Amyloid precursor protein drives down-regulation of mitochondrial oxidative phosphorylation independent of amyloid beta

Amyloid precursor protein (APP) and its extracellular domain, soluble APP alpha (sAPPα) play important physiological and neuroprotective roles. However, rare forms of familial Alzheimer’s disease are associated with mutations in APP that increase toxic amyloidogenic cleavage of APP and produce amyloid beta (Aβ) at the expense of sAPPα and other non-amyloidogenic fragments. Although mitochondrial dysfunction has become an established hallmark of neurotoxicity, the link between Aβ and mitochondrial function is unclear. In this study we investigated the effects of increased levels of neuronal APP or Aβ on mitochondrial metabolism and gene expression, in human SH-SY5Y neuroblastoma cells. Increased non-amyloidogenic processing of APP, but not Aβ, profoundly decreased respiration and enhanced glycolysis, while mitochondrial DNA (mtDNA) transcripts were decreased, without detrimental effects to cell growth. These effects cannot be ascribed to Aβ toxicity, since higher levels of endogenous Aβ in our models do not cause oxidative phosphorylation (OXPHOS) perturbations. Similarly, chemical inhibition of β-secretase decreased mitochondrial respiration, suggesting that non-amyloidogenic processing of APP may be responsible for mitochondrial changes. Our results have two important implications, the need for caution in the interpretation of mitochondrial perturbations in models where APP is overexpressed, and a potential role of sAPPα or other non-amyloid APP fragments as acute modulators of mitochondrial metabolism

Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver

<p>Abstract</p> <p>Background</p> <p>The unbound, free concentration (B_f) of unconjugated bilirubin (UCB), and not the total UCB level, has been shown to correlate with bilirubin cytotoxicity, but the key molecular mechanisms accounting for the toxic effects of UCB are largely unknown.</p> <p>Findings</p> <p>Mouse liver mitochondria increase unbound UCB oxidation, consequently increasing the apparent rate constant for unbound UCB oxidation by HRP (Kp), higher than in control and mouse brain mitochondria, emphasizing the importance of determining Kp in complete systems containing the organelles being studied. The <it>in vitro </it>effects of UCB on cytochrome <it>c </it>oxidase activity in mitochondria isolated from mouse brain and liver were studied at B_franging from 22 to 150 nM. The results show that UCB at B_fup to 60 nM did not alter mitochondrial cytochrome <it>c </it>oxidase activity, while the higher concentrations significantly inhibited the enzyme activity by 20% in both liver and brain mitochondria.</p> <p>Conclusions</p> <p>We conclude that it is essential to include the organelles being studied in the medium used in measuring both Kp and B_f. A moderately elevated, pathophysiologically-relevant B_fimpaired the cytochrome <it>c </it>oxidase activity modestly in mitochondria from mouse brain and liver.</p

Metabolic Control Analysis in a Cellular Model of Elevated MAO-B: Relevance to Parkinson’s Disease

We previously demonstrated that spare respiratory capacity of the TCA cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) was completely abolished upon increasing levels of MAO-B activity in a dopaminergic cell model system (Kumar et al., J Biol Chem 278:46432–46439, 2003). MAO-B mediated increases in H2O2 also appeared to result in direct oxidative inhibition of both mitochondrial complex I and aconitase. In order to elucidate the contribution that each of these components exerts over metabolic respiratory control as well as the impact of MAO-B elevation on their spare respiratory capacities, we performed metabolic respiratory control analysis. In addition to KGDH, we assessed the activities and substrate-mediated respiration of complex I, pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), and mitochondrial aconitase in the absence and presence of complex-specific inhibitors in specific and mixed substrate conditions in mitochondria from our MAO-B elevated cells versus controls. Data from this study indicates that Complex I and KGDH are the most sensitive to inhibition by MAO-B mediated H2O2 generation, and could be instrumental in determining the fate of mitochondrial metabolism in this cellular PD model system

Heterozygous Mutation of Drosophila Opa1 Causes the Development of Multiple Organ Abnormalities in an Age-Dependent and Organ-Specific Manner

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner

Ancient mtDNA Genetic Variants Modulate mtDNA Transcription and Replication

Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are “evolutionary silent hitchhikers”. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment

Inducing cancer indolence by targeting mitochondrial Complex I is potentiated by blocking macrophage-mediated adaptive responses

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancerstrategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Herewe use genetic ablation of this enzyme to induce indolence in two cancer types, andshow this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha(HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inabilityto respond to hypoxia, concordantly with the persistence of human oncocytomas. Wedemonstrate that CI-deficient tumors survive and carry out angiogenesis, despite theirinability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated mac-rophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneouspharmacological inhibition of CI function through metformin and macrophage infiltrationthrough PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basisfor an efficient combinatorial adjuvant therapy in clinical trials

Heterozygous Mutation of Opa1 in Drosophila Shortens Lifespan Mediated through Increased Reactive Oxygen Species Production

Optic atrophy 1 (OPA1) is a dynamin-like GTPase located in the inner mitochondrial membrane and mutations in OPA1 are associated with autosomal dominant optic atrophy (DOA). OPA1 plays important roles in mitochondrial fusion, cristae remodeling and apoptosis. Our previous study showed that dOpa1 mutation caused elevated reactive oxygen species (ROS) production and resulted in damage and death of the cone and pigment cells in Drosophila eyes. Since ROS-induced oxidative damage to the cells is one of the primary causes of aging, in this study, we examined the effects of heterozygous dOpa1 mutation on the lifespan. We found that heterozygous dOpa1 mutation caused shortened lifespan, increased susceptibility to oxidative stress and elevated production of ROS in the whole Drosophila. Antioxidant treatment partially restored lifespan in the male dOpa1 mutants, but had no effects in the females. Heterozygous dOpa1 mutation caused an impairment of respiratory chain complex activities, especially complexes II and III, and reversible decreased aconitase activity. Heterozygous dOpa1 mutation is also associated with irregular and dysmorphic mitochondria in the muscle. Our results, for the first time, demonstrate the important role of OPA1 in aging and lifespan, which is most likely mediated through augmented ROS production