UNCORRECTED PROOF Progress in Brain Research, Vol. 146 ISSN 0079-6123 Copyright ß 2003 Elsevier. All rights reserved DOI: 10.1016/S0079-6123(03)46006-8 CHAPTER 6 Pathways of survival induced by NGF and extracellular ATP after growth factor deprivation Nadia D’Ambrosi 1,2 , Barbara Murra 1 , Fabrizio Vacca 1,3 and Cinzia Volonte´ 1,4, * 1 Fondazione Santa Lucia, Rome, Italy 2 University of Rome Tor Vergata, Department of Neuroscience, Rome, Italy 3 University of Rome ‘La Sapienza’, Department of Human Physiology and Pharmacology, Rome, Italy 4 Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy Abstract: In a previous work we demonstrated that extracellular adenosine-5 0 -triphosphate (ATP), acting on P2 receptors, exerts neuritogenic and trophic effects on the phaeochromocytoma 12 (PC12) cell line. These actions are comparable to those sustained by nerve growth factor (NGF) and involve several overlapping pathways. In this work, we describe some of the mechanisms recruited by ATP and NGF in maintaining PC12 cell survival after serum deprivation. We show that both ATP and NGF upregulate the expression of the stress-induced proteins heat shock protein 70 (HSP70) and HSP90, whilst glucose-response protein 75 (GRP75) and GRP78 are not affected. In parallel with NGF, ATP prevents the cleavage and activation of caspase-2 and inhibits the release of cytochrome c from mitochondria into the cytoplasm. Finally, neither NGF, nor ATP directly modulate the expression of P2 receptors in the induction of cell survival. Our data contribute to dissect the biological mechanisms activated by extracellular purines exerting trophic actions and to establish that survival and neurite outgrowth lie on different mechanistic pathways. Keywords: P2 receptor; PC12 cells; caspase-2; cytochrome C; heat shock proteins Introduction The biological mechanisms behind differentiation, proliferation or cell death can be concurrently investi- gated by using a cellular model system such as the rat phaeochromocytoma 12 (PC12) cells (Greene and Tischler, 1976). Although the PC12 cellular model can only partially mimic the whole body complexity of an intact organism, it allows experimental control under defined culture conditions and especially a more direct investigation of the several overlapping pathways propagating a biological process. These cells share neural crest origin with sympathetic neurons and thus retain neuronal features, such as nerve growth factor (NGF)-dependency for survival and differentiation in defined serum-free conditions (Greene, 1978). In the absence of NGF and in the presence of serum, they instead maintain their proliferation properties. Finally, when PC12 cells are deprived of trophic support, they undergo rapid apoptotic death. The addition of NGF hampers such death (Batistatou and Greene, 1991; Rukenstein et al., 1991) and, moreover, induces sympathetic-like differentiation. During development, many cell populations are acutely dependent for their survival on target-derived trophic factors and neurons that fail to find the appropriate targets and sources of neurotrophic factors will eventually die (Pettmann and Henderson, 1998). Sympathetic neurons, for example, require NGF for survival during late embryogenesis and early postnatal period. Therefore, cell death constitutes an integral part of the plasticity of the nervous system (Oppenheim, 1991), also playing an important role in the removal *Correspondence to: C. Volonte´, Fondazione Santa Lucia, Via Ardeatina, 354, I-00179 Rome, Italy. Tel.: þ 39-06-5150-1557; Fax: þ 39-06-5150-1556; E-mail: cinzia@in.rm.cnr.it ARTICLE IN PRESS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48