Biomedical subjects

Illana Gozes

Publications and source records attributed to Illana Gozes.

At least 19 recordsLinked to original sources

PubMed2006

Activity-dependent neuroprotective protein (ADNP) differentially interacts with chromatin to regulate genes essential for embryogenesis.

Complete deficiency in activity-dependent neuroprotective protein (ADNP) results in neural tube closure defects and death at days 8.5-9.5 of gestation in the mouse (E8.5-9.5). To elucidate ADNP associated pathways, Affymetrix 22,690-oligonucleotide-based microarrays were used on ADNP knockout and control mouse embryos (E9) separated completely from extra embryonic tissue. Marked differences in expression profiles between ADNP-deficient embryos and ADNP-expressing embryos were discovered. Specifically, a group of dramatically up-regulated gene transcripts in the ADNP-deficient embryos were clustered into a family encoding for proteins enriched in the visceral endoderm such as apolipoproteins, cathepsins and methallotionins. In contrast, a down regulated gene cluster associated with ADNP-deficiency in the developing embryo consisted of organogenesis markers including neurogenesis (Ngfr, neurogenin1, neurod1) and heart development (Myl2). The pluripotent P19 cells were used for ADNP-chromatin-immunoprecipitation, showing direct interactions with multiple relevant gene promoters including members of the up-regulated as well as the down-regulated gene clusters. A comparison between non-differentiated and neuro-differentiated P19 cells revealed increased chromatin interaction of ADNP with chromatin from differentiated cells. These results place ADNP at a crucial point of gene regulation, repressing potential endoderm genes and enhancing genes associated with organogenesis/neurogenesis.

Animals↗

PubMed2006

Novel extended and branched N-terminal analogs of VIP.

The effects of vasoactive intestinal peptide (VIP) are primarily mediated through VPAC1 and VPAC2, receptors that are preferentially coupled to adenylate cyclase activation. As a large majority of the potent VIP antagonists have modifications in the N-terminal domain of the peptide, the effect of multiplication of this domain on VIP was examined with the aim of possibly amplifying peptide-receptor (VPAC1) activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the N-terminal domain that was obtained by either linear tandem extension or by parallel branching. Circular dichorism (CD) analysis revealed that these extended/branched peptides maintained an alpha helical structure in organic environment, similar to VIP. A specific branched VIP analog was found to be slightly more potent towards VPAC1-related cAMP production as compared to VIP. This analog could have potential therapeutic value in several disorders, similar to VIP. Two branched N-terminal VIP sequences demonstrated superior receptor binding and activation as compared to two N-terminals in tandem. The results suggest that correct alignment of the VIP N-terminal region is important for receptor binding and activation. However, increased receptor binding was not directly associated with increased cAMP production suggesting steric dynamic interactions.

Amino Acid Sequence↗

PubMed2006

VIP provides cellular protection through a specific splice variant of the PACAP receptor: a new neuroprotection target.

Vasoactive intestinal peptide (VIP) was known to provide neuroprotection. Three VIP receptors have been cloned: VPAC1, VPAC2 and PAC1. A specific splice variant of PAC1 in the third cytoplasmatic loop, hop2, was implicated in VIP-related neuroprotection. We aimed to clone the hop2 splice variant, examine its affinity to VIP and investigate whether it mediates the VIP-related neuroprotective activity. The PAC1 cDNA was cloned from rat cerebral astrocytes. Using genetic manipulation the hop2 splice variant was obtained, then inserted into an expression vector and transfected into COS-7 cells that were used for binding assays. Results showed that VIP bound the cloned hop2 splice variant. Stearyl-neurotensin(6-11) VIP(7-28) (SNH), an antagonist for VIP, was also found to bind hop2. In addition, VIP protected COS-7 cells expressing hop2 from oxidative stress. Parallel assays demonstrated that VIP increased cAMP accumulation in COS-7 cells expressing hop2. These results support the hypothesis that hop2 mediates the cytoprotective effects attributed to VIP.

Alternative Splicing↗

PubMed2006

NAP enhances neurodevelopment of newborn apolipoprotein E-deficient mice subjected to hypoxia.

Perinatal hypoxic injury is associated with significant neonatal morbidity and long-term neurodevelopmental complications. NAP, a peptide derived from ADNP (activity-dependent neuroprotective protein), has previously shown neuroprotective abilities in various adult animal models. To evaluate its neuroprotective role in neonatal hypoxic-ischemic injury, we evaluated the neurodevelopmental outcome in apolipoprotein E (ApoE)-deficient (knockout) mice (a breed prone to brain damage during hypoxic insult) exposed to postnatal global hypoxic damage with and without treatment with NAP. ApoE-deficient (n = 80) and control (C57B6) mice pups (n = 81) were exposed to postnatal global hypoxia (35 min of 8% O(2) within 24 h of birth) or room air with or without subsequent subcutaneous NAP treatment during postnatal days 1 to 14. Pups were then evaluated for neonatal motor reflex attainment, spatial learning ability in the Morris water maze, and locomotor open-field activity. The C57B6 and ApoE-deficient anoxic groups showed significantly slower achievement of neonatal reflexes, diminished locomotor activity, and diminished spatial learning ability compared with their control groups. This was more pronounced in the anoxic ApoE-deficient pups. NAP treatment had a pronounced effect on neurodevelopmental outcome in both breeds, particularly in the ApoE-deficient mice. ApoE-deficient and control mouse pups exposed to postnatal hypoxia and treated with NAP showed improvement in neurodevelopmental outcome compared with nontreated mice pups. ApoE-deficient mice show a greater susceptibility to hypoxic damage and better response to NAP treatment.

Animals↗

PubMed2006

Brain deficits associated with fetal alcohol exposure may be protected, in part, by peptides derived from activity-dependent neurotrophic factor and activity-dependent neuroprotective protein.

This review discusses the effects of prenatal alcohol exposure on the developing brain and the potential use of derived peptides from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) in neuroprotection against the insults of alcohol. Alcohol is known to impede the growth of the central nervous system and to induce neurodegeneration through cellular apoptosis. Sari et al. have shown that prenatal alcohol exposure reduced the fetal brain weight, the size of the brain regions and the number of serotonin (5-HT) neurons. Prenatal alcohol exposure compromises neural tube midline development. Sari et al. further suggested that the timing of alcohol exposure during pregnancy is critical to the induction of deficits in 5-HT neurons, as well as other types of neurons and consequently results in deficits in neural tube development. ADNF and ADNP are glial-derived proteins discovered to be induced by vasoactive intestinal peptide (VIP). These proteins are expressed during embryonic development. Functional assays and genetic manipulations have identified these proteins as highly important for neural tube closure and brain formation/development. The peptide derivatives of ADNF, ADNF-14 (VLGGGSALLRSIPA), ADNF-9 (or SALLRSIPA = SAL) and of ADNP, NAPVSIPQ = NAP have shown neuroprotective effects and have been proven to prevent brain damage associated with prenatal alcohol exposure in animals. Here, we discuss the many aspects of alcohol-associated growth restriction in the developing brain and the potential inhibition of this severe phenotype through the use of neuroprotective peptides.

Animals↗

PubMed2006

Peptide neuroprotection through specific interaction with brain tubulin.

This study aimed to identify the neuronal target for the potent neuroprotective peptide NAP. When added to pheochromocytoma cells (neuronal model), NAP was found in the intracellular milieu and was co-localized with microtubules. NAP induced neurite outgrowth and protected primary neurons against microtubule-associated ZnCl2 toxicity. Rapid microtubule reorganization into distinct microtubules ensued after NAP addition to both pheochromocytoma cells and primary cerebral cortical neurons, but not to fibrobalsts. While binding neuronal tubulin and protecting pheochromocytoma cells against oxidative stress, NAP did not bind tubulin extracted from fibroblasts, nor did it protect those cells against oxidative stress. Affinity chromatography identified the brain-specific betaIII-tubulin as a major NAP binding protein. Paclitaxel (a microtubule aggregating agent that interacts with beta-tubulin) reduced NAP tubulin binding. Thus, the underlying mechanism for the neuroprotection offered by NAP is targeting neuronal microtubules that are essential for neuronal survival and function.

Animals↗

PubMed2006

A splice variant to PACAP receptor that is involved in spermatogenesis is expressed in astrocytes.

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor, PAC1, recognizes PACAP with a higher affinity than it recognizes vasoactive intestinal peptide (VIP) and belongs to the subfamily G protein-coupled receptors. So far, more than 10 different splice variants of PAC1 have been cloned from rat tissue. Interestingly, the various PAC1 splice variants exhibit different signaling pathways. These splice variants are suggested to play a functional role mostly in the brain as well as in the testes. The present article introduces PAC1(3a) that was originally discovered in testes as another potential regulator in rat astrocytes.

Animals↗

PubMed2006

NAP, a peptide derived from the activity-dependent neuroprotective protein, modulates macrophage function.

NAP is an eight-amino acid neuroprotective peptide NAPVSIPQ; it is the smallest active element derived from the recently cloned activity-dependent neuroprotective protein (ADNP). NAP readily enters the brain from the blood. It will be important to learn whether NAP, in addition to its neuroprotective activity, also might influence immune-mediated inflammation. Here, we report that: (a) macrophages express ADNP; (b) expression of ADNP in macrophages responds to VIP; and (c) NAP downregulates the key inflammatory cytokines tumor necrosis factor (TNF-alpha), interleukin-16 (IL-16), and IL-12 in macrophages. These findings indicate that ADNP/NAP can play an important role in immune regulation as well as in neuroprotection, which may be mutually related processes.

Amino Acid Sequence↗

PubMed2006

Tubulin is the target binding site for NAP-related peptides: ADNF-9, D-NAP, and D-SAL.

The authors set out to investigate whether NAP-related peptides interact with tubulin at a NAP binding site. Previous studies have shown that the neuroprotective peptide NAP binds to tubulin. As NAP (NAPVSIPQ) shares structural similarities with ADNF-9 (SALLRSIPA), and the all-D-enantiomers, D-NAP and D-SAL, it was hypothesized that all of these peptides compete with NAP-tubulin binding. Using NAP affinity column and extracts from newborn rat brain (cerebral cortex), we now show that the above-mentioned peptides compete with NAP binding to tubulin. The identification of tubulin as a target binding site for NAP-related peptides explains, in part, the broad neuroprotective activity offered by these potent peptides.

Amino Acid Sequence↗

PubMed2006

Neurotrophic effects of the peptide NAP: a novel neuroprotective drug candidate.

This short review outlines the scientific progression from the neuropeptide vasoactive intestinal peptide as a neuroprotective agent that acts through glial cells to increase and modulate the synthesis and secretion of novel neuroprotective substances. Recent development in the studies on activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF) and short peptide derivatives of these proteins, ADNF-9 and NAP suggest that these peptides are neurotrophic and promote neurite outgrowth. These short peptides hold promise in future neuroprotective/neurotrophic drug development. Clinical development of NAP is currently in progress by Allon Therapeutics, Inc.

Animals↗

PubMed2005

PolyADP-ribosylation is involved in neurotrophic activity.

PolyADP-ribosylation is a transient posttranslational modification of proteins, mainly catalyzed by poly(ADP-ribose)polymerase-1 (PARP-1). This highly conserved nuclear protein is activated rapidly in response to DNA nick formation and promotes a fast DNA repair. Here, we examine a possible association between polyADP-ribosylation and the activity of neurotrophins and neuroprotective peptides taking part in life-or-death decisions in mammalian neurons. The presented results indicate an alternative mode of PARP-1 activation in the absence of DNA damage by neurotrophin-induced signaling mechanisms. PARP-1 was activated in rat cerebral cortical neurons briefly exposed to NGF-related nerve growth factors and to the neuroprotective peptides NAP (the peptide NAPVSIPQ, derived from the activity-dependent neuroprotective protein ADNP) and ADNF-9 (the peptide SALLRSIPA, derived from the activity-dependent neurotrophic factor ADNF) In addition, polyADP-ribosylation was involved in the neurotrophic activity of NGF-induced and NAP-induced neurite outgrowth in differentiating pheochromocytoma 12 cells as well as in the neuroprotective activity of NAP in neurons treated with the Alzheimer's disease neurotoxin beta-amyloid. A fast loosening of the highly condensed chromatin structure by polyADP-ribosylation of histone H1, which renders DNA accessible to transcription and repair, may underlie the role of polyADP-ribosylation in neurotrophic activity.

Amyloid beta-Peptides↗

PubMed2005

Neuropeptide receptor transcripts are expressed in the rat clitoris and oscillate during the estrus cycle in the rat vagina.

Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and angiotensin 2 are key neuropeptides that innervate the sexual organs. For further understanding of neuropeptide involvement in female sexual function, we investigated peptide receptor mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR) in the rat vagina and clitoris, and alteration during the shift from the proestrus to the estrus phase. VIP, angiotensin 2 and CGRP receptor subtypes transcripts were found to be expressed in the vagina and the clitoris. Significantly increased levels of angiotensin 2 and CGRP receptor subtypes transcripts were observed in the vagina as compared to the clitoris. Significant increases in the expression of the VIP receptor type 2 (VPAC2) mRNA and parallel increases in a novel VIP responsive gene, activity-dependent neuroprotective protein (ADNP) mRNA were detected in the rat vagina during the estrus phase. The expression pattern of neuropeptide receptors in the female sexual organs suggest an intimate involvement of the corresponding neuropeptides in female sexual function.

Animals↗

PubMed2005

The influence of the peptide NAP on Mac-1-deficient mice following closed head injury.

A single administration of the neuroprotective peptide NAP was previously shown to protect against death associated with closed head injury (CHI) and enhance recovery of the surviving mice. The protective effect was accompanied by down-regulation of the relative mRNA content of the complement receptor 3 (Mac-1, a marker for inflammation) as measured about a month after the injury. In contrast, the mRNA transcripts for activity-dependent neuroprotective protein (ADNP, the NAP containing protein) were shown to increase 29 days post CHI in the injured hemisphere of Mac-1 expressing mice. The present study was set out to investigate: (1) are Mac-1-deficient mice less susceptible to the adverse outcome of traumatic head injury; (2) does NAP treatment affect Mac-1-deficient mice subjected to head injury; and (3) is Mac-1 expression associated with ADNP expression. Results showed that (1) Mac-1-deficient mice were partially protected against death associated with severe head injury as compared to Mac-1 expressing mice. (2) Significant protection against death was observed in NAP-treated mice and an increase in recovery was observed in the NAP-treated Mac-1 mice 4 weeks after injury. (3) ADNP expression did not change in the Mac-1-deficient mice following head injury. Our working hypothesis is that a month following injury, gene expression in the injured brain is altered and competing proteins are expressed such as Mac-1 that is associated with inflammation and ADNP that is associated with neuroprotection. Obviously, this plasticity in gene expression is intimately interwoven with the genetic background of the animal. NAP treatment tilts the balance toward neuroprotection.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

The pregnant spontaneously hypertensive rat as a model of asymmetric intrauterine growth retardation and neurodevelopmental delay.

INTRODUCTION: Hypertension in pregnancy and vascular placental insufficiency are considered common pathogenic factors in human intrauterine growth retardation (IUGR). IUGR neonates experience higher mortality, and the surviving infants have a higher incidence of neurological and intellectual impairment. METHODS: To mimic this condition, we used pregnant spontaneously hypertensive rats (SHR) and performed biometric measurements on Embryonic Day 20, postnatal developmental reflexes, and locomotor activity evaluations. RESULTS: SHR fetuses had significant decreased body weight compared to the Wistar-Kyoto control fetuses (1.51+/-0.02 g vs. 2.05+/-0.01 g, respectively; p<0.0001), and were relatively microcephalic (2.86+/-0.04 cm vs. 3.3+/-0.03 cm, respectively; p<0.0001). Their cephalization index (head circumference/body weight) was increased (1.88+/-0.03 vs. 1.62+/-0.02, respectively; p<0.0001), indicating a "brain-sparing" process. The disproportional ratio indicated that the IUGR type in this model is asymmetric. The SHR pups exhibited a significant (p<0.04) neurodevelopmental delay in the acquisition of neonatal reflexes (righting, negative geotaxis, placing), but they spontaneously caught up with the control pups after approximately 10 days. On Day 30, the SHR pups exhibited significantly increased walking speed and distance and spent less time in quadrant than the controls (p<0.002). CONCLUSION: We speculate that the model of pregnant SHR closely simulate human IUGR caused by hypertension in pregnancy and should enable investigation of mechanisms of hypertension-mediated placenta-vascular injury as well as provide a system for preclinical evaluations of future preventive neuroprotective treatments.

Analysis of Variance↗

PubMed2005

NAP: research and development of a peptide derived from activity-dependent neuroprotective protein (ADNP).

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation. Peptide activity scanning identified NAP (NAPVSIPQ) as a small active fragment of ADNP that provides neuroprotection at very low concentrations. In cell culture, NAP has demonstrated protection against toxicity associated with the beta-amyloid peptide, N-methyl-D-aspartate, electrical blockade, the envelope protein of the AIDS virus, dopamine, H2O2, nutrient starvation and zinc overload. NAP has also provided neuroprotection in animal models of apolipoprotein E deficiency, cholinergic toxicity, closed head injury, stroke, middle aged anxiety and cognitive dysfunction. NAP binds to tubulin and facilitates microtubule assembly leading to enhanced cellular survival that is associated with fundamental cytoskeletal elements. A liquid-chromatography, mass spectrometry assay demonstrated that NAP reaches the brain after either intravenous or intranasal administration. In a battery of toxicological tests including repeated dose toxicity in rats and dogs, cardiopulmonary tests in dogs, and functional behavioral assays in rats, no adverse side effects were observed with NAP concentrations that were approximately 500-fold higher than the biologically active dose. A Phase Ia clinical trial in the US assessed the tolerability and pharmacokinetics of intranasal administration of NAP in sequential ascending doses. The results supported the safety and tolerability of a single dose of NAP administered at up to 15 mg intranasally. Furthermore, dosing was recently completed for a second Phase I clinical trial in healthy adults and elderly volunteers with an intravenous formulation of NAP. NAP is poised for further clinical development targeting several indications, including Alzheimer's disease.

Alzheimer Disease↗

PubMed2005

The peptides ADNF-9 and NAP increase survival and neurite outgrowth of rat retinal ganglion cells in vitro.

PURPOSE: Recent studies demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. We evaluated these findings in cultures of purified rat retinal ganglion cells (RGCs) using two such peptides: ADNF-9 and NAP. In a second step, the influence of these peptides on neurite outgrowth in retinal explants was investigated. METHODS: Retinal ganglion cells (RGCs) were purified from newborn (postnatal day [P]0-P2) rat retina by immunopanning with antibodies against Thy1.1 and were cultured in serum-free N2 medium for 2 days. RGCs were treated with ADNF-9 and NAP at concentrations ranging from 10(-18) to 10(-10) M. Survival was quantified by counting viable cells by phase-contrast microscopy. Retinal explants from postnatal (P9-P11) rats were cultured in three-dimensional fibrin clots in serum-free medium for 3 days. Explants were treated with 1 microM NAP or 1 microM ADNF-9. Neurite outgrowth was visualized by staining with Sudan black and quantified by measuring axonal length. RESULTS: Both peptides enhanced survival of RGCs in a dose-dependent manner. ADNF-9 showed a maximum effect at 0.1 pM with an increase in survival to 177% (95% confidence interval: 149-204) of the control level. The EC(50) was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146-189) and an EC(50) of 6.1 fM. In the explants, 1 microM ADNF-9 enhanced axonal outgrowth to 126% (118-133) and 1 microM NAP to 117% (98-137) compared with the control. CONCLUSIONS: Both peptides, ADNF-9 and NAP, not only increase RGC survival in vitro but also support neurite outgrowth in retinal explants. These peptides deserve further attention as potential neuroprotective compounds in retinal and optic nerve diseases.

Animals↗

PubMed2005

Receptors for VIP and PACAP in guinea pig cerebral cortex: effects on cyclic AMP synthesis and characterization by 125I-VIP binding.

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of 125I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of 125I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a B(max) of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of 125I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) > or = PACAP38 approximately PACAP27 approximately guinea pig VIP (gpVIP) > or = mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of 125I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific 125I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [3H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 microM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 microM, produced net increases in cAMP production in the range of 8-9% conversion, whereas 5 microM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.

Amino Acid Sequence↗