Glutamate Neurotoxicity in Rat Cerebellar Granule Cells Involves Cytochrome c Release from Mitochondria and Mitochondrial Shuttle Impairment Anna Atlante, *Sabatina Gagliardi, Ersilia Marra, ²Pietro Calissano, and ‡Salvatore Passarella Centro di Studio sui Mitocondri e Metabolismo Energetico, CNR, and *Dipartimento di Biochimica e Biologia Molecolare, Universita ` di Bari, Bari; ² Istituto di Neurobiologia, CNR, Roma; and ‡Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Universita ` del Molise, Campobasso, Italy Abstract: To gain some insight into the mechanism by which glutamate neurotoxicity takes place in cerebellar granule cells, two steps of glucose oxidation were inves- tigated: the electron flow via respiratory chain from cer- tain substrates to oxygen and the transfer of extramito- chondrial reducing equivalents via the mitochondrial shuttles. However, cytochrome c release from intact mi- tochondria was found to occur in glutamate-treated cells as detected photometrically in the supernatant of the cell homogenate suspension. As a result of cytochrome c release, an increase of the oxidation of externally added NADH was found, probably occurring via the NADH-b 5 oxidoreductase of the outer mitochondrial membrane. When the two mitochondrial shuttles glycerol 3-phos- phate/dihydroxyacetone phosphate and malate/oxalo- acetate, devoted to oxidizing externally added NADH, were reconstructed, both were found to be impaired un- der glutamate neurotoxicity. Consistent early activation in two NADH oxidizing mechanisms, i.e., lactate production and plasma membrane NADH oxidoreductase activity, was found in glutamate-treated cells. In spite of this, the increase in the cell NADH fluorescence was found to be time-dependent, an index of the progressive damage of the cell. Key Words: Neurotoxicity—Glutamate—Mito- chondria—Cytochrome c—Shuttle—Lactate. J. Neurochem. 73, 237–246 (1999). The exposure of cultured cerebellar granule cells (CGCs) to glutamate has been found to cause massive neuronal degeneration and death as revealed by both in vivo and in vitro experiments (Choi et al., 1987; Choi, 1988, 1994; Coyle and Puttfarcken, 1993). Al- though some aspects of glutamate neuronal excitotox- icity, including the statement that the initial event of this process is the extensive entry of Ca 2+ into the cell, have been exhaustively investigated, the details of how cell death takes place remain to be established. In particular, in a number of studies aimed at identi- fying the cell components involved in glutamate neu- rotoxicity (GNT), the pivotal role of mitochondria has been highlighted: it was shown that dying neurons rapidly lose their mitochondrial membrane potential and energy charge (Ankarcrona et al., 1995). Further, Ca 2+ -dependent uncoupling was found to contribute to the mitochondrial oxidative stress initiated by glu- tamate exposure (Dugan et al., 1995) with a key role in Ca 2+ homeostasis proposed for mitochondria (Budd and Nicholls, 1996; Schinder et al., 1996). Finally, we have shown the early and progressive inhibition of both glucose and succinate oxidation, in intact CGCs and cell homogenate, respectively, under GNT (At- lante et al., 1996). In light of these reports, the iden- tification of the mitochondrial target(s) and processes involved in neurotoxicity is a worthwhile goal to be pursued. As a first step in the elucidation of the role of mito- chondria in the cascade of events that lead neuronal cells to necrosis and death, we have considered two processes that play a major role in the glucose oxidation, namely, the electron flow in the respiratory chain and the trans- port of reducing equivalent from cytosol to mitochon- dria, as mediated by the mitochondrial shuttles. We show that cytochrome c (cyt c) release from the mitochondria to the extramitochondrial phase and the progressive in- hibition of mitochondrial shuttles occur within minutes as a result of glutamate exposure of CGCs. Resubmitted manuscript received October 22, 1998; final revised manuscript received February 26, 1999; accepted February 26, 1999. Address correspondence and reprint requests to Dr. S. Passarella at Centro di Studio sui Mitocondri e Metabolismo Energetico, CNR, Via Amendola, 165/A, 70126 Bari, Italy. Abbreviations used: ADK, adenylate kinase; BME, basal medium with Eagle’s salts; CGCs, cerebellar granule cells; cyt c, cytochrome c; DHAP, dihydroxyacetone phosphate; e.u., enzymatic units; GDH, glutamate de- hydrogenase; Glu-CGCs, glutamate-treated CGCs; GNT, glutamate neu- rotoxicity; G3P, glycerol 3-phosphate; G3PDH, glycerol-3-phosphate de- hydrogenase; MAL, malate; MDH, malate dehydrogenase; MK-801, (5R,10S)-(+)-5-methyl-10,11-dihydro[a,d]cyclohepten-5,10-imine hydro- gen maleate; OAA, oxaloacetate; PBS, phosphate-buffered saline; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine. 237 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry