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.
![](images/10_LDN_B.jpg)
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.
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PUBLICATIONS
- Beni-Adani
L, Gozes I, Cohen Y, Assaf Y, Brenneman DE, Eizenberg O, Trembolver
V, Shohami E. A peptide derived from activity-dependent neuroprotective
protein (ADNP) ameliorates injury response in closed head injury in
mice. J Pharmacol Exp Ther 2001;296:57-63.
- Blondel
O, Collin C, McCarran B, Zhu S, Zamonstiano R, Gozes I, Brenneman DE,
McKay RDG. A glia-derived signal regulating neuronal differentiation.
J Neurosci 2000;20:8012-8020.
- Gozes
I, Giladi E, Pinhasov A, Bardea A, Brenneman DE. Activity-dependent
neurotrophic factor: intranasal administration of femtomolar-acting
peptides improve performance in a water maze. J Phamacol Exp Ther 2000;293:1091-1098.
- Servoss
SJ, Lee SJ, Gibney G, Gozes I, Brenneman DE, Hill JM. Insulin-like
growth factor as a mediator of vasoactive intestinal peptide/activity
dependent neurotrophic factor-simulated embryonic growth. Endocrinology
2001;142:3348-3353.
- Spong
CY, Abebe D, Gozes I, Brenneman DE, Hill JM. Prevention of fetal
demise and growth restriction in a mouse model of fetal alcohol syndrome.
J Pharmacol Exp Ther 2001;297:774-779.
- White
DM, Walker S, Brenneman DE, Gozes I. CREB contributes to the increased
neurite outgrowth of sensory neurons induced by vasoactive intestinal
polypeptide and activity-dependent neurotrophic factor. Brain Res 2000;868:31-38.
- Zamostinao
R, Pinhasov A, Gelber E, Steingart RA, Seroussi E, Gilabi E, Bassan
M, Wollman Y, Eyre HJ, Mulley JC, Brenneman DE, Gozes I. Cloning
and characterization of the human activity-dependent neuroprotective
protein. J Biol Chem 2001;276:708-714.
aI. Gozes, Sakler School of Medicine,
Tel Aviv University, Tel Aviv, Israel
b H. Jaffe, NINDS, NIH
c A. Yergey, NICHD
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