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Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 μM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 μM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca 2+ -induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme gene (GBE1) that lead to the accumulation of aberrant glycogen in affected tissues, mostly in the liver. To determine whether dysfunctional glycogen metabolism in GSD IV affects other components of cellular bioenergetics, we studied mitochondrial function in heterozygous Gbe1 knockout (Gbe1 +/- ) mice. Mitochondria isolated from the livers of Gbe1 +/- mice showed elevated respiratory complex I activity and increased reactive oxygen species production, particularly by respiratory chain complex III. These observations indicate that GBE1 deficiency leads to broader rearrangements in energy metabolism and that the mechanisms underlying GSD IV pathogenesis may include more than merely mechanical cell damage caused by the presence of glycogen aggregates., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Glycogen branching enzyme (GBE1) introduces branching points in the glycogen molecule during its synthesis. Pathogenic GBE1 gene mutations lead to glycogen storage disease type IV (GSD IV), which is characterized by excessive intracellular accumulation of abnormal, poorly branched glycogen in affected tissues and organs, mostly in the liver. Using heterozygous Gbe1 knock-out mice (Gbe1 +/- ), we analyzed the effects of moderate GBE1 deficiency on oxidative stress in the liver. The livers of aged Gbe1 +/- mice (22 months old) had decreased GBE1 protein levels, which caused a mild decrease in the degree of glycogen branching, but did not affect the tissue glycogen content. GBE1 deficiency was accompanied by increased protein carbonylation and elevated oxidation of the glutathione pool, indicating the existence of oxidative stress. Furthermore, we have observed increased levels of glutathione peroxidase and decreased activity of respiratory complex I in Gbe1 +/- livers. Our data indicate that even mild changes in the degree of glycogen branching, which did not lead to excessive glycogen accumulation, may have broader effects on cellular bioenergetics and redox homeostasis. In young animals cellular homeostatic mechanisms are able to counteract those changes, while in aged tissues the changes may lead to increased oxidative stress., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mitochondria are considered the main source of reactive oxygen species (ROS) in the cell. For this reason they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O 2 . Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix, as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, converts superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H 2 O.In the chapter we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depend on the metabolic state of this organelle.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The mitochondrial respiratory chain, and in particular, complex I, is a major source of reactive oxygen species (ROS) in cells. Elevated levels of ROS are associated with an imbalance between the rate of ROS formation and the capacity of the antioxidant defense system. Increased ROS production may lead to oxidation of DNA, lipids and proteins and thus can affect fundamental cellular processes. The aim of this study was to investigate the magnitude of intracellular oxidative stress in fibroblasts of patients with Leigh syndrome with defined mutations in complex I. Moreover, we hypothesized that activation of the p66Shc protein (phosphorylation of p66Shc at Ser36 by PKCβ), being part of the oxidative stress response pathway, is partially responsible for the increased ROS production in cells with dysfunctional complex I. Characterization of bioenergetic parameters and ROS production showed that the cellular model of Leigh syndrome is described by increased intracellular oxidative stress and oxidative damage to DNA and proteins, which correlate with increased p66Shc phosphorylation at Ser36. Treatment of patients' fibroblasts with hispidin (an inhibitor of the protein kinase PKCβ), in addition to decreasing ROS production and intracellular oxidative stress, resulted in restoration of complex I activity., (Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

This work, based on in vivo and in vitro measurements, as well as in silico simulations, provides a consistent analysis of diffusion of polydisperse nanoparticles in the cytoplasm of living cells. Using the example of fluorescence correlation spectroscopy (FCS), we show the effect of polydispersity of probes on the experimental results. Although individual probes undergo normal diffusion, in the ensemble of probes, an effective broadening of the distribution of diffusion times occurs-similar to anomalous diffusion. We introduced fluorescently labeled dextrans into the cytoplasm of HeLa cells and found that cytoplasmic hydrodynamic drag, exponentially dependent on probe size, extraordinarily broadens the distribution of diffusion times across the focal volume. As a result, the in vivo FCS data were effectively fitted with the anomalous subdiffusion model while for a monodisperse probe the normal diffusion model was most suitable. Diffusion time obtained from the anomalous diffusion model corresponds to a probe whose size is determined by the weight-average molecular weight of the polymer. The apparent anomaly exponent decreases with increasing polydispersity of the probes. Our results and methodology can be applied in intracellular studies of the mobility of nanoparticles, polymers, or oligomerizing proteins.

Significance: In all cells, the endoplasmic reticulum (ER) and mitochondria are physically connected to form junctions termed mitochondria-associated membranes (MAMs). This subcellular compartment is under intense investigation because it represents a "hot spot" for the intracellular signaling of important pathways, including the synthesis of cholesterol and phospholipids, calcium homeostasis, and reactive oxygen species (ROS) generation and activity., Recent Advances: The advanced methods currently used to study this fascinating intracellular microdomain in detail have enabled the identification of the molecular composition of MAMs and their involvement within different physiopathological contexts., Critical Issues: Here, we review the knowledge regarding (i) MAMs composition in terms of protein composition, (ii) the relationship between MAMs and ROS, (iii) the involvement of MAMs in cell death programs with particular emphasis within the tumor context, (iv) the emerging role of MAMs during inflammation, and (v) the key role of MAMs alterations in selected neurological disorders., Future Directions: Whether alterations in MAMs represent a response to the disease pathogenesis or directly contribute to the disease has not yet been unequivocally established. In any case, the signaling at the MAMs represents a promising pharmacological target for several important human diseases.

The adaptor protein p66Shc links membrane receptors to intracellular signalling pathways and has the potential to respond to energy status changes and regulate mitogenic signalling. Initially reported to mediate growth signals in normal and cancer cells, p66Shc has also been recognized as a pro-apoptotic protein involved in the cellular response to oxidative stress. Moreover, it is a key element in processes such as cancer cell proliferation, tumor progression, metastasis and metabolic reprogramming. Recent findings on the role of p66Shc in the above-mentioned processes have been obtained through the use of various tumor cell types, including prostate, breast, ovarian, lung, colon, skin and thyroid cancer cells. Interestingly, the impact of p66Shc on the proliferation rate was mainly observed in prostate tumors, while its impact on metastasis was mainly found in breast cancers. In this review, we summarize the current knowledge about the possible roles of p66Shc in different cancers., (© 2015 Stichting European Society for Clinical Investigation Journal Foundation.)

The different mechanisms employed by proto-oncogenes and tumor suppressors to regulate cell death pathways are strictly linked to their localization. In addition to the canonical control of apoptosis at a transcriptional/nuclear level, intracellular zones are emerging as pivotal sites for the activities of several proapoptotic and antiapoptotic factors. Here, we review the function of the endoplasmic reticulum-mitochondria interface as a primary platform for decoding danger signals as well as a structural accommodation for several regulator or effector proteins.

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. Thus, the intracellular accumulation of ROS may not only disrupt the functions of specific tissues and organs but also lead to the premature death of the entire organism. Less severe increases in ROS levels may lead to the nonlethal oxidation of fundamental cellular components, such as proteins, phospholipids, and DNA, hence exerting a mutagenic effect that promotes oncogenesis and tumor progression. Here, we describe the use of chemical probes for the rapid detection of ROS in intact and permeabilized adherent cells by fluorescence microscopy and fluorometry. Moreover, after discussing the limitations described in the literature for the fluorescent probes presented herein, we recommend methods to assess the production of specific ROS in various fields of investigation, including the study of oncometabolism., (© 2014 Elsevier Inc. All rights reserved.)

p66Shc is an adaptor protein involved in cell proliferation and differentiation that undergoes phosphorylation at Ser36 in response to oxidative stimuli, consequently inducing a burst of reactive oxygen species (ROS), mitochondrial disruption and apoptosis. Its role during several pathologies suggests that p66Shc mitochondrial signalling can perpetuate a primary mitochondrial defect, thus contributing to the pathophysiology of that condition. Here, we show that in the fibroblasts of neuropathy, ataxia and retinitis pigmentosa (NARP) patients, the p66Shc phosphorylation pathway is significantly induced in response to intracellular oxidative stress related to disrupted ATP synthase activity and mitochondrial membrane hyperpolarisation. We postulate that the increased phosphorylation of p66Shc at Ser36 is partially responsible for further increasing ROS production, resulting in oxidative damage of proteins. Oxidative stress and p66Shc phosphorylation at Ser36 may be mitigated by antioxidant administration or the use of a p66Shc phosphorylation inhibitor. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Diabetes mellitus is a chronic disease caused by a deficiency in the production of insulin and/or by the effects of insulin resistance. Insulin deficiency leads to hyperglycemia which is the major initiator of diabetic cardiovascular complications escalating with time and driven by many complex biochemical and molecular processes. Four hypotheses, which propose mechanisms of diabetes-associated pathophysiology, are currently considered. Cardiovascular impairment may be caused by an increase in polyol pathway flux, by intracellular advanced glycation end-products formation or increased flux through the hexosamine pathway. The latter of these mechanisms involves activation of the protein kinase C. Cellular and mitochondrial metabolism alterations observed in the course of diabetes are partially associated with an excessive production of reactive oxygen species (ROS). Among many processes and factors involved in ROS production, the 66 kDa isoform of the growth factor adaptor shc (p66Shc protein) is of particular interest. This protein plays a key role in the control of mitochondria-dependent oxidative balance thus it involvement in diabetic complications and other oxidative stress based pathologies is recently intensively studied. In this review we summarize the current understanding of hyperglycemia induced cardiac mitochondrial dysfunction with an emphasis on the oxidative stress and p66Shc protein. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mitochondria are considered as the main source of reactive oxygen species (ROS) in the cell. For this reason, they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids, and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O(2). Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, convert superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H(2)O. In this chapter, we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depends on the metabolic state of this organelle.