Interrelationships of Energy Metabolism to Oxidative Damage and Neurodegeneration

Glutamate receptor involvement and oxidative stress have both been implicated in damage to neurons due to impairment of energy metabolism. Using two different neuronal in vitro model systems, an ex vivo chick retinal preparation and dopamine neurons in mesencephalic culture, the involvement and interaction of these events as early occurring contributors to irreversible neuronal damage have been examined. Consistent with previous reports, the early acute changes in the retinal preparation, as well as irreversible loss of dopamine neurons due to inhibition of metabolism, can be prevented by blocking NMDA receptors during the time of energy inhibition. Oxidative stress was suggested to be a downstream consequence and contributor to neuronal cell loss due to either glutamate receptor overstimulation or metabolic inhibition since trapping of free radicals with the cyclic nitrone spin-trapping agent MDL 102,832 (1 mM) attenuated acute excitotoxicity in the retinal preparation or loss of mesencephalic dopamine neurons due to either metabolic inhibition by the succinate dehydrogenase inhibitor, malonate, or exposure to excitotoxins. In mesencephalic culture, malonate caused an enhanced efflux of both oxidized and reduced glutathione into the medium, a significant reduction in total reduced glutathione and a significant increase in total oxidized glutathione at time points that preceded those necessary to cause toxicity. These findings provide direct evidence for early oxidative events occurring following malonate exposure and suggest that the glutathione system is important for protecting neurons during inhibition of energy metabolism. Consistent with this, lowering of glutathione by buthionine sulfoxamine (BSO) pretreatment greatly potentiated malonate toxicity in the mesencephalic dopamine population. In contrast, BSO pretreatment did not potentiate glutamate toxicity. This latter finding indicates dissimilarities in the type of oxidative stress that is generated by the two insults and suggests that the oxidative challenge during energy inhibition is not solely a downstream consequence of glutamate receptor overstimulation. Received: December 15, 1997 Accepted: February 27, 1998 Gail D. Zeevalk, PhD Robert Wood Johnson Medical School Department of Neurology, Building UBHC 675 Hoes Lane, Piscataway, NJ 08854 (USA) Tel. +1 732 235 4066, Fax +1 732 235 5295, E-Mail zeevalgd@umdnj.edu ABC Fax + 41 61 306 12 34 E-Mail karger@karger.ch www.karger. com © 1998 S. Karger AG, Basel 0378–5866/98/0205–0444$15.00/0 Accessible online at: http://BioMedNet.com/karger Excitotoxic and Oxidative Stress during Energy Inhibition Dev Neurosci 1998;20:444–453 445 Introduction Impairment or defects in energy metabolism can result in acute damage as occurs in hypoxia/ischemia or may be an underlying component of chronic long-term damage to neurons as postulated to occur in certain neurodegenerative diseases such as Parkinson’s disease (PD). Accumulating studies now confirm the existence of reduced capacity for oxidative phosphorylation in PD, reporting diminished activities for complex I, and in some cases complex II and III, ·-ketoglutarate dehydrogenase or pyruvate dehydrogenase [for discussion, see Zeevalk et al., 1995]. Understanding the cellular events involved in damage to neurons kindled by less than optimal energy metabolism may provide opportunities for intervention in both acute and chronic situations. Oxidative stress has been implicated in neuronal damage due to perturbations of energy metabolism insomuch as free radical-trapping agents ameliorate neuronal loss in models of hypoxia/ischemia [Cao and Phillis, 1994; Carney and Floyd, 1991; Phillis and Clough-Helfman, 1990; Schulz et al., 1995b; Yue et al., 1992]. These findings provide indirect evidence for a contribution by oxidative events to neuronal damage caused by impairment of energy metabolism. Markers of oxidative damage such as protein carbonyl formation or lipid peroxidation have also been described in nervous tissue challenged by metabolic inhibition [Oliver et al., 1990; Traystman et al., 1991]. Although such signs of oxidative damage provide additional support for a role for oxidative stress, evidence of oxidative stress occurring prior to irreversible neuronal damage is lacking. In addition to oxidative stress, many laboratories, including our own, have reported the involvement of glutamate receptors, and in particular, the NMDA subtype of glutamate receptor in mediating damage to neurons when energy metabolism is perturbed [for discussions see Zeevalk and Nicklas, 1991; Greene and Greenamyre, 1996]. Recent studies from several laboratories have shown that neuronal exposure to glutamate or NMDA can cause an immediate increase in reactive oxygen species (ROS) [Gunasekar and Kanthasamy, 1995; Lafon-Cazal et al., 1993; Reynolds and Hastings, 1995; Bindokas et al., 1996]. Thus, it is possible that an oxidative stress that is generated following diminished energy metabolism is a downstream consequence of a secondary activation of glutamate receptors. In this study, we examined the involvement of oxidative stress and NMDA receptors in both the early and delayed irreversible damage to neurons in two in vitro model systems, an isolated, intact chick retinal preparation and a mesencephalic culture system containing dopamine neurons, the predominant neurotransmitter population lost in PD. Our findings provide direct evidence for early oxidative changes occuring during energy impairment that precede irreversible neuronal loss and suggest that NMDA receptor involvement and oxidative stress are not simply tandem events subsequent to inhibition of energy metabolism. Material and Methods Acute Retinal Studies Embryonic (E) day 15 retina were isolated from White Leghorn chickens as described previously [Zeevalk and Nicklas, 1990]. For studies of energy impairment due to hypoglycemia, isolated retina were preincubated in a bicarbonate-buffered Krebs-Ringer (KRB) at 37°C with constant gassing with 5% CO2/95% O2 for 10 min then transferred to fresh KRB containing 5.5 mM glucose or to zero glucose containing KRB supplemented with 10 mM 2-deoxyglucose (2DG) plus or minus the NMDA antagonist MK-801 (1 ÌM). For studies of excitotoxicity, retina were incubated with either 50 ÌM NMDA or kainate (KA) plus or minus the free radical spin-trapping agent MDL 102,832 [Thomas et al., 1996]. Following 30 min of incubation, the medium was removed for analysis of GABA content and the tissue either fixed for histology or used for protein determination as described by Lowry et al. [1951]. Mesencephalic Culture and Toxin Treatment Cultures were established from E15 rat mesencephalon as previously described [Zeevalk et al., 1995]. Cells were plated at 2.5 ! 105 cells/cm2 in 24-well plates previously coated with polyornithine and serum. The culture medium consisted of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM glutamine, 10% fetal bovine serum and 10% horse serum. A complete medium change was done at 48 h. For toxicity studies, at 8 days in vitro, half the medium volume was removed and saved as conditioned medium (CM). Malonate (50 mM) plus or minus MK-801 (0.5 ÌM) or MDL 102,832 (1 mM) was added to the remaining culture medium and the cells reincubated for 24 h. For studies of excitotoxicity, glutamate (50 ÌM), NMDA (100 ÌM) or KA (100 ÌM) plus or minus MDL 102,832 (1 mM) was added to the culture medium for 1 h. For all treatments, the toxin-containing medium was then removed and the cultures returned to CM supplemented with 5 mM glucose. After a 72-hour (malonate) or 96-hour (excitotoxins) recovery period, toxicity was determined by measurement of the high-affinity uptake of 3Hdopamine. For studies in which buthionine sulfoxamine (BSO) was used to reduce glutathione, cultures had half of the medium removed on day 7. BSO (10 ÌM ) was added and after 24 h, an exposure to either malonate (50 mM, 6 h) or glutamate (10–50 ÌM, 1 h) was carried out. At the end of toxin treatment, the cultures were returned to CM supplemented with 5 mM glucose and following 96 h of recovery, dopamine uptake was determined. For studies in which oxidized and reduced glutathione (GSSG and GSH) was measured, 8-day in vitro cultures were washed and switched to KRB. Malonate (50 mM) was added and the cultures were incubated for various times as indicated. GSH and GSSG in the tissue and medium were analyzed at 1, 3 or 6 h as described below.