Ž . Developmental Brain Research 99 1997 167–175 Research report Antiserum to activity-dependent neurotrophic factor produces neuronal cell death in CNS cultures: immunological and biological specificity Illana Gozes a , Ariane Davidson a , Yehoshua Gozes b , Richard Mascolo c , Rolf Barth c , Dale Warren c , Janet Hauser c , Douglas E. Brenneman c, ) a Department of Clinical Biochemistry, Sackler School of Medicine, Tel AÕiÕ UniÕersity, Tel AÕiÕ, Israel b The Israel Institute for Biological Research, Ness Ziona, Israel c Section on DeÕelopmental and Molecular Pharmacology, Laboratory of DeÕelopmental Pharmacology, National Institute for Child Health and Human DeÕelopment, National Institutes of Health, Bethesda, MD 20892, USA Accepted 26 November 1996 Abstract Ž . Activity-dependent neurotrophic factor ADNF is a glia-derived protein that is neuroprotective at femtomolar concentrations. ADNF Ž . is released from astroglia after treatment with 0.1 nM vasoactive intestinal peptide VIP . To further assess the biological role of ADNF, Ž . antiserum was produced following sequential injections of purified ADNF into mice. Anti-ADNF ascites fluid 1:10,000 decreased neuronal survival by 45–55% in comparison to untreated cultures or those treated with control ascites. The neuronal death after anti-ADNF treatment was observed in cultures derived from the spinal cord, hippocampus or cerebral cortex at similar IC ’s. Using a 50 terminal deoxynucleotidyl transferase in situ assay to estimate apoptosis in cerebral cortical cultures, anti-ADNF was shown to produce a 70% increase in the number of labeled cells in comparison to controls. In spinal cord cultures, anti-ADNF treatment produced a 20% decrease in choline acetyltransferase activity in comparison to controls. Neuronal cell death produced by the antiserum to ADNF was prevented in cultures co-treated with purified ADNF or ADNF-15, an active peptide derived from the parent ADNF. In vitro binding between the anti-ADNF and ADNF-15 was demonstrated with size exclusion chromatography. Comparative studies with other growth Ž factors insulin-like growth factor-1, platelet-derived growth factor, nerve growth factor, epidermal growth factor, ciliary neurotrophic . growth factor, and neurotrophin-3 demonstrated that only ADNF prevented neuronal cell death associated with electrical blockade. These investigations indicated that an ADNF-like substance was present in cultures derived from multiple locations in the central nervous system and that ADNF-15 exhibited both neuroprotection and immunogenicity. ADNF appears to be both a regulator of activity-depen- dent neuronal survival and a neuroprotectant. q 1997 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Astroglia; Vasoactive intestinal peptide; Neurotrophic factor neurotrophin; Cerebral cortex 1. Introduction An increasing number of diverse neuronal growth fac- tors are being discovered. Included in this group of regula- tory molecules are trophic factors such as nerve growth Ž . w x Ž . factor NGF 29,30 , ciliary neurotrophic factor CNTF w x Ž . w x 31 , fibroblast growth factor FGF 15,40 , insulin-like Ž w x. growth factors 1 and 2 IGF 1 and 2 27,41 brain-derived Ž . w x neurotrophic factor BDNF 28 , neurotrophin-3 and neu- Ž w x w x. rotrophin-4r5 NT3 16 and NT4, 25 and glial-derived w x neurotrophic factor 32 . Furthermore, cytokines also have w x neurotrophic properties 12,34 . This expanding class of ) Ž . Corresponding author. Fax: q1 301 496-9939. w x substances includes the various interleukins 12,13,34,36 w x and leukemia inhibitory factor 35 . Although many of the classic growth factors were first recognized to play impor- tant trophic roles in neuronrtarget cell interactions, it is now clear that glial cells in the central nervous system Ž . CNS express most of these growth factorsrcytokines, and that these glial cells have significant roles during development and nerve repair. A neuroprotective, glia-derived neurotrophic protein Ž . 14,000 Da and pl 8.3 " 0.25 , was recently isolated by w x sequential chromatographic methods 14 . The protein was Ž . named activity dependent neurotrophic factor ADNF , as it protected neurons from death associated with electrical blockade. The strategy used in isolating ADNF entailed measuring changes in neuronal survival after treatment of 0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0165-3806 96 00215-5