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

Abstract

Activity-dependent neuroprotective protein (ADNP, ∼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 α-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 ∼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 Snippet NAP Reduces Tau Hyperphosphorylation and Enhances Learning in a Novel Transgenic Mouse Model

“…At the protein level, ADNP expression was assessed by Western analysis (Furman et al, 2004) using actin as a control. As expected, results showed reduced expression of ADNP at the protein level in ADNP ϩ/Ϫ mice at all ages tested.…”

“…Recent findings have shown that ADNP may colocalize with microtubules in the cytoplasm of astrocytes (Furman et al, 2004). The tubulin cytoskeleton is intimately associated with neuronal structure and function (e.g., Gozes and Sweadner, 1981), and the pathology of tau, a microtubule-associated protein that loses proper functionality in the hyperphosphorylated state, has been implicated in multiple neurodegenerative diseases related to axonal dysfunction (Mandelkow et al, 2003).…”

“…Furthermore, a potential interaction with cytoplasmic microtubules has been observed (Furman et al, 2004). Structure-activity studies identified a short peptide sequence in ADNP, NAP (NAPVSIPQ), that mimics the neuroprotective activity of the parent protein and crosses the blood brain barrier after systemic or intranasal administra-tion (Gozes et al, 2005a).…”

“…1, A and B) and that was found to be expressed in the adult subcortical domains (Stoykova and Gruss, 1994). Quantitative reverse transcription and real-time PCR was performed on individual RNA samples prepared from subcortical domains of 2-month-old male mice (ADNP Given the changes in gene expression that were observed above, the finding of ADNP as a glial protein (Bassan et al, 1999;Furman et al, 2004) and the neuroprotective activity of recombinant ADNP , it was of interest to evaluate the degree of protective support given by an astrocyte feeder layer derived from ADNP ϩ/Ϫ mice to neuronal cultures. Astrocytes were derived from newborn cerebral cortical tissues.…”

Cell-Mediated Neuroprotection in a Mouse Model of Human Tauopathy

“…Another potential mechanism for the neuroprotective effects of both the glial-derived NPC transplant and the direct astrocytes may be through secretion of activity-dependent neuroprotective protein (ADNP). Recent literature suggests this protein is secreted by astrocytes, while intranasal delivery of a peptide fragment derived from ADNP, termed NAP, has been demonstrated to increase soluble tau, reduce hyperphosphorylation, and reduce cognitive decline observed in tau transgenic mice (Furman et al, 2004;Shiryaev et al, 2009). This mechanism may be an additional effect that the exogenous transplanted astrocytes have on the endogenous populations and thus promotes neuroprotection.…”