Eliezer Giladi

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.

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.

Identification of VIP/PACAP receptors on rat astrocytes using antisense oligodeoxynucleotides.

Vasoactive intestinal peptide (VIP) has been shown to be a potent promoter of neuronal survival. Pituitary adenylate cyclase-activating peptide (PACAP), a homologous peptide, shares activity and receptor molecules with VIP. The neuroprotective effects of VIP have been shown to be mediated via astroglial-derived molecules. Utilizing a battery of antisense oligodeoxy-nucleotides directed against the multiple cloned VIP-preferring (VIP receptors 1 and 2) or PACAP-preferring receptors (six splice variants derived from the same gene transcript), the authors have demonstrated the existence of a specific PACAP receptor splice variant (PACAP4 or hop2) on astrocytes as well as a VIP type2 receptor. The identification of the receptors was achieved by incubation of the cells in the presence of the specific antisense oligodeoxynucleotide followed by radiolabeled VIP binding and displacement. Polymerase chain reaction (PCR) coupled to direct sequencing identified the expression of the PACAP4-hop2 receptor splice variant in astrocytes. Neuronal survival assays were conducted in mixed neuronal-glial cultures derived from newborn rat cerebral cortex. When these cultures were exposed to the battery of the antisense oligodeoxynucleotides, in serum-free media, only the PACAP-specific ones (e.g., hop2-specific) had an effect in decreasing neuronal cell counts. Thus, the VIP neuronal survival effect is mediated, at least in part, via a specific PACAP receptor (containing a unique insertion of 27 amino acids—the hop2 cassette). These data indicate that a hop2-like PACAP/VIP receptor is the receptor that mediates neurotropism.

Intranasal administration of NAP, a neuroprotective peptide, decreases anxiety-like behavior in aging mice in the elevated plus maze.

NAP, an 8-amino-acid peptide (NAPVSIPQ=Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln), provides neuroprotection at very low doses in a variety of animal models. Previously, acute NAP administration by the intranasal route resulted in improved performance in the Morris water maze of normal and cognitively impaired rats. In these animals, it was observed, but not quantified, that NAP exhibited an anxiolytic effect. Therefore, we have tested here the effects of chronic NAP treatment on anxiety-like behavior in mice in the elevated plus maze. Results showed that 5 months of daily (intranasal) treatment with NAP reduced anxiety, measured as the percentage of time spent in the open arms of the maze (P < 0.01). This effect was maintained after a longer (8 months) exposure to NAP. In addition, after 8 months of NAP treatment, the percentage of open arm entries out of total arms entries was significantly higher in the treated mice ( P < 0.01). Motor function indices indicated no significant differences between the groups. Furthermore, prolonged treatment with NAP (7 months) showed some beneficial effects on Morris water maze performance in the aging mice. It is concluded that NAP offers a unique combination of anxiolytic/cognitive enhancing properties observed after prolonged chronic intranasal treatment.

Vasoactive Intestinal Peptide Gene Expression from Embryos to Aging Rats

Vasoactive intestinal peptide (VIP) gene transcripts were demonstrated by RNA blot hybridization using VIP-specific RNA hybridization probes. High levels of expression were observed as early as in 16-day-old embryos. In aging rats, the VIP-mRNA levels were reduced significantly (in the cerebral cortex) as compared to 21-day-old rats. Our results suggest a role for the VIP gene protein products during embryonal development. During aging processes the decrease in VIP gene transcripts may be a consequence of either a reduction in the transcriptional activity of VIP neurons or death of VIP-producing cells.

NAP (davunetide) modifies disease progression in a mouse model of severe neurodegeneration: Protection against impairments in axonal transport

NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at days 90-100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice.

The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide.

Alzheimers disease (AD) is characterized by brain plaques containing the beta-amyloid peptide (Abeta). One approach for treating AD is by blocking Abeta aggregation. Activity-dependent neuroprotective protein contains a peptide, NAP that protects neurons in culture against Abeta toxicity. Here, NAP was shown to inhibit Abeta aggregation using: (1) fluorimetry; (2) electron microscopy; (3) high-throughput screening of Abeta deposition onto a synthetic template (synthaloid); and (4) Congo Red staining of neurons. Further assays showed biotin-NAP binding to Abeta. These results suggest that part of the neuroprotective mechanism exerted by NAP is through modulation of toxic protein folding in the extracellular milieu.

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 NAP motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through microtubule end binding proteins

The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1–3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.