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

Abstract

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.

Figures

Fig. 1

ADNP-like immunoreactivity localizes to the cytoplasm and nucleus of astrocytes. Western blot analysis of proteins extracted from astrocytes that were incubated either with (+) or without (−) VIP. Molecular weights were determined using molecular-weight markers separated with the tested samples. (A) ADNP immunoreactivity in cytoplasmic and nuclear fractions detected using α-CNAP. (B) As in A after incubation with excess of recombinant ADNP in the blocking solution (1:100, antibody:ADNP w/w). (C) Duplicate protein extracts were subjected to 10% polyacrylamide gel electrophoresis and stained with Gelcode Blue Staining Reagent, the resulting electropherogram is depicted. All experiments were repeated at least three times.

Fig. 2

Separating recombinant ADNP using SDS–PAGE. Western blot analysis of recombinant ADNP (ADNP–VP22 fusion protein). Molecular weight markers were separated alongside the tested samples. (A) Detection of recombinant ADNP (1.7 μg) with α-CNAP. (B) As in A after incubation with excess recombinant ADNP in the blocking solution (1:100, antibody:ADNP w/w). (C) Detecting recombinant ADNP with α-cMyc.

Fig. 3

Immunoprecipitation of ADNP in PC12 cells. ADNP was immunoprecipitated from PC12 cells using the α-CNAP antibody, subjected to 10% polyacrylamide gel electrophoresis and stained with Gelcode Blue Staining Reagent. Experiments were repeated three times. A representative electropherogram is shown.

Fig. 4

Tubulin, actin and ADNP-like immunoreactivity in astrocytes. (A,D) ADNP-like immunoreactivity (green) determined using α-CNAP. (B) Tubulin immunoreactivity (red) determined using α-tubulin antibodies. (C) Areas of colocalization of ADNP and tubulin are indicated in blue. (E) Actin microfilaments stained by phalloidin (red). (F) ADNP-like immunoreactivity (green) does not colocalize with actin microfilaments (red). (G) Higher magnification of C. Scale bar, 10 μm. Experiments were repeated at least three times.

Fig. 5

ADNP-like immunoreactivity in conditioned media from astrocytes. Western blot analysis on conditioned media from astrocytes incubated either with (+) or without (−) 0.1 nM VIP for 3 hours. To allow comparative analyses, 5 μg of total protein were loaded on each lane. (A) ADNP immunoreactivity determined using α-CNAP. (B) As in A after incubation with an excess of the recombinant ADNP in the blocking solution (1:100, antibody:ADNP w/w).