Karl Morten

Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7−/− erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119+/CD71High stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.

Variation in mitochondrial DNA levels in muscle from normal controls. Is depletion of mtDNA in patients with mitochondrial myopathy a distinct clinical syndrome

SummaryRecent studies have identified a group of patients with cytochrome oxidase (COX) deficiency presenting in infancy associated with a deficiency of mtDNA in muscle or other affected tissue (Moraes et al 1991). We used a novel approach to compare the level of mitochondrial (mtDNA) compared to nuclear DNA in skeletal muscle from a group of patients and controls, based on dot blots that were hybridized with a mtDNA probe labelled with35S[dCTP] and a reference nuclear DNA probe labelled with [32P]dCTP.The ratio of mtDNA to nuclear DNA varied in samples from different muscles of the same individual. Secondly, fetal muscle had very low levels of mtDNA compared to nuclear DNA, and data from older controls (cross-sectional rather than sequential) suggest that this increases rapidly over the first 3 months after birth and thereafter more slowly. Four patients with COX deficiency had levels of mtDNA that were below the age-specific range defined by ‘normal’ quadriceps muscle. The clinical features of two of these patients were similar to earlier case reports of mtDNA depletion. In three patients the clinical course was relatively benign compared to cases that have previously been described.Levels of mtDNA in skeletal muscle from some patients with other forms of muscle disease were also found to be low, suggesting that mtDNA depletion, possibly related to depletion of mitochondria, may be a relatively non-specific response of muscle to various pathological processes. However, there does appear to be a distinctive group of young patients with reduced cytochrome oxidase activity in muscle, in whom marked mtDNA depletion reflects the primary defect.

The Warburg effect: 80 years on

Influential research by Warburg and Cori in the 1920s ignited interest in how cancer cells energy generation is different from that of normal cells. They observed high glucose consumption and large amounts of lactate excretion from cancer cells compared with normal cells, which oxidised glucose using mitochondria. It was therefore assumed that cancer cells were generating energy using glycolysis rather than mitochondrial oxidative phosphorylation, and that the mitochondria were dysfunctional. Advances in research techniques since then have shown the mitochondria in cancer cells to be functional across a range of tumour types. However, different tumour populations have different bioenergetic alterations in order to meet their high energy requirement; the Warburg effect is not consistent across all cancer types. This review will discuss the metabolic reprogramming of cancer, possible explanations for the high glucose consumption in cancer cells observed by Warburg, and suggest key experimental practices we should consider when studying the metabolism of cancer.

Critical role of complex III in the early metabolic changes following myocardial infarction

AIMSnThe chronically infarcted rat heart has multiple defects in metabolism, yet the location of the primary metabolic abnormality arising after myocardial infarction is unknown. Therefore, we investigated cardiac mitochondrial metabolism shortly after infarction.nnnMETHODS AND RESULTSnMyocardial infarctions (n = 11) and sham operations (n = 9) were performed on Wistar rats, at 2 weeks cardiac function was assessed using echocardiography, and rats were grouped into failing (ejection fraction < or =45%), moderately impaired (46-60%), and sham-operated (>60%). Respiration rates were decreased by 28% in both subsarcolemmal and interfibrillar mitochondria isolated from failing hearts, compared with sham-operated controls. However, respiration rates were not impaired in mitochondria from hearts with moderately impaired function. The mitochondrial defect in the failing hearts was located within the electron transport chain (ETC), as respiration rates were suppressed to the same extent when fatty acids, ketone bodies, or glutamate were used as substrates. Complex III protein levels were decreased by 46% and complex III activity was decreased by 26%, in mitochondria from failing hearts, but all other ETC complexes were unchanged. Decreased complex III activity was accompanied by a three-fold increase in complex III-derived H(2)O(2) production, decreased cardiolipin content, and a 60% decrease in mitochondrial cytochrome c levels from failing hearts. Respiration rates, complex III activity, cardiolipin content, and reactive oxygen species generation rates correlated with ejection fraction.nnnCONCLUSIONnIn conclusion, a specific defect in complex III occurred acutely after myocardial infarction, which increased oxidative damage and impaired mitochondrial respiration. The extent of mitochondrial dysfunction in the failing heart was proportional to the degree of cardiac dysfunction induced by myocardial infarction.

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: Implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA

Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A→ G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (G→A) mtDNA mutation of Lebers hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (ρ0) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.

Differential regulation of HIF-mediated pathways increases mitochondrial metabolism and ATP production in hypoxic osteoclasts

Inappropriate osteoclast activity instigates pathological bone loss in rheumatoid arthritis. We have investigated how osteoclasts generate sufficient ATP for the energy‐intensive process of bone resorption in the hypoxic microenvironment associated with this rheumatic condition. We show that in human osteoclasts differentiated from CD14+ monocytes, hypoxia (24 h, 2% O2): (a) increases ATP production and mitochondrial electron transport chain activity (Alamar blue, O2 consumption); (b) increases glycolytic flux (glucose consumption, lactate production); and (c) increases glutamine consumption. We demonstrate that glucose, rather than glutamine, is necessary for the hypoxic increase in ATP production and also for cell survival in hypoxia. Using siRNA targeting specific isoforms of the hypoxia‐inducible transcription factor HIF (HIF‐1α, HIF‐2α), we show that employment of selected components of the HIF‐1α‐mediated metabolic switch to anaerobic respiration enables osteoclasts to rapidly increase ATP production in hypoxia, while at the same time compromising long‐term survival. We propose this atypical HIF‐driven metabolic pathway to be an adaptive mechanism to permit rapid bone resorption in the short term while ensuring curtailment of the process in the absence of re‐oxygenation. Copyright

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity.

Oxygen-sensing scaffolds for 3-dimensional cell and tissue culture

Porous membrane scaffolds are widely used materials for three-dimensional cell cultures and tissue models. Additional functional modification of such scaffolds can significantly extend their use and operational performance. Here we describe hybrid microporous polystyrene-based scaffolds impregnated with a phosphorescent O2-sensitive dye PtTFPP, optimized for live cell fluorescence microscopy and imaging of O2 distribution in cultured cells. Modified scaffolds possess high brightness, convenient spectral characteristics (534 nm excitation, 650 nm emission), stable and robust response to pO2 in phosphorescence intensity and lifetime imaging modes (>twofold response over 21/0% O2), such as confocal PLIM. They are suitable for prolonged use under standard culturing conditions without affecting cell viability, and for multi-parametric imaging analysis of cultured cells and tissue samples. We tested the O2 scaffolds with cultured cancer cells (HCT116), multicellular aggregates (PC12) and rat brain slices and showed that they can inform on tissue oxygenation at different depths and cell densities, changes in respiration activity, viability and responses to drug treatment. Using this method multiplexed with staining of dead cells (CellTox Green) and active mitochondria (TMRM), we demonstrated that decreased O2 (20-24 μM) in scaffold corresponds to highest expression of tyrosine hydroxylase in PC12 cells. Such hypoxia is also beneficial for action of hypoxia-specific anti-cancer drug tirapazamine (TPZ). Thus, O2 scaffolds allow for better control of conditions in 3D tissue cultures, and are useful for a broad range of biomaterials and physiological studies.

Mitophagy plays a central role in mitochondrial ageing

The mechanisms underlying ageing have been discussed for decades, and advances in molecular and cell biology of the last three decades have accelerated research in this area. Over this period, it has become clear that mitochondrial function, which plays a major role in many cellular pathways from ATP production to nuclear gene expression and epigenetics alterations, declines with age. The emerging concepts suggest novel mechanisms, involving mtDNA quality, mitochondrial dynamics or mitochondrial quality control. In this review, we discuss the impact of mitochondria in the ageing process, the role of mitochondria in reactive oxygen species production, in nuclear gene expression, the accumulation of mtDNA damage and the importance of mitochondrial dynamics and recycling. Declining mitophagy (mitochondrial quality control) may be an important component of human ageing.

A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA

Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism (energetic stress). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses.