ubiquinone and diphenyliodonium

Generation of reactive oxygen species (ROS) causes cellular oxidative damage and has been implicated in the etiology of Alzheimer's disease (AD). In contrast, multiple lines of evidence indicate that ROS can normally modulate long-term potentiation (LTP), a cellular model for memory formation. We recently showed that decreasing the level of superoxide through the overexpression of mitochondrial superoxide dismutase (SOD-2) prevents memory deficits in the Tg2576 mouse model of AD. In the current study, we explored whether AD-related LTP impairments could be prevented when ROS generation from mitochondria was diminished either pharmacologically or via genetic manipulation. In wild-type hippocampal slices treated with exogenous amyloid β peptide (Aβ1-42) and in slices from APP/PS1 mutant mice that model AD, LTP was impaired. The LTP impairments were prevented by MitoQ, a mitochondria-targeted antioxidant, and EUK134, an SOD and catalase mimetic. In contrast, inhibition of NADPH oxidase either by diphenyliodonium (DPI) or by genetically deleting gp91(phox), the key enzymatic component of NADPH oxidase, had no effect on Aβ-induced LTP blockade. Moreover, live staining with MitoSOX Red, a mitochondrial superoxide indicator, combined with confocal microscopy, revealed that Aβ-induced superoxide production could be blunted by MitoQ, but not DPI, in agreement with our electrophysiological findings. Finally, in transgenic mice overexpressing SOD-2, Aβ-induced LTP impairments and superoxide generation were prevented. Our data suggest a causal relationship between mitochondrial ROS imbalance and Aβ-induced impairments in hippocampal synaptic plasticity.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Biphenyl Compounds; Electrophysiological Phenomena; Hippocampus; Humans; In Vitro Techniques; Long-Term Potentiation; Membrane Glycoproteins; Mice; Mice, Transgenic; Mitochondria; NADPH Oxidase 2; NADPH Oxidases; Neuronal Plasticity; Onium Compounds; Organophosphorus Compounds; Oxidants; Reactive Oxygen Species; Superoxide Dismutase; Superoxides; Synapses; Ubiquinone

The rotenone-insensitive NADH dehydrogenase isolated from mitochondria of the procyclic form of Trypanosoma brucei has the ability to produce superoxide anions (Biochemistry 41 (2002) 3065). Superoxide production by the purified enzyme was 60% inhibited by diphenyl iodonium (DPI), stimulated significantly by ubiquinone analogues, and unaffected by metal ions. Production of reactive oxygen species (ROS) in intact cells was not affected by addition of rotenone with proline and malate as substrates; however, addition of rotenone inhibited 41% ROS production with succinate as substrate. These results suggest that complex I is not involved in production of ROS and that succinate-linked reversed electron transport occurs in trypanosome mitochondria. Superoxide formation in mitochondria with NADH as substrate was stimulated by antimycin A but was unaffected by myxothiazol plus stigmatellin, indicating that bc(1) complex is not a source of superoxide. DPI and fumarate inhibited by 68 and 36%, respectively, the rate of superoxide production with NADH as substrate. Addition of both fumarate and DPI blocked 70% superoxide production in mitochondria, a total inhibition similar to that observed with DPI addition alone. These results suggest that the rotenone-insensitive NADH dehydrogenase in addition to NADH fumarate reductase is a potential source of superoxide production in procyclic trypanosome mitochondria.

Topics: Animals; Anti-Bacterial Agents; Antimycin A; Biphenyl Compounds; Fumarates; Malates; Methacrylates; Mitochondria; NAD; NADH Dehydrogenase; Onium Compounds; Polyenes; Proline; Rotenone; Substrate Specificity; Succinic Acid; Superoxides; Thiazoles; Trypanosoma brucei brucei; Ubiquinone; Uncoupling Agents

A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.

Topics: Animals; Biphenyl Compounds; Catalysis; Dimerization; Flavin Mononucleotide; Mitochondria; Molecular Weight; NADH Dehydrogenase; Onium Compounds; Oxygen; Protein Subunits; Rotenone; Superoxides; Trypanosoma brucei brucei; Ubiquinone