Diminished glutathione levels cause spontaneous and mitochondria-mediated cell death in neurons from trisomy 16 mice: a model of Down's syndrome

It has been suggested that the increased neuronal death in cultures from trisomy 16 (Ts16) mice, a model of Down's syndrome, might result from a diminished concentration of reduced glutathione (GSH). In this study we used microfluorometric techniques to investigate the effect of GSH levels on neuronal survival in diploid and Ts16 cultures. Addition of the GSH precursors cysteine and cystine and the antioxidant tocopherol to the culture medium increased the GSH concentration up to 126.0% in diploid and up to 111.9% in Ts16 neurons. Moreover, we observed a reduced spontaneous neuronal death rate in diploid and Ts16 cultures. Following the application of 50-100 microM glutamate to culture medium, we found a GSH increase in the presence of cysteine, cystine, tocopherol, and cyclosporin A, an inhibitor of mitochondrial permeability transition (diploid, 105.8-110.8%; Ts16, 83.1-96.3%). However, only tocopherol and cyclosporin A had a protective effect on glutamate-induced neuronal death. The results suggest that reduced GSH levels affect the increase of a spontaneous and a mitochondria-mediated, cyclosporin A-sensitive type of neuronal cell death. Therefore, elevating intracellular GSH concentration may have neuroprotective effects in Down's syndrome and Alzheimer's disease.     

In vivo restoration of physiological levels of truncated TrkB.T1 receptor rescues neuronal cell death in a trisomic mouse model

Imbalances in neurotrophins or their high-affinity Trk receptors have long been reported in neurodegenerative diseases. However, a molecular link between these gene products and neuronal cell death has not been established. In the trisomy 16 (Ts16) mouse there is increased apoptosis in the cortex, and hippocampal neurons undergo accelerated cell death that cannot be rescued by administration of brain-derived neurotrophic factor (BDNF). Ts16 neurons have normal levels of the TrkB tyrosine kinase receptor but an upregulation of the TrkB.T1 truncated receptor isoform. Here we show that restoration of the physiological level of the TrkB.T1 receptor by gene targeting rescues Ts16 cortical cell and hippocampal neuronal death. Moreover, it corrects resting Ca2+ levels and restores BDNF-induced intracellular signaling mediated by full-length TrkB in Ts16 hippocampal neurons. These data provide a direct link between neuronal cell death and abnormalities in Trk neurotrophin receptor levels.     

Increased expression of NR2A subunit does not alter NMDA-evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons

The trisomy 16 (Ts16) mouse is an animal model for human trisomy 21 (Down's syndrome). The gene encoding the NR2A subunit of the NMDA receptor has been localized to mouse chromosome 16. In the present study, western blot analysis revealed a 2.5-fold increase of NR2A expression in cultured Ts16 embryonic hippocampal neurons. However, this increase did not affect the properties of NMDA-evoked currents in response to various modulators. The sensitivity of NMDA receptors to transient applications of NMDA, spermine, and Zn(2+) was investigated in murine Ts16 and control diploid cultured embryonic hippocampal neurons. Peak and steady-state currents evoked by NMDA were potentiated by spermine at concentrations < 1 mM, and inhibited by Zn(2+) in a dose-dependent and voltage-independent manner. No marked difference was observed between Ts16 and control diploid neurons for any of these modulators with regard to IC(50) and EC(50) values or voltage dependency. Additionally, inhibition by the NR2B selective inhibitor, ifenprodil, was similar. These results demonstrate that NMDA-evoked currents are not altered in cultured embryonic Ts16 neurons and suggest that Ts16 neurons contain similar functional properties of NMDA receptors as diploid control neurons despite an increased level of NR2A expression.     

Permeability Transition Pore-Mediated Mitochondrial Superoxide Flashes Regulate Cortical Neural Progenitor Differentiation

In the process of neurogenesis, neural progenitor cells (NPCs) cease dividing and differentiate into postmitotic neurons that grow dendrites and an axon, become excitable, and establish synapses with other neurons. Mitochondrial biogenesis and aerobic metabolism provide energy substrates required to support the differentiation, growth and synaptic activity of neurons. Mitochondria may also serve signaling functions and, in this regard, it was recently reported that mitochondria can generate rapid bursts of superoxide (superoxide flashes), the frequency of which changes in response to environmental conditions and signals including oxygen levels and Ca²⁺ fluxes. Here we show that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation.     

Neuronal apoptosis in mouse trisomy 16: mediation by caspases

Hippocampal neurons from the trisomy 16 (Ts16) mouse, a potential animal model of Down's syndrome (trisomy 21) and neurodegenerative disorders such as Alzheimer's disease (AD), die at an accelerated rate in vitro. Here, we present evidence that the accelerated neuronal death in Ts16 occurs by apoptosis, as has been reported for neurons in AD. First, the nuclei of dying Ts16 neurons are pyknotic and undergo DNA fragmentation, as revealed by terminal transferase-mediated dUTP nick end-labeling. Second, the accelerated death of Ts16 neurons is prevented by inhibitors of the caspase family of proteases, which are thought to act at a late, obligatory step in the apoptosis pathway. In the presence of maximally effective concentrations of caspase inhibitors, Ts16 neuron survival was indistinguishable from that of control neurons. These results suggest that overexpression of one or more genes on mouse chromosome 16 leads to caspase-mediated apoptosis in Ts16 neurons.     

Nitric oxide-induced mitochondrial dysfunction: implications for neurodegeneration

Excessive generation of nitric oxide (NO) has been implicated in the pathogenesis of several neurodegenerative disorders. Damage to the mitochondrial electron transport chain has also been implicated in these disorders. NO and its toxic metabolite peroxynitrite (ONOO(-)) can inhibit the mitochondrial respiratory chain, leading to energy failure and ultimately cell death. There appears to be a differential susceptibility of brain cell types to NO/ONOO(-), which may be influenced by factors including cellular antioxidant status and the ability to maintain energy requirements in the face of marked respiratory chain damage. Although formation of NO/ONOO(-) following cytokine exposure does not affect astrocyte survival, these molecules may diffuse out and cause mitochondrial damage to neighboring NO/ONOO(-)-sensitive cells such as neurons. Evidence suggests that NO/ONOO(-) causes release of neuronal glutamate, leading to glutamate-induced activation of neuronal NO synthase and generation of further damaging species. While neurons appear able to recover from short-term exposure to NO/ONOO(-), extending the period of exposure results in persistent damage to the respiratory chain and cell death ensues. These findings have important implications for acute infection vs. chronic neuroinflammatory disease states. The evidence for NO/ONOO(-)-mediated mitochondrial damage in neurodegenerative disorders is reviewed and potential therapeutic strategies are discussed.     

Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat

We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration ofEscherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0–0.8 Hz) in systemic arterial pressure signals. LPS also elicited progressive hypotension, and death ensued within 4 h. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochromec reductase (Complexes I + III) and cytochromec oxidase (Complex IV) in the RVLM during all three phases of endotoxemia. On the other hand, the activity of succinate cytochromec reductase (Complexes II + III) remained unaltered. We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat.     

Amyloid-β triggers the release of neuronal hexokinase 1 from mitochondria

Brain accumulation of the amyloid-β peptide (Aβ) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.     

Bioenergetic and oxidative effects of free 3-nitrotyrosine in culture: selective vulnerability of dopaminergic neurons and increased sensitivity of non-dopaminergic neurons to dopamine oxidation

Protein bound and free 3-nitrotyrosine (3NT) levels are elevated in neurodegenerative diseases and have been used as evidence for peroxynitrite generation. Intrastriatal injection of free 3NT causes dopaminergic neuron injury and represents a new mouse model of Parkinson's disease (PD). We are investigating the nature of free 3NT neurotoxicity. In primary ventral midbrain cultures, free 3NT damaged dopaminergic neurons, while adjacent non-dopaminergic neurons were unaffected. Combined treatment with free 3NT and subtoxic amounts of dopamine caused extensive death of non-dopaminergic forebrain neurons in culture. Free 3NT alone directly inhibited mitochondrial complex I, decreased ATP, sensitized neurons to mitochondrial depolarization, and increased superoxide production. Subtoxic concentrations of rotenone (instead of free 3NT) caused similar results. Additionally, free 3NT and dopamine combined increased extraneuronal hydrogen peroxide and decreased intraneuronal glutathione levels more than dopamine alone. Oxidative and bioenergetic processes have been proposed to contribute to neurodegeneration in PD. As free 3NT is a compound that is increased in PD, damages dopamine neurons in vivo and in vitro and has detrimental effects on neuronal bioenergetics, it is possible that free 3NT is an endogenous contributing factor to neuronal loss, in addition to being a marker of oxidative and nitrative processes.     

A FINE STRUCTURAL STUDY OF THE HIPPOCAMPUS AND DORSAL ROOT GANGLION IN MOUSE TRISOMY 16, A MODEL OF DOWN'S SYNDROME

Mouse trisomy 16 (Ts16) appears to provide an animal model of Down's syndrome in that a portion of mouse chromosome 16 is syntenic with part of human chromosome 21. Trisomy 21 in human beings leads to the mental retardation of Down's syndrome and in middle age, to some presenile anatomic and clinical features of Alzheimer's disease. Neural tissue from aging Ts16 mice is unavailable, however, as Ts16 mouse embryos die late in utero. We studied these embryos looking at the ultrastructure of neurons from the hippocampus and dorsal root ganglion in normal control mice embryos (diploid) and in Ts16 late embryonic litter mates after day 15 of gestation. The organelles in the Ts16 neurons looked similar to those in control neurons, fixed and processed under similar conditions. No obvious neuropathological structures were observed. These results, when compared to reports on electrophysiological abnormalities of cultured fetal Ts16 neurons and on abnormalities in neurotransmitter markers in the Ts16 fetal brain, lead us to suggest that the mental retardation of Down's syndrome is likely to result from functional and chemical defects not directly related to abnormal neuronal ultrastructure. When related to fine structural studies of transplanted embryonic Ts16 hippocampus which have been maintained for long periods of time, these results indicate that the trisomic mouse brain would not be useful as a structural model for Down's syndrome and hence presenile Alzheimer's disease, as it is not associated with any detectable morphological abnormality.     

Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport

Respiratory enzyme complex dysfunction is mechanistically involved in mitochondrial failure leading to neurodegenerative disease, but the pathway is unclear. Here, age-related differences in mitochondrial respiration were measured in both whole and permeabilized neurons from 9-month and 24-month adult rat cortex cultured in common conditions. After permeabilization, respiration increased in both ages of neurons with excess substrates. To dissect specific deficiencies in the respiratory chain, inhibitors for each respiratory chain complex were used to isolate their contributions. Relative to neurons from 9-month rats, in neurons isolated from 24-month rats, complexes I, III, and IV were more sensitive to selective inhibition. Flux control point analysis identified complex I in neurons isolated from 24-month rats as the most sensitive to endogenous substrate availability. The greatest age-related deficit in flux capacity occurred at complex IV with a 29% decrease in neurons isolated from 24-month rats relative to those from 9-month rats. The deficits in complexes I and III may contribute to a redox shift in the quinone pool within the electron transport chain, further extending these age-related deficits. Together these changes could lead to an age-related catastrophic decline in energy production and neuronal death.     

VDAC electronics: 5. Mechanism and computational model of hexokinase-dependent generation of the outer membrane potential in brain mitochondria

Glycolysis plays a key role in brain energy metabolism. The initial and rate-limiting step of brain glycolysis is catalyzed mainly by hexokinase I (HKI), the majority of which is bound to the mitochondrial outer membrane (MOM), mostly through the mitochondrial inter-membrane contact sites formed by the voltage-dependent anion channel (VDAC, outer membrane) and the adenine nucleotide translocator (ANT, inner membrane). Earlier, we proposed a mechanism for the generation of the mitochondrial outer membrane potential (OMP) as a result of partial application of the inner membrane potential (IMP) to MOM through the electrogenic ANT-VDAC-HK inter-membrane contact sites. According to this previous mechanism, the Gibbs free energy of the hexokinase reaction might modulate the generated OMP (Lemeshko, Biophys. J., 2002). In the present work, a new computational model was developed to perform thermodynamic estimations of the proposed mechanism of IMP-HKI-mediated generation of OMP. The calculated OMP was high enough to electrically regulate MOM permeability for negatively charged metabolites through free, unbound VDACs in MOM. On the other hand, the positive-inside polarity of OMP generated by the IMP-HKI-mediated mechanism is expected to protect mitochondria against elevated concentrations of cytosolic Ca²⁺. This computational analysis suggests that metabolically-dependent generation of OMP in the brain mitochondria, controlled by many factors that modulate VDAC1-HKI interaction, VDAC's voltage-gating properties and permeability, might represent one of the physiological mechanisms of regulation of the brain energy metabolism and of neuronal death resistance, and might also be involved in various neurodegenerative disorders, such as Alzheimer's disease.     

Oxidation-reduction and reactive oxygen species homeostasis in mutant plants with respiratory chain complex I dysfunction

Mutations in a mitochondrial or nuclear gene encoding respiratory chain complex I subunits lead to decreased or a total absence of complex I activity. Plant mutants with altered or lost complex I activity adapt their respiratory metabolism by inducing alternative pathways of the respiratory chain and changing energy metabolism. Apparently, complex I is a crucial component of the oxidation-reduction (redox) regulatory system in photosynthetic cells, and alternative NAD(P)H dehydrogenases of the mitochondrial electron transport chain (mtETC) cannot fully compensate for its impairment. In most cases, dysfunction of complex I is associated with lowered or unchanged hydrogen peroxide (H(2)O(2)) concentrations, but increased superoxide (O(2)(-)) levels. Higher production of reactive oxygen species (ROS) by mitochondria in the mosaic (MSC16) cucumber mutant may be related to retrograde signalling. Different effects of complex I dysfunction on H(2)O(2) and O(2)(-) levels in described mutants might result from diverse regulation of processes involved in H(2)O(2) and O(2)(-) production. Often, dysfunction of complex I did not lead to oxidative stress, but increased the capacity of the antioxidative system and enhanced stress tolerance. The new cellular homeostasis in mutants with dysfunction of complex I allows growth and development, reflecting the plasticity of plant metabolism.     

Increased NADPH oxidase-derived superoxide is involved in the neuronal cell death induced by hypoxia-ischemia in neonatal hippocampal slice cultures

Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.     

Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP)

Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q(10) of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD(+)) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.     

Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity

The biochemical changes consequent to respiratory chain inhibition and their relationship to cell death in Leishmania spp. remain elusive. Inhibitors of respiratory chain complexes I, II, and III were able to induce apoptotic death of the bloodstream form of Leishmania donovani. Complex I inhibition resulted in mitochondrial hyperpolarization that was preceded by increased superoxide production. Limitation of electron transport by thenoyltrifluoroacetone and antimycin A, inhibitors of complexes II and III, respectively, resulted in dissipation of mitochondrial membrane potential that was sensitive to cyclosporin A, a blocker of mitochondrial permeability transition pore. Further studies conducted with thenoyltrifluoroacetone showed maximal generation of hydrogen peroxide with a moderate elevation of superoxide levels. Complex III inhibition provoked superoxide generation only. Interference with complex II but not complexes I and III increased intracellular Ca(2+). A tight link between Ca(2+) and reactive oxygen species was demonstrated by antioxidant-induced diminution of the Ca(2+) increase. However, chelation of extracellular Ca(2+) could not abrogate the early increase of reactive oxygen species, providing evidence that Ca(2+) elevation was downstream to reactive oxygen species generation. Ca(2+) influx occurred through nonselective cation and L-type channels and Na(+)/Ca(2+) exchanger-like pathways. Antioxidants such as glutathione and Ca(2+) channel blockers reduced apoptotic death. This study provides a new possibility that concurrent inhibition of respiratory chain complex II with pentamidine administration increases cytotoxicity of the drug. This increased cytotoxicity was connected to a 4-fold elevation in intracellular Ca(2+) that was pooled only from intracellular sources. Therefore, inhibition of complexes I, II, and III leads to apoptosis and complex II inhibition in parallel with pentamidine administration-enhanced drug efficacy.     

NAD attenuates oxidative DNA damages induced by amyloid beta-peptide in primary rat cortical neurons

Abstract

One major pathological hallmark of Alzheimer's disease (AD) is accumulation of senile plaques in patients’ brains, mainly composed of amyloid beta-peptide (Aβ). Nicotinamide adenine dinucleotide (NAD) has emerged as a common mediator regulating energy metabolism, mitochondrial function, aging, and cell death, all of which are critically involved in neuronal demise observed in AD. In this work, we tested the hypothesis that NAD may attenuate Aβ-induced DNA damages, thereby conferring neuronal resistance to primary rat cortical cultures. We found that co-incubation of NAD dose-dependently attenuated neurotoxicity mediated by Aβ25–35 and Aβ1-42 in cultured rat cortical neurons, with the optimal protective dosage at 50 mM. NAD also abolished the formation of reactive oxygen species (ROS) induced by Aβ25-35. Furthermore, Aβs were capable of inducing oxidative DNA damages by increasing the extents of 8-hydroxy-2´-deoxyguanosine (8-OH-dG), numbers of apurinic/apyrimidinic (AP) sites, genomic DNA single-stranded breaks (SSBs), as well as DNA double-stranded breaks (DSBs)/fragmentation, which can all be attenuated upon co-incubation with NAD. Our results thus reveal a novel finding that NAD is protective against DNA damage induced by existing Aβ, leading ultimately to neuroprotection in primary cortical culture.     

The mitochondrial permeability transition pore: A molecular target for amyotrophic lateral sclerosis therapy

Effective therapies are needed for the treatment of amyotrophic lateral sclerosis (ALS), a fatal type of motor neuron disease. Morphological, biochemical, molecular genetic, and cell/animal model studies suggest that mitochondria have potentially diverse roles in neurodegenerative disease mechanisms and neuronal cell death. In human ALS, abnormalities have been found in mitochondrial structure, mitochondrial respiratory chain enzymes, and mitochondrial cell death proteins indicative of some non-classical form of programmed cell death. Mouse models of ALS are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria. This minireview summarizes work on the how malfunctioning mitochondria might contribute to neuronal death in ALS through the biophysical entity called the mitochondrial permeability pore (mPTP). The major protein components of the mPTP are enriched in mouse motor neurons. Early in the course of disease in ALS mice expressing human mutant superoxide dismutase-1, mitochondria in motor neurons undergo trafficking abnormalities and dramatic remodeling resulting in the formation of mega-mitochondria and coinciding with increased protein carbonyl formation and nitration of mPTP components. The genetic deletion of a major mPTP component, cyclophilin D, has robust effects in ALS mice by delaying disease onset and extending survival. Thus, attention should be directed to the mPTP as a rational target for the development of drugs designed to treat ALS.     

Hyperglycemia increases mitochondrial superoxide in retina and retinal cells

Oxidative stress is believed to play a significant role in the development of diabetic retinopathy. In this study, we have investigated the effects of elevated glucose concentration on the production of superoxide anion by retina and retinal cells, the cellular source of the superoxide, the effect of therapies that are known to inhibit diabetic retinopathy on the superoxide production, and the role of the superoxide in cell death in elevated glucose concentration. Superoxide release was measured from retinas collected from streptozotocin-diabetic rats (2 months) treated with or without aminoguanidine, aspirin, or vitamin E, and from transformed retinal Müller cells (rMC-1) and bovine retinal endothelial cells (BREC) incubated in normal (5 mM) and high (25 mM) glucose. Diabetes (retina) or incubation in elevated glucose concentration (rMC-1 and BREC cells) significantly increased superoxide production, primarily from mitochondria, because an inhibitor of mitochondrial electron transport chain complex II normalized superoxide production. Inhibition of reduced nicotinamine adenine dinucleotide phosphate (NADPH) oxidase or nitric oxide synthase had little or no effect on the glucose-induced increase in superoxide. Treatment of diabetic animals with aminoguanidine, aspirin, or vitamin E for 2 months significantly inhibited the diabetes-induced increase in production of superoxide in the retinas. Despite the increased production of superoxide, no increase in protein carbonyls was detected in retinal proteins from animals diabetic for 2-6 months or rMC-1 cells incubated in 25 mM glucose for 5 d unless the activities of calpain or the proteosome were inhibited. Addition of copper/zinc-containing superoxide dismutase to the media of rMC-1 and BREC cells inhibited the apoptotic death caused by elevated glucose. Diabetes-like glucose concentration increases superoxide production in retinal cells, and the superoxide contributes to impaired viability and increased cell death under those circumstances. Three therapies that inhibit the development of diabetic retinopathy all inhibit superoxide production, raising a possibility that these therapies inhibit retinopathy in part by inhibiting a hyperglycemia-induced increase in superoxide production.