Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa 3 , were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.

Age-dependent accumulation of partially deleted mitochondrial DNA (DeltamtDNA) has been suggested to contribute to aging and the development of age-associated diseases including Parkinson's disease. However, the molecular mechanisms underlying the generation and accumulation of DeltamtDNA have not been addressed in vivo. In this study, we have developed a mouse model expressing an inducible mitochondria-targeted restriction endonuclease (PstI). Using this system, we could trigger mtDNA double-strand breaks (DSBs) in adult neurons. We found that this transient event leads to the generation of a family of DeltamtDNA with features that closely resemble naturally-occurring mtDNA deletions. The formation of these deleted species is likely to be mediated by yet uncharacterized DNA repairing machineries that participate in homologous recombination and non-homologous end-joining. Furthermore, we obtained in vivo evidence that DeltamtDNAs with larger deletions accumulate faster than those with smaller deletions, implying a replicative advantage of smaller mtDNAs. These findings identify DSB, DNA repair systems and replicative advantage as likely mechanisms underlying the generation and age-associated accumulation of DeltamtDNA in mammalian neurons.

Aging is the most important risk factor for common neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Aging in the central nervous system has been associated with elevated mutation load in mitochondrial DNA, defects in mitochondrial respiration and increased oxidative damage. These observations support a 'vicious cycle' theory which states that there is a feedback mechanism connecting these events in aging and age-associated neurodegeneration. Despite being an extremely attractive hypothesis, the bulk of the evidence supporting the mitochondrial vicious cycle model comes from pharmacological experiments in which the modes of mitochondrial enzyme inhibition are far from those observed in real life. Furthermore, recent in vivo evidence does not support this model. In this review, we focus on the relationship among the components of the putative vicious cycle, with particular emphasis on the role of mitochondrial defects on oxidative stress. Mitochondrial respiratory chain and reactive oxygen species productionMitochondria, being the key players in ATP production and diverse cell signaling events, are essential organelles for the survival of eukaryotic cells. Unlike all other organelles in animals, the mitochondria have their own genome (mitochondrial DNA; mtDNA) that encodes components of the oxidative phosphorylation (OXPHOS) system. The mitochondrial OXPHOS machinery is composed of five multisubunit complexes (complex I-V). From Krebs cycle intermediates (NADH and FADH 2 ), electrons feed into complex I or II, and are transferred to complex III, then to complex IV, and finally to O 2 . The redox energy released during the electron transfer process in complexes I, III and IV is utilized to actively pump out H + from the mitochondrial matrix to the intermembrane space, generating the electrochemical gradient of H + across the inner membrane which is ultimately utilized by complex V to produce ATP [1].This elegant system for energy production, however, is not perfect. A small portion (up to 2%) of electrons passing through the electron transport chain, mostly at complex I and complex III, react with molecular oxygen and yield superoxide anion, which can be converted into other reactive oxygen species (ROS) such as hydrogen peroxide and the highly reactive hydroxyl radical through enzymatic and nonenzymatic reactions [2]. Cells are endowed with robust endogenous antioxidant systems to counteract excessive ROS. It is believed that ROS, in particular hydrogen peroxide, have physiological roles as signaling molecules [3,4]. However,

Defects in the mitochondrial cytochrome c oxidase (COX) have been associated with Alzheimer's Disease, in which the agedependent accumulation of ␤-amyloid plays an important role in synaptic dysfunction and neurodegeneration. To test the possibility that age-dependent decline in the mitochondrial respiratory function, especially COX activity, may participate in the formation and accumulation of ␤-amyloid, we generated mice expressing mutant amyloid precursor protein and mutant presenilin 1 in a neuron-specific COX-deficient background. A neuron-specific COXdeficient mouse was generated by the Cre-loxP system, in which the COX10 gene was deleted by a CamKII␣ promoter-driven Crerecombinase. COX10 is a farnesyltransferase involved in the biosynthesis of heme a, required for COX assembly and function. These KO mice showed an age-dependent COX deficiency in the cerebral cortex and hippocampus. Surprisingly, COX10 KO mice exhibited significantly fewer amyloid plaques in their brains compared with the COX-competent transgenic mice. This reduction in amyloid plaques in the KO mouse was accompanied by a reduction in A␤42 level, ␤-secretase activity, and oxidative damage. Likewise, production of reactive oxygen species from cells with partial COX activity was not elevated. Collectively, our results suggest that, contrary to previous models, a defect in neuronal COX does not increase oxidative damage nor predispose for the formation of amyloidgenic amyloid precursor protein fragments. mitochondria ͉ oxidative phosphorylation ͉ neurodegeneration A lzheimer's disease (AD) is the most prevalent age-related neurodegenerative disease, characterized by progressive brain atrophy/neuronal death that results in cognitive and memory impairment. Starting from the identification of genes responsible for familial AD, which is now known to constitute only Ͻ5% of total AD incidence, an ''amyloid hypothesis'' has been built up during the past few decades, and an enormous effort has been devoted to understand the toxic mechanism of ␤-amyloid (A␤), which forms extracellular amyloid plaques and is generated by a successive proteolytic cleavage of amyloid precursor protein (APP) by ␤-secretase and a ␥-secretase complex containing presenilins (1). Although it is not clear to what extent different A␤ species (soluble oligomers, insoluble aggregates, extracellular species, or intracellular species) contribute to neurodegeneration in vivo, recent studies demonstrated the toxicity of diverse A␤ species in vitro, in situ, and in vivo, confirming the importance of age-dependent A␤ accumulation in AD pathogenesis (2-5).Recently, age-dependent accumulation of mutations in mitochondrial DNA (mtDNA) and resulting increase in oxidative stress and impairment in mitochondrial respiratory chain, especially complex IV or cytochrome c oxidase (COX), gained attention as potential factors that could participate in the onset of sporadic AD (6). A number of studies reported a reduction in COX activity and an increase in oxidative stress in brain tissues and plate...

Neuronal oxidative phosphorylation (OXPHOS) deficiency has been associated with a variety of neurodegenerative diseases, including Parkinson’s disease and Huntington’s disease. However, it is not clear how mitochondrial dysfunction alone can lead to a preferential elimination of certain neuronal populations in vivo. We compared different types of neuronal populations undergoing the same OXPHOS deficiency to determine their relative susceptibility and mechanisms responsible for selective neuron vulnerability. We used a mouse model expressing a mitochondria-targeted restriction enzyme, PstI or mito-PstI. The expression of mito-PstI induces double-strand breaks in the mitochondrial DNA (mtDNA), leading to OXPHOS-deficiency, mostly due to mtDNA depletion. We targeted mito-PstI expression to the cortex, hippocampus, and striatum under the CamKII-α promoter. Animals undergoing long-term expression of mito-PstI displayed a selective worsening of the striatum over cortical and hippocampal areas. Mito-PstI expression and mtDNA depletion, were not worse in the striatum, but yet the latter showed the most severe defects in mitochondrial membrane potential, response to calcium, and survival. These results showed that the striatum is particularly sensitive to defects in OXPHOS possibly due to an increased reliance on OXPHOS function in this area and differences in response to physiological stimuli. These results may help explain the neuropathological features associated with Huntington’s disease, which have been associated with OXPHOS defects.