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

Published online by Cambridge University Press:  25 January 2005

SHARON FURMAN
Affiliation:
Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University
RUTH A. STEINGART
Affiliation:
Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University
SHMUEL MANDEL
Affiliation:
Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University
JANET M. HAUSER
Affiliation:
Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, Bethesda, MD 20892
DOUGLAS E. BRENNEMAN
Affiliation:
Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, Bethesda, MD 20892
ILLANA GOZES
Affiliation:
Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University

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.

Type
Research Article
Copyright
© Cambridge University Press 2005

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