Albert Pinhasov

Cloning and Characterization of the Human Activity-dependent Neuroprotective Protein*

We have recently cloned the mouse activity-dependent neuroprotective protein (ADNP). Here, we disclose the cloning of human ADNP (hADNP) from a fetal brain cDNA library. Comparative sequence analysis of these two ADNP orthologs indicated 90% identity at the mRNA level. Several single nucleotide polymorphic sites were noticed. The deduced protein structure contained nine zinc fingers, a proline-rich region, a nuclear bipartite localization signal, and a homeobox domain profile, suggesting a transcription factor function. Further comparative analysis identified an ADNP paralog (33% identity and 46% similarity), indicating that these genes belong to a novel protein family with a nine-zinc finger motif followed by a homeobox domain. The hADNP gene structure spans ∼40 kilobases and includes five exons and four introns with alternative splicing of an untranslated second exon. The hADNP gene was mapped to chromosome 20q12–13.2, a region associated with aggressive tumor growth, frequently amplified in many neoplasias, including breast, bladder, ovarian, pancreatic, and colon cancers. hADNP mRNA is abundantly expressed in distinct normal tissues, and high expression levels were encountered in malignant cells. Down-regulation of ADNP by antisense oligodeoxynucleotides up-regulated the tumor suppressor p53 and reduced the viability of intestinal cancer cells by 90%. Thus, ADNP is implicated in maintaining cell survival, perhaps through modulation of p53.

Activity-dependent neuroprotective protein: a novel gene essential for brain formation

We have recently cloned the novel homeobox-containing activity-dependent neuroprotective protein (ADNP). In the current study, mouse ADNP was shown to be expressed at the time of neural tube closure, detected at E7.5 and increased on E9.5. Expression was augmented in the brain (E12.5), sustained throughout embryogenesis and regulated by VIP. To assess the function of ADNP, knockout mice were established. Detailed analysis revealed cranial neural tube closure failure and death on E8.5-9.0 of the ADNP-knockout embryos. The expression of Oct4, a gene associated with germ-line maintenance was markedly augmented in the knockout embryos. In contrast, the expression of Pax6, a gene crucial for cerebral cortex formation, was abolished in the brain primordial tissue of the knockout embryos. Thus, Pax6 and Oct4 constitute a part of the mechanism of action of ADNP on brain formation, inhibiting germ-line division while activating morphogenesis. In conclusion, ADNP is identified here as a new key gene essential for organogenesis in the developing embryo and may be implicated as a clinical target associated with proper neurodevelopment.

A femtomolar-acting neuroprotective peptide induces increased levels of heat shock protein 60 in rat cortical neurons: a potential neuroprotective mechanism

Activity-dependent neurotrophic factor (ADNF) was recently isolated from conditioned media of astrocytes stimulated with vasoactive intestinal peptide (VIP). ADNF provided neuroprotection at femtomolar concentration against a wide variety of toxic insults. A nine amino acid peptide (ADNF-9) captured with even greater potency the neuroprotective activity exhibited by the parent protein. Utilizing Northern and Western blot analyses, it was now shown that ADNF-9 increased the expression of heat shock protein 60 (hsp60) in rat cerebral cortical cultures. In contrast, treatment with the Alzheimers toxin, the beta-amyloid peptide, reduced the amount of intracellular hsp60. Treatment with ADNF-9 prevented the reduction in hsp60 produced by the beta-amyloid peptide. The protection against the beta-amyloid peptide-associated cell death provided by ADNF-9 may be mediated in part by intracellular increases in hsp60.

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.

NAP accelerates the performance of normal rats in the water maze.

NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) has neuroprotective, memory enhancing, and neurotrophic properties. NAP is a short peptide sequence derived from the recently cloned, activity-dependent neuroprotective protein. The current study was designed to evaluate NAP activity in normal middle-aged animals to further assess NAP’s breadth of neuroprotection. NAP was administered by inhalation. Results showed that in the paradigm of the Morris water maze, assessing short-term memory, only the NAP-treated middle-aged rats and not placebotreated rats showed significant improvements by the end of the testing period. These results suggest efficacy for NAP in normal aging that is associated with accumulating environmental and genetic toxic factors.

A novel VIP responsive gene. Activity dependent neuroprotective protein.

Abstract: Activity dependent neuroprotective protein (ADNP, 828 amino acids, pI 5.99) is a glial‐derived protein that contains a femtomolar active neuroprotective peptide, NAPVSIPQ (NAP). VIP induces a two‐ to threefold increase in ADNP mRNA in astrocytes, suggesting that ADNP is a VIP‐responsive gene. ADNP is widely distributed in the mouse hippocampus, cerebellum, and cerebral cortex. VIP has been shown to possess neuroprotective activity that may be exerted through the activation of glial proteins. We suggest that ADNP may be part of the VIP protection pathway through the femtomolar‐acting NAP and through putative interaction with other macromolecules.

A vasoactive intestinal peptide receptor analog alters the expression of homeobox genes

A lipophilic analog of vasoactive intestinal peptide (VIP), stearyl-Nle(17)-neurotensin(6-11)VIP(7-28) (SNH), that inhibited lung cancer growth, has been previously described. The mechanism of SNH inhibition of cancer growth is still being elucidated. The present study examined the effects of SNH on homeobox genes in the colon cancer cell line HT 29 that expresses VIP receptors. Homeobox genes contain a characteristic DNA sequence, coding for a stretch of 61 amino acid homeodomain that binds specific DNA motifs. While the HOX gene family contains a single homeodomain, the POU gene family contains an additional DNA binding homeodomain. HT 29 cells were incubated with SNH; RNA was extracted and subjected to reverse-transcription-polymerase chain reaction (RT-PCR) with primers that matched the conserved area of the various HOX or POU genes. The PCR products that were altered by SNH treatment were sequenced. Three candidate SNH-responsive genes, the HOX A4, the HOX B5 and the PUO V transcription factor I (Oct-3) were identified. Semi-quantitative RT-PCR with specific primers confirmed the increase in HOX A4 and the decrease in Oct-3 expression levels following SNH treatment. Thus, the HOX A4 and the Oct-3 homeobox genes may partially mediate SNH activity on cancer cells.

Intranasal Delivery of Bioactive Peptides or Peptide Analogues Enhances Spatial Memory and Protects Against Cholinergic Deficits

Studies utilizing the 28 amino acid vasoactive intestinal peptide (VIP), or glial-derived VIP-associated proteins as templates for future drug design originated from two lines of experimental results: 1] The findings of increased expression of the VIP gene (Bodner et al.,1985) during synapse formation (Gozes et al., 1987) and its decreased synthesis with aging (Gozes et al.,1988). 2] The findings of neuroprotective activities for VIP against electrical blockade (Brenneman and Eiden, 1986) that are mediated by glial cells (Brenneman et al.,1987; Brenneman et al.,1990) expressing high affinity VIP receptors (Gozes et al.,1991).