Ariane Davidson

Complete Sequence of a Novel Protein Containing a Femtomolar‐Activity‐Dependent Neuroprotective Peptide

Abstract : The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pl 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity‐dependent neurotrophic factor ; (2) a glutaredoxin active site ; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuron—astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the β‐amyloid peptide (the Alzheimers disease neurotoxin), N‐methyl‐D‐aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E‐deficient mice accelerated the acquisition of developmental reflexes and prevented short‐term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E‐deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.

Protection against developmental retardation in apolipoprotein E-deficient mice by a fatty neuropeptide: Implications for early treatment of Alzheimer's disease

Stearyl-Nle17-VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100-fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, mice deficient in apolipoprotein E (ApoE), a molecule associated with the etiology of Alzheimers disease, served as a model to investigate the developmental and protective effects of SNV. In comparison to control animals, the deficient mice exhibited (a) reduced amounts of VIP messenger RNA; (b) decreased cholinergic activity (c) significant retardation in the acquisition of developmental milestones: forelimb placing behavior and cliff avoidance behavior; and (d) learning and memory impairments. Daily injections of SNV to ApoE-deficient newborn pups resulted in increased cholinergic activity and marked improvements in the time of acquisition of behavioral milestones, with peptide-treated animals developing as fast as control animals and exhibiting improved cognitive functions after cessation of peptide treatment. Specificity was demonstrated in that treatment with a related peptide (PACAP), pituitary adenylate cyclase-activating peptide, produced only limited amelioration. As certain genotypes of ApoE increase the probability of Alzheimers disease, early counseling and preventive treatments may now offer an important route for therapeutics design.

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.

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.

A Novel Signaling Molecule for Neuropeptide Action: Activity‐dependent Neuroprotective Protein

Abstract: The complete coding sequence of a novel protein (828 amino acids, pI 5.99), a potential new mediator of vasoactive intestinal peptide (VIP) activity was recently revealed. The expression of this molecule, activity‐dependent neuroprotective protein (ADNP), was augmented in the presence of VIP, in cerebral cortical astrocytes. The mRNA transcripts encoding ADNP were enriched in the mouse hippocampus and cerebellum. The protein deduced sequence contained the following: (1) a unique peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity‐dependent neurotrophic factor (ADNF) and exhibiting neuroprotection in vitro and in vivo; (2) a glutaredoxin active site; and (3) a classical zinc binding domain. Comparative studies suggested that the peptide, NAPVSIPQ (NAP), was more efficacious than peptides derived from ADNF. ADNP, a potential mediator of VIP‐associated neuronal survival, and the new peptide, a potential lead compound for drug design, are discussed below.

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.

Native Xenopus oocytes express two types of muscarinic receptors

We have recently described two types of muscarinic responses in native Xenopus oocytes of different donors (common and variant) that display qualitative and quantitative differences (Lupu‐Meiri et al., 1990). Here we characterized the muscarinic receptors mediating these two types. The anti‐muscarinic toxins from Dentroaspis significantly inhibited responses in oocytes of common donors, but had little effect on responses in oocytes of variant donors, possibly indicating expression of different receptor subtypes. Using specific muscarinic antagonists, we found that oocytes of common donors exhibit a pattern compatible with the M3 subtype of muscarinic receptors, while oocytes of variant donors appear to possess receptors of the M1 subtype. To more directly determine the subtypes of muscarinic receptors in oocytes of both populations of donors, we have microinjected antisense oligonucleotides into native oocytes. Antisense oligonucleotides to unique sequences in the N‐terminal and the third cytoplasmic loop of M3 muscarinic receptors caused a significant inhibition of the response of common oocytes, but had virtually no effect on responses in oocytes of variant donors. Conversely, oligonucleotides complementary to the unique sequences of the m1 muscarinic receptors inhibited the response in variant oocytes, but not in oocytes of common donors. We conclude that native Xenopus oocytes of different donors phenotypically express either M3‐like (majority) or M1‐like (minority) muscarinic receptor subtypes. The differences in receptor subtype expression may explain the different characteristics of responses in the two populations.

Locomotor Activity Causes a Rapid Up-regulation of Vasoactive Intestinal Peptide in the Rat Hippocampus

Vasoactive intestinal peptide (VIP) expression is restricted to interneurons in the hippocampus of normal adult rats. However, 3–6 hours after a 60‐minute walk in an activity wheel, VIP was transiently expressed in most pyramidal and granular neurons of the hippocampus. Locomotion was also associated with a dramatic increase in VIP immunoreactivity in the motor cortex, primarily in bipolar cells. Reverse transcriptase‐polymerase chain reaction analysis indicated that VIP mRNA increases transiently by more than twofold, before the increases in peptide immunoreactivity in both the hippocampus and motor cortex. By comparison, another marker of inhibitory interneurons, glutamate decarboxylase, did not change its expression pattern after locomotion. The calcium binding protein, calbindin‐D28K, normally expressed in interneurons, was now found also in glial cells of the hippocampus and motor cortex. Another marker of enhanced electrical activity, the immediate early gene, c‐Fos, was expressed in pyramidal and granular neurons at 3 hours but not at 6 hours after locomotion. These results suggest that mapping of peptide expression in the brain of a docile, inactive rat may not reflect the real distribution and functions of a peptide in an active animal. Hippocampus 1999;9:534–541.

Vasoactive intestinal peptide. Link between electrical activity and glia-mediated neurotrophism.

Abstract: Vasoactive intestinal peptide has neurotrophic and neuroprotective properties that influence the survival of activity‐dependent neurons in the central nervous system. Investigations of the mechanism of this neurotrophic peptide indicated that these actions are contingent on interactions with astroglia. The complex mixture of neurotrophic mediators released from astroglia include cytokines, a protease inhibitor, and activity‐dependent neurotrophic factor, a protein with apparent structural similarities to hsp60. Investigations of ADNF resulted in the discovery of active peptides of extraordinary potency and broad neuroprotective properties. These studies indicate that a nine‐amino acid core peptide of ADNF had significantly greater neuroprotective properties in comparison to the parent growth factor and these advantages identify ADNF‐9 as an attractive lead compound for drug development.

The effects of vascular intrauterine growth retardation on cortical astrocytes

OBJECTIVE We sought to determine the pathogenesis of neurodevelopmental impairments in survivors of intrauterine growth retardation (IUGR). METHODS We used an experimental rabbit vascular IUGR model. We ligated 25% of uteroplacental vessels (partial ischemia) of one-half of the fetuses on day 25 at the end of the third trimester. We then determined hemispheral DNA and protein levels, and used glial fibrillary acidic protein (GFAP) staining to count the labeled astrocytes at the superficial cortical layers. RESULTS Ischemic fetuses were significantly smaller than control fetuses and presented a disproportionately small body and a relatively larger head compared with the normal body/head ratio, confirming the study model as that of asymmetric IUGR. Hemispheral DNA was unchanged in IUGR fetuses, but they had decreased brain weight, hemispheral protein content, and a reduced number of mature (GFAP-positive) cortical astrocytes compared with control fetuses. CONCLUSION Vascular IUGR, as demonstrated in our asymmetric IUGR model, adversely affected brain growth, cell size, and cortical astrocytes maturation. In view of the neurotrophic and neuroprotective functions of astrocytes, a reduced number of mature astrocytes during this critical period of development may be implicated in the pathogenesis of the neurodevelopmental impairments observed in IUGR.