Liver X receptor activation mitigates oxysterol-induced dysfunction in fetoplacental endothelial cells.

Maintaining the homeostasis of the placental vasculature is of paramount importance for ensuring normal fetal growth and development. Any disruption in this balance can lead to perinatal morbidity. Several studies have uncovered an association between high levels of oxidized cholesterol (oxysterols), and complications during pregnancy, including gestational diabetes mellitus (GDM) and preeclampsia (PE). These complications often coincide with disturbances in placental vascular function. Here, we investigate the role of two oxysterols (7-ketocholesterol, 7ß-hydroxycholesterol) in (dys)function of primary fetoplacental endothelial cells (fpEC). Our findings reveal that oxysterols exert a disruptive influence on fpEC function by elevating the production of reactive oxygen species (ROS) and interfering with mitochondrial transmembrane potential, leading to its depolarization. Moreover, oxysterol-treated fpEC exhibited alterations in intracellular calcium (Ca2+) levels, resulting in the reorganization of cell junctions and a corresponding increase in membrane stiffness and vascular permeability. Additionally, we observed an enhanced adhesion of THP-1 monocytes to fpEC following oxysterol treatment. We explored the influence of activating the Liver X Receptor (LXR) with the synthetic agonist T0901317 (TO) on oxysterol-induced endothelial dysfunction in fpEC. Our results demonstrate that LXR activation effectively reversed oxysterol-induced ROS generation, monocyte adhesion, and cell junction permeability in fpEC. Although the effects on mitochondrial depolarization and calcium mobilization did not reach statistical significance, a strong trend towards stabilization of calcium mobilization was evident in LXR-activated cells. Taken together, our results suggest that high levels of systemic oxysterols link to placental vascular dysfunction and LXR agonists may alleviate their impact on fetoplacental vasculature.

Synergy of uncoupling proteins (1 and 2) with mitochondrial Ca2+ uptake machinery potentiate mitochondrial uncoupling.

Mitochondrial uncoupling proteins UCP1 and UCP2 have a structural homology of app. 60%. They execute their mitochondria uncoupling function through different molecular mechanisms. Non-shivering thermogenesis by UCP1 is mediated through a transmembrane dissipation of the proton motive force to create heat during sympathetic stimulation. UCP2, on the other hand, modulates through the interaction with methylated MICU1 the permeability of the cristae junction, which acts as an isolator for the cristae-located mitochondrial membrane potential. In this mini-review, we discuss and compare the recently described molecular mechanism of UCP1 in brown adipose tissue and UCP2 in aged and cancer non-excitable cells that contribute to mitochondrial uncoupling, and the synergistic effects of both UCPs with the mitochondrial Ca2+ uptake machinery.

MFN2 mediates ER-mitochondrial coupling during ER stress through specialized stable contact sites.

Endoplasmic reticulum (ER) functions critically depend on a suitable ATP supply to fuel ER chaperons and protein trafficking. A disruption of the ability of the ER to traffic and fold proteins leads to ER stress and the unfolded protein response (UPR). Using structured illumination super-resolution microscopy, we revealed increased stability and lifetime of mitochondrial associated ER membranes (MAM) during ER stress. The consequent increase of basal mitochondrial Ca2+ leads to increased TCA cycle activity and enhanced mitochondrial membrane potential, OXPHOS, and ATP generation during ER stress. Subsequently, OXPHOS derived ATP trafficking towards the ER was increased. We found that the increased lifetime and stability of MAMs during ER stress depended on the mitochondrial fusion protein Mitofusin2 (MFN2). Knockdown of MFN2 blunted mitochondrial Ca2+ effect during ER stress, switched mitochondrial F1FO-ATPase activity into reverse mode, and strongly reduced the ATP supply for the ER during ER stress. These findings suggest a critical role of MFN2-dependent MAM stability and lifetime during ER stress to compensate UPR by strengthening ER ATP supply by the mitochondria.

MICU1 controls spatial membrane potential gradients and guides Ca2+ fluxes within mitochondrial substructures.

Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca2+ control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.

Sigma-1 Receptor Modulation by Ligands Coordinates Cancer Cell Energy Metabolism.

Sigma-1 receptor (S1R) is an important endoplasmic reticulum chaperone with various functions in health and disease. The purpose of the current work was to elucidate the involvement of S1R in cancer energy metabolism under its basal, activated, and inactivated states. For this, two cancer cell lines that differentially express S1R were treated with S1R agonist, (+)-SKF10047, and antagonist, BD1047. The effects of the agonist and antagonist on cancer energy metabolism were studied using single-cell fluorescence microscopy analysis of real-time ion and metabolite fluxes. Our experiments revealed that S1R activation by agonist increases mitochondrial bioenergetics of cancer cells while decreasing their reliance on aerobic glycolysis. S1R antagonist did not have a major impact on mitochondrial bioenergetics of tested cell lines but increased aerobic glycolysis of S1R expressing cancer cell line. Our findings suggest that S1R plays an important role in cancer energy metabolism and that S1R ligands can serve as tools to modulate it.

Lysophosphatidic Acid Receptor 5 (LPA5) Knockout Ameliorates the Neuroinflammatory Response In Vivo and Modifies the Inflammatory and Metabolic Landscape of Primary Microglia In Vitro.

Systemic inflammation induces alterations in the finely tuned micromilieu of the brain that is continuously monitored by microglia. In the CNS, these changes include increased synthesis of the bioactive lipid lysophosphatidic acid (LPA), a ligand for the six members of the LPA receptor family (LPA1-6). In mouse and human microglia, LPA5 belongs to a set of receptors that cooperatively detect danger signals in the brain. Engagement of LPA5 by LPA polarizes microglia toward a pro-inflammatory phenotype. Therefore, we studied the consequences of global LPA5 knockout (-/-) on neuroinflammatory parameters in a mouse endotoxemia model and in primary microglia exposed to LPA in vitro. A single endotoxin injection (5 mg/kg body weight) resulted in lower circulating concentrations of TNFα and IL-1ß and significantly reduced gene expression of IL-6 and CXCL2 in the brain of LPS-injected LPA5-/- mice. LPA5 deficiency improved sickness behavior and energy deficits produced by low-dose (1.4 mg LPS/kg body weight) chronic LPS treatment. LPA5-/- microglia secreted lower concentrations of pro-inflammatory cyto-/chemokines in response to LPA and showed higher maximal mitochondrial respiration under basal and LPA-activated conditions, further accompanied by lower lactate release, decreased NADPH and GSH synthesis, and inhibited NO production. Collectively, our data suggest that LPA5 promotes neuroinflammation by transmiting pro-inflammatory signals during endotoxemia through microglial activation induced by LPA.

Citrin mediated metabolic rewiring in response to altered basal subcellular Ca2+ homeostasis.

In contrast to long-term metabolic reprogramming, metabolic rewiring represents an instant and reversible cellular adaptation to physiological or pathological stress. Ca2+ signals of distinct spatio-temporal patterns control a plethora of signaling processes and can determine basal cellular metabolic setting, however, Ca2+ signals that define metabolic rewiring have not been conclusively identified and characterized. Here, we reveal the existence of a basal Ca2+ flux originating from extracellular space and delivered to mitochondria by Ca2+ leakage from inositol triphosphate receptors in mitochondria-associated membranes. This Ca2+ flux primes mitochondrial metabolism by maintaining glycolysis and keeping mitochondria energized for ATP production. We identified citrin, a well-defined Ca2+-binding component of malate-aspartate shuttle in the mitochondrial intermembrane space, as predominant target of this basal Ca2+ regulation. Our data emphasize that any manipulation of this ubiquitous Ca2+ system has the potency to initiate metabolic rewiring as an instant and reversible cellular adaptation to physiological or pathological stress.

Near-UV Light Induced ROS Production Initiates Spatial Ca2+ Spiking to Fire NFATc3 Translocation.

Ca2+-dependent gene regulation controls several functions to determine the fate of the cells. Proteins of the nuclear factor of activated T-cells (NFAT) family are Ca2+ sensitive transcription factors that control the cell growth, proliferation and insulin secretion in ß-cells. Translocation of NFAT proteins to the nucleus occurs in a sequence of events that starts with activating calmodulin-dependent phosphatase calcineurin in a Ca2+-dependent manner, which dephosphorylates the NFAT proteins and leads to their translocation to the nucleus. Here, we examined the role of IP3-generating agonists and near-UV light in the induction of NFATc3 migration to the nucleus in the pancreatic ß-cell line INS-1. Our results show that IP3 generation yields cytosolic Ca2+ rise and NFATc3 translocation. Moreover, near-UV light exposure generates reactive oxygen species (ROS), resulting in cytosolic Ca2+ spiking via the L-type Ca2+ channel and triggers NFATc3 translocation to the nucleus. Using the mitochondria as a Ca2+ buffering tool, we showed that ROS-induced cytosolic Ca2+ spiking, not the ROS themselves, was the triggering mechanism of nuclear import of NFATc3. Collectively, this study reveals the mechanism of near-UV light induced NFATc3 migration.

Sigma-1 Receptor Promotes Mitochondrial Bioenergetics by Orchestrating ER Ca2+ Leak during Early ER Stress.

The endoplasmic reticulum (ER) is a complex, multifunctional organelle of eukaryotic cells and responsible for the trafficking and processing of nearly 30% of all human proteins. Any disturbance to these processes can cause ER stress, which initiates an adaptive mechanism called unfolded protein response (UPR) to restore ER functions and homeostasis. Mitochondrial ATP production is necessary to meet the high energy demand of the UPR, while the molecular mechanisms of ER to mitochondria crosstalk under such stress conditions remain mainly enigmatic. Thus, better understanding the regulation of mitochondrial bioenergetics during ER stress is essential to combat many pathologies involving ER stress, the UPR, and mitochondria. This article investigates the role of Sigma-1 Receptor (S1R), an ER chaperone, has in enhancing mitochondrial bioenergetics during early ER stress using human neuroblastoma cell lines. Our results show that inducing ER stress with tunicamycin, a known ER stressor, greatly enhances mitochondrial bioenergetics in a time- and S1R-dependent manner. This is achieved by enhanced ER Ca2+ leak directed towards mitochondria by S1R during the early phase of ER stress. Our data point to the importance of S1R in promoting mitochondrial bioenergetics and maintaining balanced H2O2 metabolism during early ER stress.

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.

The contribution of uncoupling protein 2 to mitochondrial Ca2+ homeostasis in health and disease - A short revisit.

Considering the versatile functions attributed to uncoupling protein 2 (UCP2) in health and disease, a profound understanding of the protein's molecular actions under physiological and pathophysiological conditions is indispensable. This review aims to revisit and shed light on the fundamental molecular functions of UCP2 in mitochondria, with particular emphasis on its intricate role in regulating mitochondrial calcium (Ca2+) uptake. UCP2's modulating effect on various vital processes in mitochondria makes it a crucial regulator of mitochondrial homeostasis in health and disease.

Tracking intra- and inter-organelle signaling of mitochondria.

Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca2+ and fundamentally contributing to various signaling pathways. Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine-tuned by intra- and interorganelle H+ , K+ , Na+ , and Ca2+ signaling. In this review, we provide an up-to-date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting-edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.

Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli.

BACKGROUND: DNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2'-deoxyguanosine-5'-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. METHODS: Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. RESULTS: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6-4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. DISCUSSION: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.

Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases.