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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.

The unique features of the mitochondrial genome, such as its high copy number and lack of defined mechanisms of recombination, have hampered efforts to manipulate its sequence to create specific mutations in mouse mtDNA. As such, the generation of in vivo mouse models of mtDNA disease has proved technically challenging. This chapter describes a unique approach to create mitochondrial oxidative phosphorylation (OXPHOS) defects in mouse ES cells by transferring mtDNA from different murid species into Mus musculus domesticus ES cells using cytoplasmic hybrid ("cybrid") fusion. The resulting "xenocybrid" ES cells carry OXPHOS defects of varying severity, and can be utilized to generate live mouse models of mtDNA disease.

Mitochondrial DNA (mtDNA) is replicated throughout life in postmitotic cells, resulting in higher levels of somatic mutation than in nuclear genes. However, controversy remains as to the importance of low-level mtDNA somatic mutants in cancerous and normal human tissues. To capture somatic mtDNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippocampus and cortex brain biopsies. We analyzed the whole mtDNA genome from 120 cybrid clones derived from four individual donors by chemical cleavage of mismatch and Sanger sequencing, scanning around two million base pairs. Seventeen different somatic point mutations were identified, including eight coding region mutations, four of which result in frameshifts. Examination of one cybrid clone with a novel m.2949_2953delCTATT mutation in MT-RNR2 (which encodes mitochondrial 16S rRNA) revealed a severe disruption of mtDNA-encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT-ND1, previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mtDNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mtDNA mutants in vitro to study effects on mitochondrial respiratory chain function., (© 2014 WILEY PERIODICALS, INC.)

Mitochondrial oxidative phosphorylation (OXPHOS) dysfunction is implicated in a growing spectrum of diseases, from neurodegeneration to cancer. Where tissues or transformed cells are available, respirometry and enzymology allow a sophisticated analysis of OXPHOS with modest-cost equipment. The isolation of organelle fractions is also invaluable for determining association of proteins of interest. Here we revisit and consolidate methods to measure whole cell mitochondrial ATP synthesis, respiration, isolation of mitochondria from cultured cells and tissues, and OXPHOS enzymology. We also explain common pitfalls, guide optimisation of the methods for new users, and provide full laboratory protocols in Supplementary materials., (Copyright © 2014 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Mitochondria play an integral role in cell death signaling, yet how mitochondrial defects disrupt this important function is not well understood. We have used a mouse L-cell fibroblast model harboring Rattus norvegicus mtDNA (Rn xenocybrids) to examine the effects of multiple oxidative phosphorylation (OXPHOS) defects on reactive oxygen species (ROS) generation and cell death signaling. Blue native-PAGE analyses of Rn xenocybrids revealed defects in OXPHOS complex biogenesis with reduced steady-state levels of complexes I, III and IV. Isolated Rn xenocybrid mitochondria exhibited deficiencies in complex II+III and III activities, with CIII-stimulated ROS generation 66% higher than in control mitochondria. Rn xenocybrid cells were resistant to staurosporine-induced cell death, but exhibited a four-fold increase in sensitivity to ceramide-induced cell death that was caspase-3 independent and did not induce chromosomal DNA degradation. Furthermore, ceramide directly inhibited Rn xenocybrid complex II+III activity by 97%, although this inhibition could be completely abolished by exogenous decylubiquinone. Ceramide also induced a further increase in ROS output from Rn xenocybrid complex III by 42%. These results suggest that the interaction of ceramide with OXPHOS complex III is significantly enhanced by the presence of the xenotypic Rattus cytochrome b in complex III, likely due to the increased affinity for ceramide at the ubiquinone binding site. We propose a novel mechanism of altered mitochondrial cell death signaling due to mtDNA mutations whereby ceramide directly induces OXPHOS complex ROS generation to initiate cell death pathways., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies.

Oxidative phosphorylation (OXPHOS) drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homoeostasis. In diabetic kidney disease (DKD), mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics have not been previously documented. In the present study, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks), a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells (PTECs) of diabetic kidneys were clearly apparent, but no changes in urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide (H2O2) generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary kidney injury molecule-1 (KIM-1) excretion, where an increase in the Complex I-linked oxygen consumption rate (OCR), in the context of a decrease in kidney ATP, indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of DKD. [ABSTRACT FROM AUTHOR]

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease. [ABSTRACT FROM AUTHOR]

ABSTRACT Mitochondrial DNA (mt DNA) is replicated throughout life in postmitotic cells, resulting in higher levels of somatic mutation than in nuclear genes. However, controversy remains as to the importance of low-level mt DNA somatic mutants in cancerous and normal human tissues. To capture somatic mt DNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippocampus and cortex brain biopsies. We analyzed the whole mt DNA genome from 120 cybrid clones derived from four individual donors by chemical cleavage of mismatch and Sanger sequencing, scanning around two million base pairs. Seventeen different somatic point mutations were identified, including eight coding region mutations, four of which result in frameshifts. Examination of one cybrid clone with a novel m.2949_2953del CTATT mutation in MT- RNR2 (which encodes mitochondrial 16 S r RNA) revealed a severe disruption of mt DNA-encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT- ND1, previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mt DNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mt DNA mutants in vitro to study effects on mitochondrial respiratory chain function. [ABSTRACT FROM AUTHOR]

Abstract: Background: Diabetes is a metabolic syndrome that results in chronically increased blood glucose (hyperglycaemia) due to defects either in insulin secretion consequent to the loss of beta cells in the pancreas (type 1) or to loss of insulin sensitivity in target organs in the presence of normal insulin secretion (type 2). Long term hyperglycaemia can lead to a number of serious health-threatening pathologies, or complications, especially in the kidney, heart, retina and peripheral nervous system. Scope of review: Here we summarise the current literature on the role of the mitochondria in complications associated with diabetes, and the limitations and potential of rodent models to explore new modalities to limit complication severity. Major conclusions: Prolonged hyperglycaemia results in perturbation of catabolic pathways and in an over-production of ROS by the mitochondria, which in turn may play a role in the development of diabetic complications. Furthermore, current models don't offer a comprehensive recapitulation of these complications. General significance: The onset of complications associated with type 1 diabetes can be varied, even with tightly controlled blood glucose levels. The potential role of inherited, mild mitochondrial dysfunction in accelerating diabetic complications, both in type 1 and 2 diabetes, remains unexplored. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research. [Copyright &y& Elsevier]

Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers. [ABSTRACT FROM AUTHOR]

Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies. [ABSTRACT FROM AUTHOR]

Energy in the form of ATP is continually produced by all cells for normal growth and function. Anaerobic glycolysis can provide enough ATP for some cells, but energetic cells such as cardiomyocytes and neurons require a more efficient ATP supply, which can only be provided by mitochondrial oxidative phosphorylation. Invented by bacteria that became symbiotically associated with other bacteria to form eukaryotic cells billions of years ago, oxidative phosphorylation carries with it a genetic legacy that is unique. The mitochondrial oxidative phosphorylation complexes are assembled from protein subunits encoded by both the mitochondrial genome (mtDNA) and the nuclear genome (nDNA, located in the chromosomes). The mtDNA is a remnant genome of the bacterial progenitor of mitochondria, and (unlike the biparental diploidy that characterizes the nuclear genome) is present in thousands of copies per cell, is replicated through life, and is inherited (cytoplasmically) only from the female parent. Oxidative phosphorylation comprises five multimeric enzyme complexes that act as a redox pathway, passing electrons from oxidizable intermediates produced by the metabolism of food to molecular oxygen in the mitochondrial mat-rix, while producing an electrochemical gradient by pumping protons into the intermembranal space. The proton (hydrogen ion) gradient across the inner mitochondrial membrane is used by the H+- transporting ATP synthase to produce ATP from ADP and inorganic phosphate, with the protons released into the mitochondrial matrix then combining with electronated oxygen to form water. Many of the details regarding the control of the synthesis of oxidative phosphorylation enzyme complexes remain to be elucidated. Transmitochondrial cell culture systems have been developed so that defective oxidative phosphorylation can be studied in a controlled nuclear background. Such systems may soon enable the development of mtDNA ‘knockout’ mice in order to better model mtDNA transmission and mitochondrial disease. [ABSTRACT FROM PUBLISHER]

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease. [ABSTRACT FROM AUTHOR]

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) must be assembled precisely from 45 protein subunits for it to function correctly. One of its mitochondrial DNA (mtDNA) encoded subunits, ND1, is incorporated during the early stages of complex I assembly. However, little is known about how mutations in ND1 affect this assembly process. We found that in human 143B cybrid cells carrying a homoplasmic MT-ND1 mutation, ND1 protein could not be translated. As a result, the early stages of complex I assembly were disrupted, with mature complex I undetectable and complex I-linked respiration severely reduced to 2.0% of control levels. Interestingly, complex IV (ferrocytochrome c:oxygen oxidoreductase) steady-state levels were also reduced to 40.3%, possibly due to its diminished stability in the absence of respiratory supercomplex formation. This was in comparison with 143B cybrid controls (that contained wild-type mtDNA on the same nuclear background), which exhibited normal complex I, complex IV, and supercomplex assembly. We conclude that the loss of ND1 stalls complex I assembly during the early stages of its biogenesis, which not only results in the loss of mature complex I but also disrupts the stability of complex IV and the respiratory supercomplex to cause mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Abstract: The similarities between glaucoma and mitochondrial optic neuropathies have driven a growing interest in exploring mitochondrial function in glaucoma. The specific loss of retinal ganglion cells is a common feature of mitochondrial diseases – not only the classic mitochondrial optic neuropathies of Leber’s Hereditary Optic Neuropathy and Autosomal Dominant Optic Atrophy – but also occurring together with more severe central nervous system involvement in many other syndromic mitochondrial diseases. The retinal ganglion cell, due to peculiar structural and energetic constraints, appears acutely susceptible to mitochondrial dysfunction. Mitochondrial function is also well known to decline with aging in post-mitotic tissues including neurons. Because age is a risk factor for glaucoma this adds another impetus to investigating mitochondria in this common and heterogeneous neurodegenerative disease. Mitochondrial function may be impaired by either nuclear gene or mitochondrial DNA genetic risk factors, by mechanical stress or chronic hypoperfusion consequent to the commonly raised intraocular pressure in glaucomatous eyes, or by toxic xenobiotic or even light-induced oxidative stress. If primary or secondary mitochondrial dysfunction is further established as contributing to glaucoma pathogenesis, emerging therapies aimed at optimizing mitochondrial function represent potentially exciting new clinical treatments that may slow retinal ganglion cell and vision loss in glaucoma. [Copyright &y& Elsevier]