Mitochondrial sodium/calcium exchanger (NCLX) regulates basal and starvation-induced autophagy through calcium signaling.

Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporters and efflux by Na+ /Ca2+ exchangers (NCLX). Here, we describe a novel relationship among NCLX, intracellular Ca2+ , and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.

Goldilocks calcium concentrations and the regulation of oxidative phosphorylation: Too much, too little, or just right.

Calcium (Ca2+) is a key regulator in diverse intracellular signaling pathways and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture, and cell death. Moderate Ca2+ within the organelle, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. Here, we aimed to determine in a quantitative manner if extra- or intramitochondrial Ca2+ modulates oxidative phosphorylation in mouse liver mitochondria and intact hepatocyte cell lines. To do so, we monitored the effects of more modest versus supraphysiological increases in cytosolic and mitochondrial Ca2+ on oxygen consumption rates. Isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low (2.4 ± 0.6 µM) and medium (22.0 ± 2.4 µM) Ca2+ concentrations in the presence of complex I-linked substrates pyruvate plus malate and α-ketoglutarate, respectively, but not complex II-linked succinate. In intact cells, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific "just right" concentrations that activate oxidative phosphorylation.

Mitochondrial Ca2+ handling as a cell signaling hub: lessons from astrocyte function.

Astrocytes are a heterogenous population of macroglial cells spread throughout the central nervous system with diverse functions, expression signatures, and intricate morphologies. Their subcellular compartments contain a distinct range of mitochondria, with functional microdomains exhibiting widespread activities, such as controlling local metabolism and Ca2+ signaling. Ca2+ is an ion of utmost importance, both physiologically and pathologically, and participates in critical central nervous system processes, including synaptic plasticity, neuron-astrocyte integration, excitotoxicity, and mitochondrial physiology and metabolism. The mitochondrial Ca2+ handling system is formed by the mitochondrial Ca2+ uniporter complex (MCUc), which mediates Ca2+ influx, and the mitochondrial Na+/Ca2+ exchanger (NCLX), responsible for most mitochondrial Ca2+ efflux, as well as additional components, including the mitochondrial permeability transition pore (mtPTP). Over the last decades, mitochondrial Ca2+ handling has been shown to be key for brain homeostasis, acting centrally in physiopathological processes such as astrogliosis, astrocyte-neuron activity integration, energy metabolism control, and neurodegeneration. In this review, we discuss the current state of knowledge regarding the mitochondrial Ca2+ handling system molecular composition, highlighting its impact on astrocytic homeostasis.

Mitochondrial sodium/calcium exchanger NCLX regulates glycolysis in astrocytes, impacting on cognitive performance.

Intracellular Ca2+ concentrations are strictly controlled by plasma membrane transporters, the endoplasmic reticulum, and mitochondria, in which Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUc), while efflux occurs mainly through the mitochondrial Na+ /Ca2+ exchanger (NCLX). RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared with neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+ -dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.

Mitochondrial morphology regulates organellar Ca2+ uptake and changes cellular Ca2+ homeostasis.

Changes in mitochondrial size and shape have been implicated in several physiologic processes, but their role in mitochondrial Ca2+ uptake regulation and overall cellular Ca2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics toward increased fusion through expression of a dominant negative (DN) form of the fission protein [dynamin-related protein 1 (DRP1)] markedly increased both mitochondrial Ca2+ retention capacity and Ca2+ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion-promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin (MFN)-2 strongly reduced the mitochondrial Ca2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in mitochondrial calcium uniporter expression, inner membrane potentials, or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as indicated by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium-replenishing process of store-operated calcium entry was impaired in MFN2 knockdown cells, whereas DRP1-DN-promoted fusion resulted in faster cytosolic Ca2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca2+ uptake, which impacts cellular Ca2+ homeostasis.-Kowaltowski, A. J., Menezes-Filho, S. L., Assali, E. A., Gonçalves, I. G., Cabral-Costa, J. V., Abreu, P., Miller, N., Nolasco, P., Laurindo, F. R. M., Bruni-Cardoso, A., Shirihai, O. Mitochondrial morphology regulates organellar Ca2+ uptake and changes cellular Ca2+ homeostasis.

The Role of Steroid Hormones in the Modulation of Neuroinflammation by Dietary Interventions.

Steroid hormones, such as sex hormones and glucocorticoids, have been demonstrated to play a role in different cellular processes in the central nervous system, ranging from neurodevelopment to neurodegeneration. Environmental factors, such as calorie intake or fasting frequency, may also impact on such processes, indicating the importance of external factors in the development and preservation of a healthy brain. The hypothalamic-pituitary-adrenal axis and glucocorticoid activity play a role in neurodegenerative processes, including in disorders such as in Alzheimer's and Parkinson's diseases. Sex hormones have also been shown to modulate cognitive functioning. Inflammation is a common feature in neurodegenerative disorders, and sex hormones/glucocorticoids can act to regulate inflammatory processes. Intermittent fasting can protect the brain against cognitive decline that is induced by an inflammatory stimulus. On the other hand, obesity increases susceptibility to inflammation, while metabolic syndromes, such as diabetes, are associated with neurodegeneration. Consequently, given that gonadal and/or adrenal steroids may significantly impact the pathophysiology of neurodegeneration, via their effect on inflammatory processes, this review focuses on how environmental factors, such as calorie intake and intermittent fasting, acting through their modulation of steroid hormones, impact on inflammation that contributes to cognitive and neurodegenerative processes.

Longevity Pathways (mTOR, SIRT, Insulin/IGF-1) as Key Modulatory Targets on Aging and Neurodegeneration.

Recent data from epidemiologic studies have shown that the majority of the public health costs are related to age-related disorders, and most of these diseases can lead to neuronal death. The specific signaling mechanisms underpinning neurodegeneration and aging are incompletely understood. Much work has been directed to the search for the etiology of neurodegeneration and aging and to new therapeutic strategies, including not only drugs but also non-pharmacological approaches, such as physical exercise and low-calorie dietary intake. The most important processes in aging-associated conditions, including neurodegeneration, include the mammalian (or mechanistic target of rapamycin (mTOR, sirtuin (SIRT and insulin/insulin growth factor 1 signaling (IIS pathways. These longevity pathways are involved in an array of different processes, including metabolism, cognition, stress response and brain plasticity. In this review we focus on the current advances involving the mTOR, SIRT and IIS longevity pathways during the course of healthy aging processes and neurodegenerative diseases, bringing new insights in the form of a better understanding of the signaling mechanisms underpinning neurodegeneration and how these differ from physiological normal aging processes. This also provides new targets for the therapeutic management and/or prevention of these devastating age-related disorders.

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K+ channel activity and reactive oxygen species generation.

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H(2)O(2) release using different substrates and ATP-sensitive K(+) transport activities are increased in mitochondria from animals on high fat diets. The increase in H(2)O(2) release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K(+) channels, indicating it was not related to an observed increase in K(+) transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K(+) transport in mitochondria can be modulated by diet.