Abstract. In the light of both the major role played by released cytochrome c in apoptosis of a variety of cells and the availability of cerebellar granule cells as a model system to investigate apoptosis as a function of time from induction to cell death, we review data aimed at elucidating the events dealing with cytochrome c release from mitochondria as well as its role outside mitochondria. We report cytochrome c release in the apoptosis time course as dependent on the function of both the antioxidant and proteolytic systems. We show that, beside the role played by cytochrome c in participating in apoptosome formation and in triggering the caspase cascade, at least in cerebellar granule cells, released cytochrome c can maintain its ability to work as an electron carrier, being a scavenger of reactive oxygen species and an electron donor to cytochrome oxidase, thus driving the ATP synthesis. Mitochondria are sites of cellular energy production which may also influence life and death decisions by initiating or inhibiting cell death. Mitochondrial depolarization and the subsequent release of pro-apoptotic factors, including cytochrome c (cyt c), have been suggested to be required for the activation of a cell death program in some forms of neuronal apoptosis. Despite the large body of literature dealing with cyt c release from mitochondria in cells undergoing apoptosis (1-9) and its role in caspase activation (10, 11), knowledge of the relationship between apoptosis and cyt c remains incomplete. For instance, it has not been well established: a) whether cyt c release occurs from uncoupled/damaged mitochondria or cyt c release takes place before any mitochondrial dysfunction, b) how cyt c release is regulated, c) the release time course, d) whether and how the cytosolic level of released cyt c changes during apoptosis, and 5) the role played by the released cyt c in apoptosis. Since the apoptosis pathways can differ from each other, depending on the cells investigated as well as on the manner by which apoptosis is induced, a variety of response is expected. The above points can be described by using as a model system rat cerebellar granule cells (CGCs). Dissociated CGCs from early postnatal rats can be maintained in serum-containing medium in the presence of elevated potassium levels (25mM) (12) or by adding a low concentration of N-methyl-D-aspartic acid (NMDA) to the culture medium (13). Both a low concentration of NMDA and depolarization are assumed to mimic endogenous excitatory activity (14) with survival promotion mediated by increases in intracellular calcium level. Dissociated cerebellar granule cells develop characteristics of mature cerebellar granule cells in vivo, including an extensive neurite network, expression of excitatory amino acids receptor and production and release of L-glutamate (14). CGCs undergo apoptosis after serum removal, in the presence of low K + concentrations (5mM) (15), while over stimulation of glutamate receptors on granule cells leads to excitotoxic death (16-18). Therefore, these neurons are a versatile system to dissect the mechanisms which are common to apoptosis and necrosis and those which are unique. Apoptotic cell death, which is accompanied by DNA fragmentation and is dependent on cell biosynthesis (15, 19), probably mimics the naturally occurring death of 20-30% of granule cells (20), which is important for matching the number of granule cells with Purkinje cells, which occurs during the third to fifth postnatal weeks (21, 22). 335 Correspondence to: Prof Salvatore Passarella, Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via De Sanctis - 86100 Campobasso, Italy. Tel: +39 (0874) 404671-243, Fax: +39 (0874) 404678, e-mail: passarel@unimol.it Key Words: Apoptosis, cerebellar granule cells, cytochrome c, oxidative stress. in vivo 18: 335-344 (2004) Review Apoptosis and Cytochrome c Release in Cerebellar Granule Cells ANTONELLA BOBBA 1 , ANNA ATLANTE 1 , LIDIA de BARI 1 , SALVATORE PASSARELLA 2 and ERSILIA MARRA 1 1 Istituto di Biomembrane e Bioenergetica, CNR, Via G. Amendola, 165/A - 70126 Bari; 2 Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via De Sanctis, 86100 Campobasso, Italy 0258-851X/2004 $2.00+.40