A femtomolar-acting neuroprotective peptide induces increased levels of heat shock protein 60 in rat cortical neurons: a potential neuroprotective mechanism Rachel Zamostiano a , Albert Pinhasov a , Merav Bassan a , Orly Perl a , Ruth A. Steingart a , Roy Atlas a , Douglas E. Brenneman b , Illana Gozes a, * a Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel b Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, NICHD, NIH, Bethesda, MD 20892, USA Received 17 July 1998; received in revised form 19 December 1998; accepted 28 January 1999 Abstract Activity-dependent neurotrophic factor (ADNF) was recently isolated from conditioned media of astrocytes stimulated with vasoactive intestinal peptide (VIP). ADNF provided neuroprotection at femtomolar concentration against a wide variety of toxic insults. A nine amino acid peptide (ADNF-9) captured with even greater potency the neuroprotective activity exhibited by the parent protein. Utilizing Northern and Western blot analyses, it was now shown that ADNF-9 increased the expression of heat shock protein 60 (hsp60) in rat cerebral cortical cultures. In contrast, treatment with the Alzheimer’s toxin, the b-amyloid peptide, reduced the amount of intracellular hsp60. Treatment with ADNF-9 prevented the reduction in hsp60 produced by the b-amyloid peptide. The protection against the b-amyloid peptide-associated cell death provided by ADNF-9 may be mediated in part by intracellular increases in hsp60.  1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: hsp60; Activity-dependent neurotrophic factor; Stress proteins; Neurons Activity-dependent neurotrophic factor (ADNF) [3] is unique among the neuroprotective compounds in that it acts at femtomolar concentration [3], has structural similar- ity to heat shock protein 60 (hsp60) [3,7], and the entire protein’s neuroprotective activity is mimicked by short pep- tides derived from it [3,5,7,10]. ADNF constitutes a part of the neuroprotective milieu secreted from astroglial cells in the presence of the neuro- peptide vasoactive intestinal peptide (VIP) [4,17]. The active site of ADNF has recently been shown to be asso- ciated with a stretch of nine amino acids, ADNF-9. ADNF-9 protected neurons against death associated with electrical blockade (tetrodotoxin), excitotoxicity (N-methyl-D-aspar- tate, NMDA) and the Alzheimer’s neurotoxin (the b-amy- loid peptid [2,6], amino acids 25–35 [5]). Hsp60 is a stress protein associated with protein folding and hence supports cell survival [12]. Exposure of neurons to an elevated temperature is sufficient to induce the heat shock response and to provide neuroprotection [14]. The current study investigated whether part of the activity of ADNF-9 is mediated through intracellular increases in hsp60. Peptide synthesis and purification of ADNF-9 (SAL- LRSIPA) was conducted as previously described [5]. Cerebral cortical cultures from newborn rats were used [3,5,8,10]. Neurons were plated on a bed of astrocytes and cultures were maintained for 1 week. Peptides ADNF-9 (10 -15 M, dissolved in DMSO, 1 mg/10 ml, followed by serial dilutions in saline) and b-amyloid (amino acids 25– 35, 25 mM) were added for a 3-h incubation period at 37°C/ 10% CO 2 . RNA was extracted from the cultures utilizing RNAzolB (Biotex, Friendswood, TX) and subjected to Northern blot hybridizations [9]. The hsp60 mRNA was detected by Northern blot hybridization utilizing a radio- active probe. The probe was obtained by reverse transcrip- Neuroscience Letters 264 (1999) 9–12 0304-3940/99/$ - see front matter  1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(99)00168-8 * Corresponding author. Tel.: +972-3-640-7240; fax: +972-3-640- 8541; e-mail: igozes@post.tau.ac.il