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Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca²⁺ targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca²⁺-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca²⁺] ([Ca²⁺]c), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca²⁺-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca²⁺]c but promoted mitochondrial fragmentation at high [Ca²⁺]c. These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca²⁺]c-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.

NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles. NAD and NADP play fundamentally different roles in cellular metabolism, and yet these pyridine nucleotides cannot be distinguished spectroscopically in living cells. Blacker et al. demonstrate that fluorescence lifetime imaging can be used to quantify NADPH/NADH balance in cultured cells and in the mammalian cochlea.

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 > 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 > G mutation. Heteroplasmic mtDNA mutations cause disease in humans. Here, Chung et al find the PI3K-Akt-mTORC1 pathway constitutively activated in cells with the heteroplasmic m.3243 A > G mutation, and inhibition of the pathway cell autonomously reduces mutant mtDNA load and rescues mitochondrial bioenergetics.

Mitochondrial Ca 2+ uptake has a key role in cellular Ca 2+ homeostasis. Excessive matrix Ca 2+ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH 2 -linked substrates. The functional consequences of Ca 2+ -induced mPTP opening were assessed by Ca 2+ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca 2+ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca 2+ overload.

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. Synopsis MCU is the highly selective channel responsible for mitochondrial Ca 2+ uptake. MCU expression is shown here to correlate with breast cancer progression. Depletion of MCU reduces tumor growth and metastasis formation via suppression of ROS production and inhibition of HIF‐1α signaling. MCU expression correlates with breast tumor size and lymph node infiltration MCU silencing reduces TNBC cell migration and invasion MCU deletion reduces in vivo tumor growth and metastasis formation MCU silencing inhibits ROS–HIF‐1α signaling route Graphical Abstract MCU is the highly selective channel responsible for mitochondrial Ca 2+ uptake. MCU expression is shown here to correlate with breast cancer progression. Depletion of MCU reduces tumor growth and metastasis formation via suppression of ROS production and inhibition of HIF‐1α signaling.

Heterozygous mutations of the lysosomal enzyme glucocerebrosidase (GBA1) represent the major genetic risk for Parkinson’s disease (PD), while homozygous GBA1 mutations cause Gaucher disease, a lysosomal storage disorder, which may involve severe neurodegeneration. We have previously demonstrated impaired autophagy and proteasomal degradation pathways and mitochondrial dysfunction in neurons from GBA1 knockout (gba1−/−) mice. We now show that stimulation with physiological glutamate concentrations causes pathological [Ca2+]c responses and delayed calcium deregulation, collapse of mitochondrial membrane potential and an irreversible fall in the ATP/ADP ratio. Mitochondrial Ca2+ uptake was reduced in gba1−/− cells as was expression of the mitochondrial calcium uniporter. The rate of free radical generation was increased in gba1−/− neurons. Behavior of gba1+/− neurons was similar to gba1−/− in terms of all variables, consistent with a contribution of these mechanisms to the pathogenesis of PD. These data signpost reduced bioenergetic capacity and [Ca2+]c dysregulation as mechanisms driving neurodegeneration.

Ryanodine receptor 1-related myopathies (RYR1-RM) are caused by RYR1 gene variants and comprise a wide spectrum of histopathological manifestations. Here, we focus on patients carrying RYR1 variants and muscle histopathology consistent with central core disease (CCD) or multi-minicore disease (MmD). RNA-sequencing analyses of skeletal muscle biopsies obtained from both CCD and MmD patients and from healthy controls were performed to better understand the molecular pathways activated by RYR1 variants. Our analyses revealed that, beyond the well-established role of RYR1 in calcium homeostasis, broader cellular pathways are implicated. In CCD, differentially expressed genes were enriched for pathways related to oxidative stress response, SMAD signalling, and apoptosis, consistent with the role of intracellular calcium dysregulation in promoting mitochondrial dysfunction and cell death. In contrast, MmD patients exhibited enrichment of pathways related to immune activation. This was corroborated by the upregulation of GTPase-regulating genes and the down-regulation of transcriptional repressors such as ZFP36 and ATN1. When considering all RYR1-RM patients collectively, Wnt signalling, immune-related pathways, and oxidative phosphorylation emerged as shared enriched pathways, indicating possible convergent mechanisms across histopathological phenotypes. Our study suggests that complex gene regulation driven by RYR1 variants may be a unifying feature in CCD and MmD, offering new insight into potential therapeutic targets.

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial. Vps34 is a lipid kinase conserved from yeast to humans and involved in in intracellular vesicular trafficking and autophagy. Here Bilanges et al. show that inhibition of this kinase in mice improves glucose tolerance and diet-induced steatosis by modulating mitochondrial respiration and metabolism.

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Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca 2+ uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca 2+ -induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca 2+ -induced membrane depolarisation and Ca 2+ retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature.