Isolated Cerebral and Cerebellar Mitochondria Produce Free Radicals when Exposed to Elevated Ca²⁺ and Na⁺: Implications for Neurodegeneration

Abstract

The evidence is compelling that free radicals, plus increases in free cytosolic Ca2+ and Na+, figure prominently in neuronal death after exposure to glutamate and dicarboxylic excitotoxins such as NMDA and kainate. However, neither the source of these radicals nor the direct connection between Ca2+ mobilization and radical production has been well defined. Electron paramagnetic resonance studies reported here indicate that intact mitochondria isolated from adult rat cerebral cortex and cerebellum generate extremely reactive hydroxyl (•OH) radicals, plus ascorbyl and other carbon‐centered radicals when exposed to 2.5 µM Ca2+, 14 mM Na+, plus elevated ADP under normoxic conditions, circumstances that prevail in the cytoplasm of neurons during excitotoxin‐induced neurodegeneration. In a feed‐forward cycle, exposure of isolated mitochondria to •OH significantly increases subsequent radical production five‐ to 16‐fold (average = 8.8 ± 1.6 SE, n = 6, p > 0.01) with succinate as substrate, and also selectively impairs function of NADH‐CoQ dehydrogenase activity (electron transport complex 1). These effects are also reflected by respiration rates that are reduced 48% with complex 1 substrates, but increased 27% with complex 2 substrate, after •OH exposure. Comparable complex 1 dysfunction is observed in mitochondria isolated from the substantia nigra of Parkinson's disease patients, from platelets of Huntington's disease patients, and from neocortex of Alzheimer's disease patients. Mitochondrial radical production provides a testable model, based on oxyradical toxicity, oxidative enzyme inactivation, and mitochondrial dysfunction, for the final common pathway of neuronal necrosis during excitotoxicity, and in a host of neurodegenerative disorders.

Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

“…Where indicated, 5 mM succinate or 5 mM ␣-ketoglutarate A were included into the medium. The sequence of additions was as follows: mitochondria (0.1-0.125 mg/ml) were added into the incubation medium and incubated for 2 min, then phosphorylation was inhibited with 1. activity is stimulated by Ca 2ϩ and ADP may perhaps account for previous findings that mitochondrial ROS production was increased by Ca 2ϩ (Dykens, 1994;Kowaltowski et al, 1995Kowaltowski et al, , 1996Kowaltowski et al, , 1998a, Ca 2ϩ in the presence of rotenone and succinate , and by ADP (Barja, 1999). The results presented in the accompanying report by Tretter and Adam-Vizi (2004) demonstrate that Ca 2ϩ activated ROS production by isolated KGDHC both in the presence and in the absence of pyridine nucleotides.…”

“…The finding that H 2 O 2 production is frequently reported as being almost absent in the presence of succinate and rotenone (Barja, 1999;Liu et al, 2002) is intriguing because the intramitochondrial NADPH/NADP ϩ ratio under such conditions is high. Stimulatory effects of ADP (Barja, 1999) and Ca 2ϩ (Dykens, 1994;Kowaltowski et al, 1995Kowaltowski et al, , 1996Kowaltowski et al, , 1998a on mitochondrial ROS production are also puzzling because both Ca 2ϩ uptake/retention and ADPinduced oxidative phosphorylation dissipate energy and would be expected to decrease the level of reduction of complex I and hence the ROS production. It also appears that the stimulatory effect of the complex I inhibitor rotenone on ROS production that possibly originates from a site within complex I is species and tissue dependent; ROS stimulation by rotenone varies from ϳ300% in guinea pig to 0% in horse heart submitochondrial particles (Herrero and Barja, 2000) and in whole intact rat heart mitochondria (Chen et al, 2003) to inhibition of ROS production in mouse kidney mitochondria (Kwong and Sohal, 1998).…”

“…Reactive oxygen species (ROS) are thought to contribute to neuronal cell death caused by ischemia, excitotoxicity, and various acute and chronic neurological disorders (Dykens, 1994;Fiskum et al, 1999;Murphy et al, 1999;Fiskum, 2000;Nicholls and Budd, 2000). A compelling body of evidence indicates that mitochondria are the major source of ROS in normal tissues and under a variety of neurodegenerative conditions .…”

Cellular and Molecular Pathways of Ischemic Neuronal Death

“…An increase of oxygen during reperfusion induces the uncoupling of the electron flow from the mitochondria electron transport chain, causing the accumulation of free radicals. During hypoxic-ischemic injury, Ca 2+ is accumulated in the cytosol (see above) and enters into mitochondria, which results in the production of mitochondrial free radicals (Hasegawa et al, 1993;Dykens, 1994; Dugan et al, 1995;Giulivi et al, 1995;Piantodosi and Zhang, 1996). Excess Ca 2+ in the mitochondria interrupts the electron transport chain and collapses the mitochondrial membrane potential (Zhang et al, 1990;Rego et al, 2000).…”

Ischemic Cardiomyopathy Following Seizure Induction by Domoic Acid

“…14 Overstimulation of ionotropic glutamate receptors (iGluR), namely N-methyl-D-aspartate (NMDA) receptors, provokes pathophysiological increases in intracellular Ca 2ϩ and consequently leads to the activation of several overlapping Ca 2ϩ -dependent processes, such as the generation of mitochondrial reactive oxygen species and loss of cellular viability. [15][16][17][18][19][20] DOM has been shown to be 8 to 10 times more potent than kainic acid in rodents, producing a similar reproducible pattern of behavioral toxicity culminating in seizures. 8,21,22 Studies conducted in rats, mice, and cynomolgus monkeys have shown a consistent pattern of DOM-induced damage to the hippocampal pyramidal neurons, as well as to the thalamic, amygdalar, entorhinal, cortical, and septal neurons after DOM administration (0.22 to 4.4 mg/kg i.p.).…”