ADNP differential nucleus/cytoplasm localization in neurons suggests multiple roles in neuronal differentiation and maintenance.

Complete deficiency in activity-dependent neuroprotective protein (ADNP) results in neural tube closure defects and death at gestation day 9 in mice. ADNP-deficient embryos exhibit dramatic increases in gene transcripts associated with lipid metabolism coupled to reduction in organogenesis/neurogenesis-related transcripts. In the pluripotent teratocarcinoma cell line P19, ADNP was shown to interact with specific chromatin regions in the neurodifferentiated state, which was associated with binding to the heterochromatin protein 1 alpha. In this study, using P19 cells as a differentiation model, we showed that ADNP expression and cytoplasm/nucleus distribution is unique in neuronal-differentiated cells compared to cardiovascular and nondifferentiated pluripotent cells. ADNP-like immunohistochemical localization to the neuronal cytoplasm and neurites was shown in this study not only in the cellular model but also in the brain cerebral cortex and olfactory bulb. Small hairpin RNA ADNP downregulation was used to further investigate ADNP involvement in p19 neurodifferentiation. An approximately 80% robust reduction in ADNP led to a substantial reduction in embryoid body formation and a significant reduction (approximately 50%) in neurite numbers. These results position ADNP in direct association with neuronal cell differentiation and maturation.

Silencing of the ADNP-family member, ADNP2, results in changes in cellular viability under oxidative stress.

Activity-dependent neuroprotective protein (ADNP) 2 (KIAA0863; ZNF508) gene, a homeobox-profile containing gene, was identified in a screen for homologous proteins to ADNP. The human ADNP2 contains 1131 amino acid residues with a molecular weight of 122.8 KDa. In silico analysis indicated that ortholgs to ADNP2 exist in different phyla, suggesting that ADNP2 might be evolutionary conserved. Here, we began to explore the molecular and functional characterization of ADNP2. Results showed that the mouse ADNP2 mRNA is ubiquitously expressed in distinct normal tissues with increased expression in the brain, particularly in the cerebral cortex. During development, a relatively high level of ADNP2 gene expression was found in the embryonic mouse brain and was sustained throughout embryogenesis and adulthood. An increase in the mRNA was detected in differentiated P19 neuronal/glial-like cells as compared with the non-differentiated cells. To gain insight into ADNP2 function, ADNP2-deficient cell lines were established by the RNA silencing (small interfering RNA) technology. ADNP2 deficiency significantly changed the toxicity induced by hydrogen peroxide in P19 embryonic carcinoma cells, similar to what would be predicted for ADNP deficiency. These findings represent an initial characterization of ADNP2 and suggest that this gene product may have an important function in brain by playing a role in cellular survival pathways.

Activity-dependent neuroprotective protein constitutes a novel element in the SWI/SNF chromatin remodeling complex.

Complete deficiency in activity-dependent neuroprotective protein (ADNP), a heterochromatin 1-binding protein, results in dramatic changes in gene expression, neural tube closure defects, and death at gestation day 9 in mice. To further understand the cellular roles played by ADNP, the HEK293 human embryonic kidney cell line that allows efficient transfection with recombinant DNA was used as a model for the identification of ADNP-interacting proteins. Recombinant green fluorescent protein (GFP)-ADNP was localized to cell nuclei. When nuclear extracts were subjected to immunoprecipitation with specific GFP antibodies followed by polyacrylamide gel electrophoresis, several minor protein bands were observed in addition to GFP-ADNP. In-gel protein digests followed by mass spectrometry identified BRG1, BAF250a, and BAF170, all components of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex, as proteins that co-immunoprecipitate with ADNP. These results were verified utilizing BRG1 antibodies. ADNP short hairpin RNA down-regulation resulted in microtubule reorganization and changes in cell morphology including reduction in cell process formation and cell number. These morphological changes are closely associated with the SWI/SNF complex multifunctionality. Taken together, the current study uncovers a molecular basis for the essential function of the ADNP gene and protein.

Activity-dependent neuroprotective protein snippet NAP reduces tau hyperphosphorylation and enhances learning in a novel transgenic mouse model.

Activity-dependent neuroprotective protein (ADNP) differentially interacts with chromatin to regulate essential genes. Because complete ADNP deficiency is embryonic lethal, the outcome of partial ADNP deficiency was examined. ADNP(+/-) mice exhibited cognitive deficits, significant increases in phosphorylated tau, tangle-like structures, and neurodegeneration compared with ADNP(+/+) mice. Increased tau hyperphosphorylation is known to cause memory impairments in neurodegenerative diseases associated with tauopathies, including the most prevalent Alzheimer's disease. The current results suggest that ADNP is an essential protein for brain function and plays a role in normal cognitive performance. ADNP-deficient mice offer an ideal paradigm for evaluation of cognitive enhancers. NAP (NAPVSIPQ) is a peptide derived from ADNP that interacts with microtubules and provides potent neuroprotection. NAP treatment partially ameliorated cognitive deficits and reduced tau hyperphosphorylation in the ADNP(+/-) mice. NAP is currently in phase II clinical trials assessing effects on mild cognitive impairment.

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.

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes.

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.

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.