Mitochondria couple cellular metabolic state with Ca(2+) transport processes. They therefore control not only their own intra-organelle Ca(2+), but they also influence the entire cellular network of ...cellular Ca(2+) signaling, including the endoplasmic reticulum, the plasma membrane, and the nucleus. Through the detailed study of mitochondrial roles in Ca(2+) signaling, a remarkable picture of inter-organelle communication has emerged. We here review the ways in which this system provides integrity and flexibility for the cell to cope with the countless demands throughout its life cycle and discuss briefly the mechanisms through which it can also drive cell death.
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.
Mitochondrial Ca
uptake has a key role in cellular Ca
homeostasis. Excessive matrix Ca
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
-linked substrates. The functional consequences of Ca
-induced mPTP opening were assessed by Ca
retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca
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
overload.
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.
Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular ...mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium (Ca
2+
c) is a potent inducer of macroautophagy. Various Ca
2+ mobilizing agents (vitamin D
3 compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca
2+/calmodulin-dependent kinase kinase-β and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the Ca
2+
ER and attenuates agonist-induced Ca
2+ fluxes. Thus, an increase in the Ca
2+
c serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.
Aging has been linked to several degenerative processes that, through the accumulation of molecular and cellular damage, can progressively lead to cell dysfunction and organ failure. Human aging is ...linked with a higher risk for individuals to develop cancer, neurodegenerative, cardiovascular, and metabolic disorders. The understanding of the molecular basis of aging and associated diseases has been one major challenge of scientific research over the last decades. Mitochondria, the center of oxidative metabolism and principal site of reactive oxygen species (ROS) production, are crucial both in health and in pathogenesis of many diseases. Redox signaling is important for the modulation of cell functions and several studies indicate a dual role for ROS in cell physiology. In fact, high concentrations of ROS are pathogenic and can cause severe damage to cell and organelle membranes, DNA, and proteins. On the other hand, moderate amounts of ROS are essential for the maintenance of several biological processes, including gene expression. In this review, we provide an update regarding the key roles of ROS-mitochondria cross talk in different fundamental physiological or pathological situations accompanying aging and highlighting that mitochondrial ROS may be a decisive target in clinical practice.
Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain degree of ER damage, this response triggers ...apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized. However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that, during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca²⁺ uptake. Accordingly, uncoupling of the organelles or blocking Ca²⁺ transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca²⁺ transfer, which, by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.
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 Ca
responses and delayed calcium deregulation, collapse of mitochondrial membrane potential and an irreversible fall in the ATP/ADP ratio. Mitochondrial Ca
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 Ca
dysregulation as mechanisms driving neurodegeneration.
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