Associate editor: S.D. Brain
Activity-dependent neuroprotective protein: From gene to drug candidate

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Abstract

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation. The gene encoding ADNP is highly conserved and abundantly expressed in the brain. ADNP contains a homeobox profile and a peptide motif providing neuroprotection against a variety of cytotoxic insults. ADNP mRNA and protein expression responds to brain injury and oscillates as a function of the estrus cycle. The plastic nature of ADNP expression is correlated with brain protection and an association between neuroendocrine regulation and neuroprotection is put forth with ADNP as a focal point. Further understanding of neuroprotective molecules should pave the path to better diagnostics and therapies. In this respect, structure–activity studies have identified a short 8 amino acid peptide in ADNP/NAPVSIPQ (NAP) that provides potent neuroprotection. NAP is currently in clinical development for neuroprotection.

Section snippets

Introduction: neuroendocrine regulation and neuroprotection

Neurotrophic and neuroprotective mechanisms modulate developmental processes as well as recovery from acute and chronic neural injuries, aging, and death. The development of the nervous system involves cell migration, differentiation (at the level of the single cell into neurons and glia), pruning, and death of certain cell populations. These processes are not limited to embryonic development but they also appear to occur in the adult brain after acute or chronic injury. Brain plasticity during

Activity-dependent neuroprotective protein discovery and structure

ADNP was originally cloned from P19 mouse carcinoma cells differentiated (in the presence of retinoic acid) into neuroglial cells (Bassan et al., 1999). Subsequently, human ADNP was cloned from a fetal brain cDNA library (Zamostiano et al., 2001). Comparative sequence analysis of these 2 ADNP orthologues indicated 90% identity at the mRNA level, suggesting a very high evolutionary conservation, which also extended to the rat sequence (Sigalov et al., 2000). The deduced protein structure

Function

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%.

Activity-dependent neuroprotective protein expression in rodents

Northern blot hybridizations have identified a unique 5.5-kb mouse ADNP mRNA in the mouse brain. Further hybridizations have identified ADNP mRNA in rat astrocytes. Comparison of tissues revealed an enrichment in brain-derived structures (hippocampus and cerebellum as well as midbrain and cerebral cortex) and low abundance in the lung, kidney, and intestine with slight increases in the testis. When considering the level of the actin mRNA signal (used as an internal standard), the enrichment in

Activity-dependent neuroprotective protein cellular distribution

The subcellular distribution of ADNP was assessed through cell fractionation, gel electrophoresis, immunoblotting, and immunocytochemistry. An antibody directed to the NAP peptide fragment was used (Furman et al., 2004). To assess antibody specificity, recombinant ADNP was used as a competitive ligand (Steingart & Gozes, 2006). ADNP-like immunoreactivity was found in both the cytoplasmic and in the nuclear cell fractions of astrocytes.

In the nucleus, the primary structure of ADNP contains

Activity-dependent neuroprotective protein/NAPVSIPQ, microtubules, and cellular protection

The original discovery of ADNP identified an 8-amino acid fragment, NAP as a domain in ADNP that confers neuroprotection (Bassan et al., 1999). Further studies identified tubulin as a NAP target for astrocyte (Divinski et al., 2004) and neuronal protection (Divinski et al., 2006). Changes in microtubule organization following incubation with NAP parallel decreases in the relative amount of the hyperphosphorylated the tubulin associated unit (tau) (Gozes & Divinski, 2004). Hyperphosphorylated

NAPVSIPQ

NAP was recently reviewed as a neuroprotective drug candidate (Gozes et al., 2005a). 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. In ex vivo whole embryo cultures, NAP mediated protection from ethanol-induced neural tube defects (Chen et al., 2005). NAP has also provided neuroprotection in animal

Concluding remarks and open questions

Turning a stone in biology and medical research unravels unknown mysteries and discovers new horizons for further research and development. The discovery of ADNP led to the discovery of KIAA0863 (Zamostiano et al., 2001) and the relationship of these 2 family members deserves further attention. As indicated in the Section 2, KIAA0863 and ADNP share structural similarities; however, KIAA0863 does not contain the neuroprotective NAP motif. Therefore, an open question is, does KIAA0863 function as

Note added in Proof

We have recently published a relevant new paper that adds insights to ADNP activities (Mandel et al., in press).

Acknowledgments

This work was supported by the United States–Israel Binational Science Foundation, the Israel Science Foundation, and the Lily and Avraham Gildor Chair for the Investigation of Growth Factors, the Dr. Diana and Zelman Elton (Elbaum) Laboratory for Molecular Neuroendocrinology and Allon Therapeutics, Inc. NAP is in clinical development at Allon Therapeutics Inc. Additional support was obtained from the National Institute of Child Health and Human Development, the National Institute on Aging, and

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