit unique monotonic dose responses that differ widely in potency. The biological activity of these peaks is inhibited by a distinct group of the antisera generated against ADNF peptides. Thus, the breadth of the MADLI spectrum for ADNF may be attributed to the existence of multiple components that are similar in size and tightly associated. Antisera developed against ADNF-derived peptides have been used to assess ADNF immunoreactivity in sections of the neonatal brain. These studies revealed that the most dense localization of ADNF-like protein was the brain stem, particularly in cells of the trigeminal ganglion, reticular formation, and parabrachial/facial nuclei. In fibers, the greatest abundance of ADNF-like protein was found in the hindbrain, with significant amounts localized in the caudate putamen, olfactory tubercle, and, to a lesser extent, the cerebral cortex and hippocampus.

Activity-Dependent Neurotrophic Factor
Hill, Li, Hauser, McCune, Spong, Brenneman in collaboration with Gozes,a Jaffe,b Yergeyc
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 levels and to develop therapeutic agents based on peptides derived from ADNF. Although initially thought to be a single 14 kDa protein, ADNF is now known to exist as a complex of at least three components. MALDI (Matrix Assisted Laser Desorption/Ionization) and SELDI (Surface Enhanced Laser Desorption/Ionization) mass spectrometric/time of flight analyses have yielded additional information on the ADNF complex. With these techniques, we determined that the molecular weight of the ADNF complex was 14,590 Daltons. Furthermore, both the MALDI and SELDI spectra of ADNF exhibited an unusually broad peak molecular range (in excess of 1,000 Daltons), an observation consistent with the conclusion that ADNF is an array of proteins or glycoproteins that are stably associated to form a functional unit. Further support for this molecular model is the separation of three component peaks of ADNF by N-linked carbohydrate (N-CHO) capillary electrophoresis. The three peaks isolated from the N-CHO column exhibit unique monotonic dose responses that differ widely in potency. The biological activity of these peaks is inhibited by a distinct group of the antisera generated against ADNF peptides. Thus, the breadth of the MADLI spectrum for ADNF may be attributed to the existence of multiple components that are similar in size and tightly associated. Antisera developed against ADNF-derived peptides have been used to assess ADNF immunoreactivity in sections of the neonatal brain. These studies revealed that the most dense localization of ADNF-like protein was the brain stem, particularly in cells of the trigeminal ganglion, reticular formation, and parabrachial/facial nuclei. In fibers, the greatest abundance of ADNF-like protein was found in the hindbrain, with significant amounts localized in the caudate putamen, olfactory tubercle, and, to a lesser extent, the cerebral cortex and hippocampus.

Neuroprotective Peptides
Spong, Goodwin, Hauser, Brenneman in collaboration with Gozesa
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. Further exploration of the pharmacology of ADNF-9 (agonist to ADNF) and NAP (agonist to ADNP)showed, surprisingly, that ADNF-9 and NAP are fully active in the D-isomer form as well as in the L-isomer form. This observation was initially made in studies of apoptotic death produced by tetrodotoxin in cerebral cortical cultures. Importantly, the combination of D-ADNF-9 and D-NAP significantly increased the range of concentrations at which the peptides exhibited effective neuroprotection. The studies with D-isomer peptides were extended to the prevention of embryonic death in a model of fetal alcohol syndrome. The studies indicated that both intraperitoneally and orally administered D-ADNF-9 was efficacious in preventing fetal death produced by prenatal alcohol treatment. The studies also revealed that the peptides from these glial proteins exhibit protective properties through nonchiral mechanisms. Furthermore, the discovery of orally active forms of the peptides increases their potential as lead compounds for drug development.

VIP and the Neural Tube
Hill, McCune, Sahir, Brenneman
The growth-promoting action of VIP is evident in the early postimplantation period of embryogenesis. VIP receptors are localized to the neural tube at this stage of development, with explanted neural tubesused as a model system to study VIP-mediated regulation of gene expression and neurotrophin release. We used the GEArray system to monitor signal transduction pathways and cytokines in control and VIP-stimulated neural tubes. The following cDNAs were up-regulated after VIP treatment: GRO1, BAX, p21, and p57. A number of cytokines were down-regulated after VIP treatment: GCSF, IL-10, TGF-B1, TNF-alpha, TNF-beta, and p53. The constellation of changes mediated by VIP demonstrates the complexity of cytokine and pathway genes regulated by VIP at this critical period of development. Thus, the growth-promoting action of VIP has been correlated with both the activation of gene expression and the release of recognized mediators of nervous system development. PACAP-38, a peptide that belongs to the VIP family of peptides, was found to inhibit the growth of embryonic day 9.5 mouse embryos in culture. The stimulatory action of VIP on growth is therefore not a general feature of this group of peptides.