Janet Hauser

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes.

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.

Antiserum to activity-dependent neurotrophic factor produces neuronal cell death in CNS cultures: immunological and biological specificity.

Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. ADNF is released from astroglia after treatment with 0.1 nM vasoactive intestinal peptide (VIP). To further assess the biological role of ADNF, antiserum was produced following sequential injections of purified ADNF into mice. Anti-ADNF ascites fluid (1:10,000) decreased neuronal survival by 45-55% in comparison to untreated cultures or those treated with control ascites. The neuronal death after anti-ADNF treatment was observed in cultures derived from the spinal cord, hippocampus or cerebral cortex at similar IC50s. Using a terminal deoxynucleotidyl transferase in situ assay to estimate apoptosis in cerebral cortical cultures, anti-ADNF was shown to produce a 70% increase in the number of labeled cells in comparison to controls. In spinal cord cultures, anti-ADNF treatment produced a 20% decrease in choline acetyltransferase activity in comparison to controls. Neuronal cell death produced by the antiserum to ADNF was prevented in cultures co-treated with purified ADNF or ADNF-15, an active peptide derived from the parent ADNF. In vitro binding between the anti-ADNF and ADNF-15 was demonstrated with size exclusion chromatography. Comparative studies with other growth factors (insulin-like growth factor-1, platelet-derived growth factor, nerve growth factor, epidermal growth factor, ciliary neurotrophic growth factor, and neurotrophin-3) demonstrated that only ADNF prevented neuronal cell death associated with electrical blockade. These investigations indicated that an ADNF-like substance was present in cultures derived from multiple locations in the central nervous system and that ADNF-15 exhibited both neuroprotection and immunogenicity. ADNF appears to be both a regulator of activity-dependent neuronal survival and a neuroprotectant.

VIP and d-ala-peptide T-amide release chemokines which prevent HIV-1 GP120-induced neuronal death

Vasoactive intestinal peptide (VIP) and DAPTA (D-ala(1)-peptide T-amide, a gp120-derived octapeptide homologous to VIP) prevent neuronal cell death produced by five variants of HIV-1 (human immunodeficiency virus) envelope protein (gp120). VIP or DAPTA treatment of astrocyte cultures resulted in the release of macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES, beta chemokines known to block gp120 interactions with microglial chemokine receptors. In rat cerebral cortical cultures, gp120-induced neuronal killing was partially or completely prevented by chemokines that stimulate the CXCR4, CCR3 or CCR5 chemokine receptors. Chemokines exhibited marked differences in potency and efficacy in preventing toxicity associated with five gp120 variants (LAV/BRU, CM243, RF, SF2, and MN). RANTES had the broadest and most potent inhibition (IC(50)<3 pM for RF isolate). An octapeptide derived from RANTES also exhibited neuroprotection from gp120 (RF isolate) toxicity (IC(50)=0.3 microM). Treatment with chemokines alone had no detectable effect on neuronal cell number. However, antiserum to MIP-1alpha produced neuronal cell death that was prevented by co-treatment with MIP-1alpha, suggesting that this endogenous chemokine exerts a tonic regulation important to neuronal survival. The neuroprotective action of VIP on gp120 was attenuated by co-treatment with anti-MIP-1alpha. These studies suggest that the neuroprotective action of VIP is linked in part to its release of MIP-1alpha. Furthermore, neuroprotection produced by chemokines is dependent on both the type of chemokine and the variant structure of gp120 and may be relevant to drug strategies for the treatment of AIDS dementia.

Complex array of cytokines released by vasoactive intestinal peptide

A complex mixture of five cytokines has been shown to be released by vasoactive intestinal peptide (VIP). Cytokines were measured in paired samples of culture medium and astroglial cytosol by capillary electrophoresis. This is the first description of VIP-mediated release for TNF-alpha, IL-3, G-CSF and M-CSF from astrocyte cultures. Kinetic studies after VIP treatment demonstrated a gradual but incomplete depletion of cytosolic cytokine levels, with differences observed among the cytokines. Significant increases in release were apparent within 15-30 min for all cytokines. As the recognized VIP receptors (VPAC1 and VPAC2) are linked to adenylate cyclase and also interact with pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), both this homologous peptide and 8-bromo cAMP were investigated and compared to VIP-mediated release. Treatment with 1 mM 8-bromo cAMP produced cytokine release similar in amount to 0.1 nM PACAP-38, but significantly less (<50%) in comparison to 0.1 nM VIP. PACAP-38 and VIP exhibited similar EC(50)s for the release of G-CSF and TNF-alpha; however, the maximal release was 4-6 times greater for VIP than for PACAP-38. This similarity in potency suggested a VPAC-like receptor; however, the greater efficacy for VIP in comparison to PACAP-38, combined with a lack of cAMP production at subnanomolar concentrations of VIP, suggested a mechanism not currently associated with VPAC receptors. For M-CSF, IL-3 and IL-6, the EC(50)s of VIP were 3-30 times more potent than those of PACAP-38 in producing release. These studies suggested that multiple mechanisms mediate cytokine release in astrocytes: (1) a low efficacy release produced by PACAP-38 that is cAMP-mediated and (2) a high efficacy, VIP-preferring mechanism that was not linked to cAMP. In summary, subnanomolar concentrations of VIP released a complex array of cytokines from astrocytes that may contribute to the mitogenic and neurotrophic properties of this neuropeptide in the central nervous system.

The identification of secreted heat shock 60 -like protein from rat glial cells and a human neuroblastoma cell line

The intracellular stress-induced proteins provide protection against toxic insults. Here, a 60,000-Da heat shock 60 (hsp60)-like protein was detected, with five different antibodies, in conditioned media derived from rat cortical astrocytes and a human neuroblastoma cell line. Extracellular neuroblastoma hsp60-like immunoreactivity was increased 3-fold in the presence of the neuropeptide vasoactive intestinal peptide (VIP) and was augmented 2-fold after temperature elevation. Intracellular hsp60 immunoreactivity was reduced 2-3-fold in the presence of VIP; this reduction was attenuated in the presence of brefeldin A, an inhibitor of protein secretion. In contrast, the activity of lactate dehydrogenase (LDH), an intracellular marker, did not change in the presence of VIP. Essentially no extracellular LDH activity was detected, indicating no cellular damage. A novel aspect for stress proteins having extracellular protective roles is suggested.

The expression of activity-dependent neuroprotective protein (ADNP) is regulated by brain damage and treatment of mice with the ADNP derived peptide, NAP, reduces the severity of traumatic head injury.

NAP is a short octapeptide sequence (single letter code, NAPVSIPQ) that protects neurons against a wide variety of insults. The NAP sequence was identified by peptide structure/function scanning of activity-dependent neuroprotective protein (ADNP), a gene product essential for brain formation. To further evaluate the in vivo efficacy of NAP neuroprotection we used a mouse model of head trauma; a condition that presents a risk factor for the development of Alzheimers disease in injured patients. In the mouse model, NAP treatment (prophylactic or curative) indicated improvement in longitudinal clinical, biochemical and anatomical outcomes. Furthermore, closed head injury was associated with a delayed increase in the expression of the immune cell surface glycoprotein Mac-1 (CD11B antigen) at the injury site that was decreased in NAP-treated mice. Additional experiments with Mac-1-deficient mice suggested partial protection against death related to severe head injury. NAP protection in Mac-1-deficient mice against adverse clinical outcome was concomitant with the time period when increases in Mac-1 transcripts were observed in the Mac-1 expressing mice ( approximately four weeks after the injury). The expression of ADNP (the NAP parent protein) was also increased at the injured brain site four weeks after the traumatic event, only in Mac-1 expressing mice. Here, using immunocytochemistry, we localized the increase in ADNP to microglia and astrocyte-like cells. The increase in ADNP in injured brains is now suggested to be a part of an endogenous compensatory mechanism and NAP treatment provides an additional protection. Toxicology studies suggest NAP as safe for further clinical development.

Chemokine release is associated with the protective action of PACAP-38 against HIV envelope protein neurotoxicity.

The envelope protein (gp120) of the human immunodeficiency virus produces neuronal cell death in cultures that can be prevented by co-treatment with pituitary adenylate activating peptide-38 (PACAP-38) or chemokines. To investigate the hypothesis that a functional relationship exists between these two protectants, the release of chemokines was measured in rat astrocyte cultures after PACAP-38 treatment. Chemokine analyses were performed by immunoaffinity capillary electrophoresis. Bell-shaped dose-responses for PACAP-mediated release of chemokines into the culture medium were observed with EC(50)s of 3 x 10(15) M (RANTES: regulated upon activation normal T cell expressed and secreted), 3 x 10(-11) M (MIP-1 beta) and 10(-7)M (MIP-1 alpha). In addition, PACAP-mediated depletion of chemokines from cultured astrocytes exhibited inverted bell-shaped curves, with similar EC(50)s to those observed for chemokine measurements of the medium. Comparative studies with structurally related peptides (vasoactive intestinal peptide [VIP] and secretin) revealed that PACAP was the most potent secretagogue for RANTES on astrocyte cultures. Gp120-mediated neuronal cell death was prevented by co-treatment with PACAP-38, although the efficacy of protection varied significantly among the gp120 isolates. A bi-model dose-response was observed with EC(50)s of 3 x 10(-15) and 3 x 10(-11) M. Co-treatment with neutralizing antiserum to RANTES attenuated PACAP-mediated protection from toxicity associated with gp120. In contrast to previous studies with VIP and gp120 toxicity, co-treatment with anti-MIP-1 alpha did not affect PACAP-induced protection. These studies support the hypothesis that PACAP produces neuroprotection from gp120 toxicity, in part, through the release of RANTES and this mechanism is distinct from that observed with VIP.

VIP Neurotrophism in the Central Nervous System: Multiple Effectors and Identification of a Femtomolar-Acting Neuroprotective Peptide

Abstract: Vasoactive intestinal peptide has neurotrophic and growth‐regulating properties. As in the case of many neurotrophic molecules, VIP also has neuroprotective properties, including the prevention of cell death associated with excitotoxicity (NMDA), beta‐amyloid peptide, and gp120, the neurotoxic envelope protein from the human immunodeficiency virus. The neurotrophic and neuroprotective properties are mediated in part through the action of glial‐derived substances released by VIP. These substance include cytokines, pro tease nexin I, and ADNF, a novel neuroprotective protein with structural similarities to heat‐shock protein 60. Antiserum against ADNF produced neu ronal cell death and an increase in apoptotic neurons in cell culture. A 14 amino acid peptide (ADNF‐14) derived from ADNF has been discovered that mimics the survival‐promoting action of the parent protein. These studies support the conclusion that VIP, PACAP, and associated molecules are both important regulators of neurodevelopment and strong candidates for therapeutic development for the treatment of neurodegenerative disease.

Sexual dimorphism of activity-dependent neuroprotective protein in the mouse arcuate nucleus

Activity-dependent neuroprotective protein (ADNP) is a highly conserved vasoactive intestinal peptide (VIP) responsive gene that is expressed abundantly in the brain and in the body and is essential for brain formation and embryonic development. Since, VIP exhibits sexual dimorphism in the hypothalamus, the potential differential expression of ADNP in male and female mice was investigated. Real-time polymerase chain reaction revealed sexual dimorphism in ADNP mRNA expression as well as fluctuations within the estrus cycle. Immunohistochemistry with an antibody to ADNP showed specific staining in the arcuate nucleus of the hypothalamus. ADNP-like immunoreactivity in the arcuate nucleus also exhibited fluctuations during the estrus cycle. Here, brain sections at proestrus were the most immunoreactive and brain sections at estrus--the least. Furthermore, male arcuate nucleus ADNP-like immunoreactivity was significantly lower than that of the female estrus. Many neuropeptides, neurotransmitters and proteins are localized to the arcuate nucleus where they contribute to the regulation of reproductive cyclicity and energy homeostasis. The results presented here suggest that ADNP has a part in the estrus cycle as an affecter or an effector.

Blockage of VIP during mouse embryogenesis modifies adult behavior and results in permanent changes in brain chemistry

Vasoactive intestinal peptide (VIP) regulates growth and development during the early postimplantation period of mouse embryogenesis. Blockage of VIP with a VIP antagonist during this period results in growth restriction, microcephaly, and developmental delays. Similar treatment of neonatal rodents also causes developmental delays and impaired diurnal rhythms, and the adult brains of these animals exhibit neuronal dystrophy and increased VIP binding. These data suggest that blockage of VIP during the development of the nervous system can result in permanent changes to the brain. In the current study, pregnant mice were treated with a VIP antagonist during embryonic days 8 through 10. The adult male offspring were examined in tests of novelty, paired activity, and social recognition. Brain tissue was examined for several measures of chemistry and gene expression of VIP and related compounds. Glial cells from the cortex of freated newborn mice were plated with neurons and examined for VIP binding and their ability to enhance neuronal survival. Treated adult male mice exhibited increased anxiety-like behavior and deficits in social behavior. Brain tissue exhibited regionally specific changes in VIP chemistry and a trend toward increased gene expression of VIP and related compounds that reached statistical significance in the VIP receptor, VPAC-1, in the female cortex. When compared to control astrocytes, astrocytes from treated cerebral cortex produced further increases in neuronal survival with excess synaptic connections and reduced VIP binding. In conclusion, impaired VIP activity during mouse embryogenesis resulted in permanent changes to both adult brain chemistry/cell biology and behavior with aspects of autism-like social deficits.