Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD ؉ /NADH redox couple on H 2 O 2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate؉malate alone do generate only small amounts of H 2 O 2 (0.04 ؎ 0.02 nmol H 2 O 2 /min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 ؎ 0.25 nmol H 2 O 2 / min/mg) or in the presence of the respiratory substrate succinate alone (0.80 ؎ 0.27 nmol H 2 O 2 /min/mg). The maximal rate of H 2 O 2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 ؎ 0.07 nmol H 2 O 2 /min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chaininhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to ؊295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H 2 O 2 induced increased H 2 O 2 production at this site we propose that reactive oxygen species can activate a selfaccelerating vicious cycle causing mitochondrial damage and neuronal cell death.Superoxide anion, a product of one-electron reduction of oxygen, is the by-product of normal functioning of the mitochondrial respiratory chain (1). It has been reported, that this radical is generated by complexes I and III of the mitochondrial respiratory chain and readily converted to H 2 O 2 by mitochondrial Mn-superoxide dismutase (2-6). There is substantial evidence that superoxide and H 2 O 2 contribute to the pathogenesis of certain neurodegenerative diseases (7,8). However, there is considerable disagreement in recent literature concerning generated amounts and sites of superoxide production by isolated mitochondria. In contrast to the well documented production of superoxide at center "o" of antimycin A-inhibited complex III (2, 9, 10), the exact site and the total contribution of reactive oxygen species (ROS) 1 generation in complex I has not been established so far. Although certain investigators (11, 12) suggested low potential iron-sulfur clusters as potential sites, others (6) proposed flavine mononucleotide (FMN) to be the producer of superoxide being responsible for the H 2 O 2 generation by brain mitochondria. In addition, there are substantial controversies regarding the exact amounts of ROS production at the different sites of respirato...

BackgroundParkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD.Methodology/Principal FindingsNow we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of α-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons.ConclusionThus, aging Pink1−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5′-3′exonuclease MGME1, elimination of the 3′-5′exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.

No abstract

Mitochondrial function is a key determinant of both excitability and viability of neurons. Here, we demonstrate seizure-dependent changes in mitochondrial oxidative phosphorylation in the epileptic rat hippocampus. The intense pathological neuronal activity in pilocarpine-treated rats exhibiting spontaneous seizures resulted in a selective decline of the activities of NADH-CoQ oxidoreductase (complex I of the respiratory chain) and cytochrome c oxidase (complex IV of respiratory chain) in the CA3 and CA1 hippocampal pyramidal subfields. In line with these findings, high-resolution respirometry revealed an increased flux control of complex I on respiration in the CA1 and CA3 subfields and decreased maximal respiration rates in the more severely affected CA3 subfield. Imaging of mitochondrial membrane potential using rhodamine 123 showed a lowered mitochondrial membrane potential in both pyramidal subfields. In contrast to the CA1 and CA3 subfields, mitochondrial oxidative phosphorylation was unaltered in the dentate gyrus and the parahippocampal gyrus. The changes of oxidative phosphorylation in the epileptic rat hippocampus cannot be attributed to oxidative enzyme modifications but are very likely related to a decrease in mitochondrial DNA copy number as shown in the more severely affected CA3 subfield and in cultured PC12 cells partially depleted of mitochondrial DNA. Thus, our results demonstrate that seizure activity downregulates the expression of mitochondrial-encoded enzymes of oxidative phosphorylation. This mechanism could be invoked during diverse forms of pathological neuronal activity and could severely affect both excitability and viability of hippocampal pyramidal neurons.