Rewiring cell signalling pathways in pathogenic mtDNA mutations

Mitochondria generate the energy to sustain cell viability and serve as a hub for cell signalling. Their own genome (mtDNA) encodes genes critical for oxidative phosphorylation. Mutations of mtDNA cause major disease and disability with a wide range of presentations and severity. We review here an emerging body of data suggesting that changes in cell metabolism and signalling pathways in response to the presence of mtDNA mutations play a key role in shaping disease presentation and progression. Understanding the impact of mtDNA mutations on cellular energy homeostasis and signalling pathways seems fundamental to identify novel therapeutic interventions with the potential to improve the prognosis for patients with primary mitochondrial disease

Simultaneous measurement of mitochondrial calcium and mitochondrial membrane potential in live cells by fluorescent microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca(2+)) buffers to tightly regulate intracellular Ca(2+) concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester Ca(2+). The influx of Ca(2+) into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨm, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. We describe here a method for simultaneously measuring mitochondria Ca(2+) uptake and ΔΨm in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca(2+) can be measured using the fluorescent Ca(2+) indicator Fluo-4, AM, with measurement of ΔΨm using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca(2+) and ΔΨm simultaneously. Using the sequential addition of Ca(2+) aliquots, mitochondrial Ca(2+) uptake can be monitored, and the concentration at which Ca(2+) induces mitochondrial membrane permeability transition and the loss of ΔΨm determined

MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology

While the analysis of mitochondrial morphology has emerged as a key tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a challenging task and bottleneck for statistically robust conclusions. Here, we present Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet in a toolbox for Windows and Linux operating systems that combines segmentation with morphological analysis

Selective mitochondrial antioxidant MitoTEMPO reduces renal dysfunction and systemic inflammation in experimental sepsis in rats

BACKGROUND: Excess mitochondrial reactive oxygen species (mROS) in sepsis is associated with organ failure, in part by generating inflammation through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. We determined the impact of a mitochondrial-targeted antioxidant (MitoTEMPO) on mitochondrial dysfunction in renal proximal tubular epithelial cells, peritoneal immune cell function ex vivo, and organ dysfunction in a rat model of sepsis. METHODS: The effects of MitoTEMPO were assessed ex vivo using adenosine triphosphate and lipopolysaccharide-stimulated rat peritoneal immune cells and fresh rat kidney slices exposed to serum from septic rats. We assessed mROS production and phagocytotic capacity (flow cytometry), mitochondrial functionality (multiphoton imaging, respirometry), and NLRP3 inflammasome activation in cell culture. The effect of MitoTEMPO on organ dysfunction was evaluated in a rat model of faecal peritonitis. RESULTS: MitoTEMPO decreased septic serum-induced mROS (P0.05). In vivo, compared with untreated septic animals, MitoTEMPO reduced systemic IL-1β (P=0.01), reduced renal oxidative stress as determined by urine isoprostane levels (P=0.04), and ameliorated renal dysfunction (reduced serum urea (P<0.001) and creatinine (P=0.05). CONCLUSIONS: Reduction of mROS by a mitochondria-targeted antioxidant reduced IL-1β, and protected mitochondrial, cellular, and organ functionality after septic insults

The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1α

Triple-negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca(2+) uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca(2+) uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU-silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia-inducible factor-1α (HIF-1α) is reduced, suggesting a signaling role for mROS and HIF-1α, downstream of mitochondrial Ca(2+) Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF-1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca(2+) uptake is a potential novel therapeutic target for clinical intervention

The spectrum of neurodevelopmental, neuromuscular and neurodegenerative disorders due to defective autophagy

Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. Additionally, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases.Peer reviewe

Imaging Mitochondrial Calcium Fluxes with Fluorescent Probes and Single- or Two-Photon Confocal Microscopy

The concentration of calcium ions in the mitochondria has been shown to affect its function, modulating respiratory activity at low levels and causing lethal damage at high concentrations. The rhodamine series of dyes can be used to measure mitochondrial calcium concentration, but the reliability of measurements depends upon correct partitioning of the dye within to the mitochondria. Methods are described to aid verification and quantification of the mitochondrial calcium concentration using single- or two-photon confocal microscopy. The method of linear unmixing to separate fluorescent signals based on either differing excitation or emission spectra is outlined and for the purposes of illustration is applied to the separation of rhod-2 signals originating from the dye within the mitochondria and nucleoli

Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation

Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy – tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A &gt; G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A &gt; G mutation

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

From the cell membrane to the nucleus: unearthing transport mechanisms for Dynein

Mutations in the motor protein cytoplasmic dynein have been found to cause Charcot-Marie-Tooth disease, spinal muscular atrophy, and severe intellectual disabilities in humans. In mouse models, neurodegeneration is observed. We sought to develop a novel model which could incorporate the effects of mutations on distance travelled and velocity. A mechanical model for the dynein mediated transport of endosomes is derived from first principles and solved numerically. The effects of variations in model parameter values are analysed to find those that have a significant impact on velocity and distance travelled. The model successfully describes the processivity of dynein and matches qualitatively the velocity profiles observed in experiments