Martyn A. Sharpe

β-Amyloid inhibits integrated mitochondrial respiration and key enzyme activities

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), α‐ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post‐mortem Alzheimers disease brain. β‐Amyloid is strongly implicated in Alzheimers pathology and can be formed intracellularly in neurones. We have investigated the possibility that β‐amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the β‐amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimers brain. Mitochondrial respiration, electron transport chain complex activities, α‐ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. β‐Amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, α‐ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by β‐amyloid. The Km of cytochrome oxidase for reduced cytochrome c was raised by β‐amyloid. We conclude that β‐amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimers disease.

β-Amyloid fragment 25-35 causes mitochondrial dysfunction in primary cortical neurons

β-Amyloid deposition and compromised energy metabolism both occur in vulnerable brain regions in Alzheimers disease. It is not known whether β-amyloid is the cause of impairment of energy metabolism, nor whether impaired energy metabolism is specific to neurons. Our results, using primary neuronal cultures, show that 24-h incubation with Aβ25–35 caused a generalized decrease in the specific activity of mitochondrial enzymes per milligram of cellular protein, induced mitochondrial swelling, and decreased total mitochondrial number. Incubation with Aβ25–35 decreased ATP concentration to 58% of control in neurons and 71% of control in astrocytes. Levels of reduced glutathione were also lowered by Aβ25–35 in both neurons (from 5.1 to 2.9 nmol/mg protein) and astrocytes (from 25.2 to 14.9 nmol/mg protein). We conclude that 24-h treatment with extracellular Aβ25–35 causes mitochondrial dysfunction in both astrocytes and neurons, the latter being more seriously affected. In astrocytes mitochondrial impairment was confined to complex I inhibition, whereas in neurons a generalized loss of mitochondria was seen.

Astrocyte-Derived Nitric Oxide Causes Both Reversible and Irreversible Damage to the Neuronal Mitochondrial Respiratory Chain

Cytokine‐stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO‐), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO‐ and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO‐ causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24‐h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte‐derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO‐producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO‐producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48‐h coculture with NO‐producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short‐term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic‐derived NO for longer periods causes permanent damage to neuronal complex IV.

Interaction of peroxynitrite with mitochondrial cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of enzyme activity

Purified mitochondrial cytochrome coxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear hemea 3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a 3nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreasedV max and a raised K m for oxygen. Under these conditions, the redox state of cytochromea is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 μm peroxynitrite to 10 μm fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochromea 3, partial reduction of cytochromea, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 μm) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a anda 3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase.

Cytochrome c oxidase rapidly metabolises nitric oxide to nitrite

Previous studies have shown that the addition of nitric oxide to cytochrome c oxidase rapidly generates spectral changes compatible with the formation of nitrite at the binuclear haem:copper centre. Here we directly demonstrate nitrite release following nitric oxide addition to the enzyme. The nitrite complex is kinetically inactive and the off rate for nitrite was found to be slow (0.024 min−1). However, the presence of reductants enhances the off rate and enables cytochrome oxidase to catalyse the rapid oxidation of nitric oxide to nitrite free in solution. This may play a major role in the mitochondrial metabolism of nitric oxide.

Nitric oxide ejects electrons from the binuclear centre of cytochrome c oxidase by reacting with oxidised copper: a general mechanism for the interaction of copper proteins with nitric oxide?

Small increases in NO concentration can inhibit mitochondrial oxygen consumption by reacting at the binuclear haem a 3/CuB oxygen reduction site of cytochrome c oxidase. Here we demonstrate that under normal turnover conditions NO reacts initially with the oxidised CuB rather than the haem a 3. We propose that hydration of an initial Cu+/NO+ complex forms nitrite, a proton and CuB +; the latter ejects an electron from the binuclear centre and results in the observed (100 s−1) reduction of other electron transfer centres in the enzyme (haem a and CuA). These reactions may have implications for the interactions of NO with other copper proteins.

Fibrillar beta-amyloid peptide Aβ1–40 activates microglial proliferation via stimulating TNF-α release and H2O2 derived from NADPH oxidase: a cell culture study

BackgroundAlzheimers disease is characterized by the accumulation of neuritic plaques, containing activated microglia and β-amyloid peptides (Aβ). Fibrillar Aβ can activate microglia, resulting in production of toxic and inflammatory mediators like hydrogen peroxide, nitric oxide, and cytokines. We have recently found that microglial proliferation is regulated by hydrogen peroxide derived from NADPH oxidase. Thus, in this study, we investigated whether Aβ can stimulate microglial proliferation and cytokine production via activation of NADPH oxidase to produce hydrogen peroxide.MethodsPrimary mixed glial cultures were prepared from the cerebral cortices of 7-day-old Wistar rats. At confluency, microglial cells were isolated by tapping, replated, and treated either with or without Aβ. Hydrogen peroxide production by cells was measured with Amplex Red and peroxidase. Microglial proliferation was assessed under a microscope 0, 24 and 48 hours after plating. TNF-α and IL-1β levels in the culture medium were assessed by ELISA.ResultsWe found that 1 μM fibrillar (but not soluble) Aβ1–40 peptide induced microglial proliferation and caused release of hydrogen peroxide, TNF-α and IL-1β from microglial cells. Proliferation was prevented by the NADPH oxidase inhibitor apocynin (10 μM), by the hydrogen peroxide-degrading enzyme catalase (60 U/ml), and by its mimetics EUK-8 and EUK-134 (20 μM); as well as by an antibody against TNF-α and by a soluble TNF receptor inhibitor. Production of TNF-α and IL-1β, measured after 24 hours of Aβ treatment, was also prevented by apocynin, catalase and EUKs, but the early release (measured after 1 hour of Aβ treatment) of TNF-α was insensitive to apocynin or catalase.ConclusionThese results indicate that Aβ1–40-induced microglial proliferation is mediated both by microglial release of TNF-α and production of hydrogen peroxide from NADPH oxidase. This suggests that TNF-α and NADPH oxidase, and its products, are potential targets to prevent Aβ-induced inflammatory neurodegeneration.

Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage.

Haemolytic events, such as those following rhabdomyolysis and subarachnoid haemorrhage, often result in pathological complications such as vasoconstriction. Haem-protein cross-linked myoglobin and haemoglobin are generated by ferric-ferryl redox cycling, and thus can be used as markers of oxidative stress. We have found haem-protein cross-linked myoglobin in the urine of patients suffering from rhabdomyolysis and haem-protein cross-linked haemoglobin in the cerebrospinal fluid of patients following subarachnoid haemorrhage. These findings provide strong evidence that these respiratory haem proteins can be involved in powerful oxidation processes in vivo. We have previously proposed that these oxidation processes in rhabdomyolysis include the formation of potent vasoconstrictor molecules, generated by the myoglobin-catalysed oxidation of membranes, inducing nephrotoxicity and renal failure. Haem-protein cross-linked haemoglobin in cerebrospinal fluid suggests that a similar mechanism of lipid oxidation is present and that this may provide a mechanistic basis for the delayed vasospasm that follows subarachnoid haemorrhage.

A chemically explicit model for the mechanism of proton pumping in heme–copper oxidases

A mechanism for proton pumping is described that is based on chemiosmotic principles and the detailed molecular structures now available for cytochrome oxidases. The importance of conserved water positions and a step-wise gated process of proton translocation is emphasized, where discrete electron transfer events are coupled to proton uptake and expulsion. The trajectory of each pumped proton is the same for all four substrate electrons. An essential role for the His-Tyr cross-linked species is discussed, in gating of the D- and K-channels and as an acceptor/donor of electrons and protons at the binuclear center.

Superoxide dismutase mimetics with catalase activity reduce the organ injury in hemorrhagic shock.

Reactive oxygen species (ROS) contribute to the multiple organ failure (MOF) in hemorrhagic shock. Here we investigate the effects of two superoxide dismutase (SOD) mimetics with catalase activity (EUK-8 and EUK-134) on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock in the anesthetised rat. Hemorrhage (sufficient to lower mean arterial blood pressure to 45 mmHg for 90 min) and subsequent resuscitation with shed blood resulted (within 4 h after resuscitation) in a delayed fall in blood pressure, liver injury and renal dysfunction as well as pancreatic injury. Treatment of rats on resuscitation with EUK-8 (3 mg/kg i.v. bolus followed by 3 mg/kg/h i.v. infusion) significantly attenuated liver injury, renal dysfunction and pancreatic injury caused by hemorrhage and resuscitation. Administration of EUK-134 (3 mg/kg i.v. bolus followed by 3 mg/kg/h) reduced the liver injury and renal dysfunction (but not the pancreatic injury) caused by hemorrhagic shock. However, neither EUK-8 nor EUK-134 reduced the delayed circulatory failure associated with hemorrhagic shock. Thus, we propose that an enhanced formation of ROS contributes to the MOF in hemorrhagic shock, and that membrane-permeable SOD-mimetics with catalase activity, such as EUK-8 or EUK-134, may represent a novel therapeutic approach for the therapy of hemorrhagic shock.