Increase In Mitochondrial Ca2 Research Articles

Nutrient-Sensitising Drug Repurposing Screen Identifies Lomerizine As a Mitochondrial Metabolism Inhibitor in Chronic Myeloid Leukaemia

In chronic myeloid leukaemia (CML), the persistence of leukaemic stem cells (LSCs) following first line treatment with tyrosine kinase inhibitors (TKIs) such as imatinib, can lead to disease relapse. We have previously demonstrated that therapy-resistant CML LSCs rely on oxidative phosphorylation (OXPHOS) for survival and that targeting mitochondrial metabolism sensitises CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy, or are associated with adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. To identify clinically applicable compounds that target cells dependent on mitochondrial metabolism, we used a nutrient-sensitised repurposing screening strategy. Upon replacement of glucose with galactose as the only sugar source in cell culture media, there is a shift in cellular metabolism towards OXPHOS (as cells are unable to effectively use galactose for glycolysis). Therefore, CML cells grown in galactose-containing media are more sensitive to inhibition of mitochondrial respiration than glucose-grown cells. Using this approach, we screened total of >1,100 unique and structurally diverse FDA-approved drugs in CML cells, cultured in the presence of 11mM glucose or 11mM galactose. This uncovered lomerizine dihydrochloride, a L-type Ca2+ channel blocker used as an anti-migraine drug, as a candidate compound effective against OXPHOS-dependent CML cells. Subsequently, patient derived CML CD34+ cells were shown to have increased ER mass (the main intracellular Ca2+ store) and an increase in mitochondrial Ca2+ levels compared with normal cells. Moreover, transcriptional analysis revealed that Ca2+ channel genes such as voltage-gated CACNA1D and receptor-activated TRPC6 are significantly upregulated in CML LSCs (CD34+CD38-) when compared with normal haematopoietic stem cells (HSCs), which was not reverted following imatinib treatment. Therefore, we hypothesised that the observed increase in mitochondrial metabolism in CML LSCs was dependent on CACNA1D and TRPC6 activity, and the increase in mitochondrial Ca2+ levels, which could be inhibited by lomerizine treatment. Notably, CRISPR-Cas9-mediated CACNA1D or TRPC6 knockout significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and decreased cellular oxygen consumption rate, indicative of reduction in OXPHOS. Similarly, while imatinib treatment did not affect intracellular Ca2+ levels in primary CML CD34+ cells, lomerizine treatment significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and mitochondrial respiration/OXPHOS. Applying liquid chromatography-mass spectrometry and isotope assisted metabolomics, we traced labelled nutrients (glucose and palmitate) in CD34+ cells. This revealed a significant reduction in activity of mitochondria-located Ca2+ dependent dehydrogenases and overall reduction in tricarboxylic acid (TCA) cycle activity in primary CML cells following lomerizine treatment. Of clinical relevance, using combination of cell surface markers, cell division trackers and functional assays, including long-term culture initiating cell (LTC-IC) assay, we demonstrate that lomerizine treatment, alone or in combination with imatinib, targeted CML LSCs when cultured in physiological medium in vitro, without affecting normal HSCs. Notably, combination treatment with imatinib and lomerizine reduced CML tumour burden, targeted CML LSCs and extended survival in xenotransplantation model of human CML. In summary, we reveal for the first time that Ca2+ regulation is disrupted in CML stem cells and influx via CACNA1D and TRPC6 is crucial for maintaining high mitochondrial Ca2+ levels and enhanced mitochondrial OXPHOS. This renders CML stem cells sensitive to lomerizine, an FDA-approved Ca2+ inhibitor identified following metabolism-specific drug-repurposing screening.

Deficiency of connexin43 increases arrhythmias due to increases in Ca2+ and ROS within the mitochondria

Abstract Introduction Connexin43 (Cx43) is a major connexin that forms gap junctions in the hearts and plays important roles in the occurrence of reentrant arrhythmias. Cx43 also exits as hemichannels in the inner mitochondrial membrane (mCx43), but it has not yet been established whether mCx43 are involved in the occurrence of arrhythmias. Purpose To examine what roles mCx43 plays in the occurrence of arrhythmias and whether mitochondrial Ca2+ and ROS are involved in their occurrence. Methods To generate cardiac-specific Cx43-deficient (Cx43-/-) mice, Cx43flox/flox mice were crossed with α-myosin heavy chain cre+/- mice. The resulting offspring, Cx43-/- mice and their littermates (Cx43+/+ mice) were used. Trabeculae were dissected from right ventricles of mouse hearts. Force was measured with a strain gauge, mitochondrial Ca2+ with rhod-2, and mitochondrial ROS with MitoSox Red. The spatial distribution of rhod-2 and MitoSox Red was imaged using confocal microscopy, and it was similar to that of MitoTracker Green within trabeculae, meaning that rhod-2 and MitoSox Red are mainly loaded within the mitochondria. To determine the amount of ROS production, MitoSox Red fluorescence was measured before (Fl) and after the addition of 10 mM H2O2 (FlH2O2), and the ratio of Fl to FlH2O2 was calculated. To estimate arrhythmia susceptibility, the minimal extracellular Ca2+ concentration (Cao), at which arrhythmias were induced by electrical stimulation, was determined (Caomin). Arrhythmias were induced by electrical stimulation with 0.3-s stimulus intervals for 30 s. Results Most of Cx43-/- mice died within 8 weeks (p<0.01). Body and heart weights and tibial length of Cx43-/- mice were similar to those of Cx43+/+ mice. Cx43 was present in the inner mitochondrial membrane in Cx43+/+ mice but not in Cx43-/- mice. Developed force of Cx43-/- mice was similar to that of Cx43+/+ mice at Cao ranging from 0.2 to 6 mM. Rhod-2 fluorescence in Cx43-/- mice was higher than that of Cx43+/+ mice, especially at higher Cao such as 4 and 6 mM (n=7, p<0.01), and the Caomin in Cx43-/- mice was lower than that in Cx43+/+ mice (n=5, p<0.01), suggesting that Cx43-/- mice shows higher arrhythmia susceptibility with higher mitochondrial Ca2+. In Cx43-/- mice, Ru360 (5 μM), a mitochondrial calcium uniporter inhibitor, increased the Caomin, that is, decreased arrhythmia susceptibility with decreases in rhod-2 and MitoSox Red fluorescence (n=6, p<0.05). Additionally, antioxidant peptide SS-31 (50 µM) and N-acetyl-L-cysteine (1 mM) increased the Caomin in Cx43-/- mice (n=5, p<0.05), and the ratio of Fl to FlH2O2 in Cx43-/- mice was larger that that in Cx43+/+ mice, suggesting that an increase in ROS production plays important roles in the occurrence of arrhythmias in Cx43-/- mice. Conclusions Deficiency of Cx43 increases arrhythmia susceptibility with increases in mitochondrial Ca2+ and ROS, providing an attractive therapeutic target for improving arrhythmias in diseased hearts.

Abstract 15068: Connexin43 Concerns Arrhythmias Through Changes in Mitochondrial Ca 2+ and ROS

Introduction: Connexin43 (Cx43) is a connexin that forms gap junctions in hearts and exists as hemichannels in the inner mitochondrial membrane (mCx43). It is still unknown whether mCx43 is involved in the occurrence of arrhythmias. Thus, we examined whether deficiency of Cx43 increases arrhythmias and what roles mitochondrial Ca 2+ and ROS play in the occurrence of arrhythmias. Methods: Cardiac-specific Cx43-deficient ( Cx43 -/- ) mice were generated by crossing Cx43 flox/flox mice with α-myosin heavy chain cre +/- mice. The resulting offspring, Cx43 -/- mice and their littermates ( Cx43 +/+ mice), were used. Trabeculae were dissected from mouse hearts. Force was measured with a strain gauge, mitochondrial Ca 2+ with rhod-2, and mitochondrial ROS with MitoSox Red. The spatial distribution of rhod-2 and MitoSox Red imaged with confocal microscopy was similar to that of MitoTracker Green within trabeculae, meaning that rhod-2 and MitoSox Red are loaded within mitochondria. MitoSox Red fluorescence was measured before (Fl) and after the addition of 10 mM H 2 O 2 (FlH 2 O 2 ), and the ratio of Fl to FlH 2 O 2 was calculated. To estimate arrhythmogenesis, the minimal extracellular Ca 2+ concentration (Cao), at which arrhythmias were induced by electrical stimulation (0.3-s stimulus intervals for 30 s), was determined (Cao min ). Results: Cx43 -/- mice died within 8 weeks (p<0.01). Cx43 was present in the inner mitochondrial membrane in Cx43 +/+ mice but not in Cx43 -/- mice. In Cx43 -/- mice, rhod-2 fluorescence was higher than that of Cx43 +/+ mice (n=7, p<0.01), while developed force of Cx43 -/- mice was similar to that of Cx43 +/+ mice. The Cao min in Cx43 -/- mice was lower than that in Cx43 +/+ mice (n=5, p<0.01), suggesting that Cx43 -/- mice shows increased arrhythmogenesis. In Cx43 -/- mice, Ru360 (5 μM), a mitochondrial calcium uniporter inhibitor, increased the Cao min , that is, decreased arrhythmogenesis, with decreases in rhod-2 and MitoSox Red fluorescence (n=6, p<0.05). Antioxidant peptide SS-31 (50 μM) and N-acetyl-L-cysteine (1 mM) increased the Cao min in Cx43 -/- mice (n=5, p<0.05), and the ratio of Fl to FlH 2 O 2 in Cx43 -/- mice was larger than that in Cx43 +/+ mice. Conclusions: Deficiency of Cx43 increases arrhythmogenesis with increases in mitochondrial Ca 2+ and ROS.

α-Synuclein-dependent increases in PIP5K1γ drive inositol signaling to promote neurotoxicity

Anomalous aggregation of α-synuclein (α-Syn) is a pathological hallmark of many degenerative synucleinopathies including Lewy body dementia (LBD) and Parkinson's disease (PD). Despite its strong link to disease, the precise molecular mechanisms that link α-Syn aggregation to neurodegeneration have yet to be elucidated. Here, we find that elevated α-Syn leads to an increase in the plasma membrane (PM) phosphoinositide PI(4,5)P2, which precipitates α-Syn aggregation and drives toxic increases in mitochondrial Ca2+ and reactive oxygen species leading to neuronal death. Upstream of this toxic signaling pathway is PIP5K1γ, whose abundance and localization is enhanced at the PM by α-Syn-dependent increases in ARF6. Selective inhibition of PIP5K1γ or knockout of ARF6 in neurons rescues α-Syn aggregation and cellular phenotypes of toxicity. Collectively, our data suggest that modulation of phosphoinositide metabolism may be a therapeutic target to slow neurodegeneration for PD and other related neurodegenerative disorders.

Increased mitochondrial free Ca2+ during ischemia is suppressed, but not eliminated by, germline deletion of the mitochondrial Ca2+ uniporter

Mitochondrial Ca2+ overload is proposed to regulate cell death via opening of the mitochondrial permeability transition pore. It is hypothesized that inhibition of the mitochondrial Ca2+ uniporter (MCU) will prevent Ca2+ accumulation during ischemia/reperfusion and thereby reduce cell death. To address this, we evaluate mitochondrial Ca2+ in ex-vivo-perfused hearts from germline MCU-knockout (KO) and wild-type (WT) mice using transmural spectroscopy. Matrix Ca2+ levels are measured with a genetically encoded, red fluorescent Ca2+ indicator (R-GECO1) using an adeno-associated viral vector (AAV9) for delivery. Due to the pH sensitivity of R-GECO1 and the known fall in pH during ischemia, hearts are glycogen depleted to decrease the ischemic fall in pH. At 20min of ischemia, there is significantly less mitochondrial Ca2+ in MCU-KO hearts compared with MCU-WT controls. However, an increase in mitochondrial Ca2+ is present in MCU-KO hearts, suggesting that mitochondrial Ca2+ overload during ischemia is not solely dependent on MCU.

Ru360 protects against vitrification-induced oocyte meiotic defects by restoring mitochondrial function

Oocyte vitrification has been widely application in female fertility preservation. Recent studies found that vitrification of immature (germinal vesicle stage, GV) oocytes increased the risk of aneuploidy during meiotic maturation; however, the underlying mechanisms and the strategies to prevent this defect remain unexplored. In this study, we found that vitrification of GV oocytes decreased the first polarbody extrusion rate (90.51 ± 1.04% vs. 63.89 ± 1.39%, p < 0.05) and increased the aneuploid rate (2.50% vs. 20.00%, p < 0.05), accompanied with a series of defects during meiotic maturation, including aberrant spindle morphology, chromosome misalignment, incorrect Kinetochore-Microtubule attachments (KT-MTs) and weakened spindle assembly checkpoint protein complex (SAC) function. We also found that vitrification disrupted mitochondrial function by increasing mitochondrial Ca2+ levels. Importantly, inhibition of mitochondrial Ca2+ entry by 1 μM Ru360 significantly restored mitochondrial function and rescued the meiotic defects, indicating that the increase of mitochondrial Ca2+, at least, was a cause of meiotic defects in vitrified oocytes. These results shed light on the molecular mechanisms of oocyte vitrification-induced adverse effects of meiotic maturation and provided a potential strategy to improve oocyte cryopreservation protocols further.

Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle.

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43-/-) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/- mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/- mice (cCx43-/- mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2',7'-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43-/- mice died suddenly within 8weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43-/- mice. In cCx43-/- mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43-/- mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43-/- mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.

Afatinib triggers a Ni2+ -resistant Ca2+ influx pathway in A549 non-small cell lung cancer cells.

Afatinib is used to treat non-small cell lung cancer cells (NSCLC), and its mechanism involves irreversible inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. In this study, we examined if afatinib had cytotoxic action against NSCLC other than inhibition of tyrosine kinase. Afatinib (1-30 μM) caused apoptotic death in A549 NSCLC in a concentration-dependent manner. Afatinib triggered Ca2+ influx without causing Ca2+ release, and the Ca2+ influx was unaffected by sodium orthovanadate (SOV, an inhibitor of tyrosine phosphatase), suggesting that afatinib-triggered Ca2+ response was unrelated to its inhibition of tyrosine kinase. Addition of afatinib also promoted Mn2+ influx. Ca2+ influx triggered by afatinib was resistant to SKF96365 and ruthenium red (two general blockers of TRP channels) and, unexpectedly, Ni2+ (a non-specific Ca2+ channel blocker). Afatinib caused an increase in mitochondrial Ca2+ level, an initial mitochondrial hyperpolarization (4 h) and followed by mitochondrial potential collapse (24-48 h). Afatinib-induced cell death was slightly but significantly alleviated in low extracellular Ca2+ condition or under pharmacological block of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A. Therefore, in addition to tyrosine kinase inhibition as a major anti-cancer mechanism of afatinib, stimulation of an atypical Ca2+ influx pathway, mitochondrial Ca2+ overload, and potential collapse in part contribute to afatinib-induced cell death.

Effects of high glutamate concentrations on mitochondria of human neuroblastoma SH-SY5Y cells

The excess amount of glutamate in neurons is associated with the excitotoxicity and neurodegenerative diseases. Glutamate induces neurotoxicity primarily by immense influx of Ca2+arising from overstimulation of the NMDA subtype of glutamate receptors. The neuronal death induced by the overstimulation of glutamate receptors depends critically on a sustained increase in mitochondrial Ca2+influx and impairment in mitochondrial functions. The mitochondrial impairment is an important contributor to the glutamate-induced neuronal toxicity and thus provides an important target for the intervention. The present study investigates the effects of high glutamate concentrations on mitochondrial functions. Here, we have shown that the higher concentration of glutamate treatment caused a significant elevation in the N-methyl-D-aspartate (NMDA) receptors expression and elevated the intra-mitochondrial calcium accumulation in SHSY5Y neuronal cells. As a result of an accumulation of intra-mitochondrial calcium, there is a concentration-dependent elevation in ROS in the mitochondria. Tyrosine nitration of several mitochondrial proteins was increased while the mitochondrial membrane potential was dissipated. Furthermore, glutamate treatments also resulted in mitochondrial membrane permeability transition. These findings suggest that treatment of high glutamate concentration causes impairment of mitochondrial functions by an increase in intra-mitochondrial calcium, ROS production, dissipation of mitochondrial membrane potential and mitochondrial permeability transition pore opening in human neuroblastoma SHSY5Y cells.

APP Genetic Deficiency Alters Intracellular Ca2+ Homeostasis and Delays Axonal Degeneration in Dorsal Root Ganglion Sensory Neurons.

The activation of self-destructive cellular programs helps sculpt the nervous system during development, but the molecular mechanisms used are not fully understood. Prior studies have investigated the role of the APP in the developmental degeneration of sensory neurons with contradictory results. In this work, we sought to elucidate the impact of APP deletion in the development of the sensory nervous system in vivo and in vitro. Our in vivo data show an increase in the number of sciatic nerve axons in adult male and female APP-null mice, consistent with the hypothesis that APP plays a pro-degenerative role in the development of peripheral axons. In vitro, we show that genetic deletion of APP delays axonal degeneration triggered by nerve growth factor deprivation, indicating that APP does play a pro-degenerative role. Interestingly, APP depletion does not affect caspase-3 levels but significantly attenuates the rise of axoplasmic Ca2+ that occurs during degeneration. We examined intracellular Ca2+ mechanisms that could be involved and found that APP-null DRG neurons had increased Ca2+ levels within the endoplasmic reticulum and enhanced store-operated Ca2+ entry. We also observed that DRG axons lacking APP have more mitochondria than their WT counterparts, but these display a lower mitochondrial membrane potential. Finally, we present evidence that APP deficiency causes an increase in mitochondrial Ca2+ buffering capacity. Our results support the hypothesis that APP plays a pro-degenerative role in the developmental degeneration of DRG sensory neurons, and unveil the importance of APP in the regulation of calcium signaling in sensory neurons.SIGNIFICANCE STATEMENT The nervous system goes through a phase of pruning and programmed neuronal cell death during development to reach maturity. In such context, the role played by the APP in the peripheral nervous system has been controversial, ranging from pro-survival to pro-degenerative. Here we present evidence in vivo and in vitro supporting the pro-degenerative role of APP, demonstrating the ability of APP to alter intracellular Ca2+ homeostasis and mitochondria, critical players of programmed cell death. This work provides a better understanding of the physiological function of APP and its implication in developmental neuronal death in the nervous system.

Lack of TRPV1 aggravates obesity-associated hypertension through the disturbance of mitochondrial Ca2+ homeostasis in brown adipose tissue

The combination of obesity and hypertension is associated with high morbidity and mortality; however, the mechanism underlying obesity-induced hypertension remains unclear. In this study, we detected the possible effects of TRPV1, a previously identified antihypertensive calcium (Ca2+) channel in adipose tissue, on the occurrence of obesity and hypertension in mice lacking UCP1, a spontaneously genetically manipulated obesity model, by generating TRPV1 and UCP1 double knockout mice. In these mice, obesity and hypertension appeared earlier and were more severe than in mice with the knockout of UCP1 or TRPV1 alone. The knockout of TRPV1 in UCP1 knockout mice further reduced functional brown adipose tissue (BAT) generation; decreased resting oxygen consumption, heat production, and locomotor activities; and was accompanied by severe mitochondrial respiratory dysfunction in BAT. Mechanistically, TRPV1, UCP1, and LETM1 acted as a complex to maintain an appropriate mitochondrial Ca2+ level, and TRPV1 knockout caused a compensatory increase in mitochondrial Ca2+ uptake via LETM1 activation. However, the compensatory response was blocked in UCP1−/− mice, resulting in dramatically reduced mitochondrial Ca2+ uptake and higher production of ATP and oxidative stress. This study provides in vivo evidence for the critical role of BAT mitochondrial Ca2+ homeostasis in obesity-associated hypertension and indicates that the TRPV1/UCP1/LETM1 complex may be an alternative intervention target.

Vascular calcium signalling and ageing.

Changes in cellular Ca2+ levels have major influences on vascular function and blood pressure regulation. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) orchestrate vascular activity in distinct ways, often involving highly specific fluctuations in Ca2+ signalling. Ageing is a major risk factor for cardiovascular diseases, but the impact of ageing per se on vascular Ca2+ signalling has received insufficient attention. We reviewed the literature for age-related changes in Ca2+ signalling in relation to vascular structure and function. Vascular tone dysregulation in several vascular beds has been linked to abnormal expression or activity of SMC voltage-gated Ca2+ channels, Ca2+ -activated K+ channels or TRPC6 channels. Some of these effects were linked to altered caveolae density, microRNA expression or 20-HETE abundance. Intracellular store Ca2+ handling was suppressed in ageing mainly via reduced expression of intracellular Ca2+ release channels, and Ca2+ reuptake or efflux pumps. An increase in mitochondrial Ca2+ uptake, leading to oxidative stress, could also play a role in SMC hypercontractility and structural remodelling in ageing. In ECs, ageing entailed diverse effects on spontaneous and evoked Ca2+ transients, as well as structural changes at the EC-SMC interface. The concerted effects of altered Ca2+ signalling on myogenic tone, endothelium-dependent vasodilatation, and vascular structure are likely to contribute to blood pressure dysregulation and blood flow distribution deficits in critical organs. With the increase in the world's ageing population, future studies should be directed at solving specific ageing-induced Ca2+ signalling deficits to combat the imminent accelerated vascular ageing and increased risk of cardiovascular diseases.

IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease

Ca2+ is the most ubiquitous second messenger in neurons whose spatial and temporal elevations are tightly controlled to initiate and orchestrate diverse intracellular signaling cascades. Numerous neuropathologies result from mutations or alterations in Ca2+ handling proteins; thus, elucidating molecular pathways that shape Ca2+ signaling is imperative. Here, we report that loss-of-function, knockout, or neurodegenerative disease-causing mutations in the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1), initiate a damaging signaling cascade that alters the expression and nanoscale distribution of IP3R type 1 (IP3R1) in endoplasmic reticulum membranes. These alterations detrimentally increase Gq-protein coupled receptor-stimulated Ca2+ release and spontaneous IP3R1 Ca2+ activity, leading to mitochondrial Ca2+ cytotoxicity. Mechanistically, we find that SREBP-dependent increases in Presenilin 1 (PS1) underlie functional and expressional changes in IP3R1. Accordingly, expression of PS1 mutants recapitulate, while PS1 knockout abrogates Ca2+ phenotypes. These data present a signaling axis that links the NPC1 lysosomal cholesterol transporter to the damaging redistribution and activity of IP3R1 that precipitates cell death in NPC1 disease and suggests that NPC1 is a nanostructural disease.

IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca2+ through the noncanonical STAT3 pathway

Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca2+ Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca2+-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.

Mitostasis, Calcium and Free Radicals in Health, Aging and Neurodegeneration.

Mitochondria play key roles in ATP supply, calcium homeostasis, redox balance control and apoptosis, which in neurons are fundamental for neurotransmission and to allow synaptic plasticity. Their functional integrity is maintained by mitostasis, a process that involves mitochondrial transport, anchoring, fusion and fission processes regulated by different signaling pathways but mainly by the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α also favors Ca2+ homeostasis, reduces oxidative stress, modulates inflammatory processes and mobilizes mitochondria to where they are needed. To achieve their functions, mitochondria are tightly connected to the endoplasmic reticulum (ER) through specialized structures of the ER termed mitochondria-associated membranes (MAMs), which facilitate the communication between these two organelles mainly to aim Ca2+ buffering. Alterations in mitochondrial activity enhance reactive oxygen species (ROS) production, disturbing the physiological metabolism and causing cell damage. Furthermore, cytosolic Ca2+ overload results in an increase in mitochondrial Ca2+, resulting in mitochondrial dysfunction and the induction of mitochondrial permeability transition pore (mPTP) opening, leading to mitochondrial swelling and cell death through apoptosis as demonstrated in several neuropathologies. In summary, mitochondrial homeostasis is critical to maintain neuronal function; in fact, their regulation aims to improve neuronal viability and to protect against aging and neurodegenerative diseases.

Forsythiasides-Rich Extract From Forsythiae Fructus Inhibits Mast Cell Degranulation by Enhancing Mitochondrial Ca2+ Uptake.

Mast cells (MCs) activated via IgE/FcεRI or MAS-related G protein coupled receptor (Mrgpr)-mediated pathway can release granules that play prominent roles in hypersensitivity reactions. Forsythiae Fructus, a well-known traditional Chinese medicine, has been clinically used for allergic diseases. Although previous studies indicated that Forsythiae Fructus extract inhibited compound 48/80-induced histamine release from MCs, its effect on IgE-dependent MC degranulation and possible underlying mechanisms remain to be explored. Herein, we prepared the forsythiasides-rich extract (FRE) and investigated its action on MC degranulation and explored its underlying mechanism. Our data showed that FRE could dampen IgE/FcεRI- and Mrgpr-mediated MC degranulation in vitro and in vivo. Mechanism study indicated that FRE decreased cytosolic Ca2+ (Ca2+ [c]) level rapidly and reversibly. Moreover, FRE decreased Ca2+ [c] of MCs independent of plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). While, along with Ca2+ [c] decrease, the increase of mitochondrial Ca2+ (Ca2+ [m]) occurred simultaneously in FRE-treated RBL-2H3 cells. In the isolated mitochondria, FRE also promoted the subcellular organelle to uptake more extramitochondrial Ca2+. In conclusion, by increasing Ca2+ [m] uptake, FRE decreases Ca2+ [c] level to suppress MC degranulation. Our findings may provide theoretical support for the clinical application of Forsythiae Fructus on allergy and other MC-involved diseases.

Parvalbumin affects skeletal muscle trophism through modulation of mitochondrial calcium uptake.

SummaryParvalbumin (PV) is a cytosolic Ca2+-binding protein highly expressed in fast skeletal muscle, contributing to an increased relaxation rate. Moreover, PV is an “atrogene” downregulated in most muscle atrophy conditions. Here, we exploit mice lacking PV to explore the link between the two PV functions. Surprisingly, PV ablation partially counteracts muscle loss after denervation. Furthermore, acute PV downregulation is accompanied by hypertrophy and upregulation by atrophy. PV ablation has a minor impact on sarcoplasmic reticulum but is associated with increased mitochondrial Ca2+ uptake, mitochondrial size and number, and contacts with Ca2+ release sites. Mitochondrial calcium uniporter (MCU) silencing abolishes the hypertrophic effect of PV ablation, suggesting that mitochondrial Ca2+ uptake is required for hypertrophy. In turn, an increase of mitochondrial Ca2+ is required to enhance expression of the pro-hypertrophy gene PGC-1α4, whose silencing blocks hypertrophy due to PV ablation. These results reveal how PV links cytosolic Ca2+ control to mitochondrial adaptations, leading to muscle mass regulation.

Mitochondrial N-methyl-d-aspartate receptor activation enhances bioenergetics by calcium-dependent and –Independent mechanisms

Our laboratory has demonstrated that functional N-methyl-d-aspartate-like receptors are present on neuronal mitochondria (NMDAm). This novel site gates the influx of Ca2+ and causes a several-fold increase in ATP levels. Although elevations in ATP in other cell types have been linked to increases in mitochondrial Ca2+, it has not been established whether the same holds true for calcium uptake via NMDAm. In this study, we have investigated the effect of NMDAm activation on a variety of bioenergetic parameters. Our findings suggest that mitochondrial bioenergetics are not only modulated by NMDAm activation in a Ca2+-dependent but also in a Ca2+-independent manner.

The mitochondrial calcium homeostasis orchestra plays its symphony: Skeletal muscle is the guest of honor.

Skeletal muscle mitochondria are placed in close proximity of the sarcoplasmic reticulum (SR), the main intracellular Ca2+ store. During muscle activity, excitation of sarcolemma and of T-tubule triggers the release of Ca2+ from the SR initiating myofiber contraction. The rise in cytosolic Ca2+ determines the opening of the mitochondrial calcium uniporter (MCU), the highly selective channel of the inner mitochondrial membrane (IMM), causing a robust increase in mitochondrial Ca2+ uptake. The Ca2+-dependent activation of TCA cycle enzymes increases the synthesis of ATP required for SERCA activity. Thus, Ca2+ is transported back into the SR and cytosolic [Ca2+] returns to resting levels eventually leading to muscle relaxation. In recent years, thanks to the molecular identification of MCU complex components, the role of mitochondrial Ca2+ uptake in the pathophysiology of skeletal muscle has been uncovered. In this chapter, we will introduce the reader to a general overview of mitochondrial Ca2+ accumulation. We will tackle the key molecular players and the cellular and pathophysiological consequences of mitochondrial Ca2+ dyshomeostasis. In the second part of the chapter, we will discuss novel findings on the physiological role of mitochondrial Ca2+ uptake in skeletal muscle. Finally, we will examine the involvement of mitochondrial Ca2+ signaling in muscle diseases.

NCLX prevents cell death during adrenergic activation of the brown adipose tissue

A sharp increase in mitochondrial Ca2+ marks the activation of brown adipose tissue (BAT) thermogenesis, yet the mechanisms preventing Ca2+ deleterious effects are poorly understood. Here, we show that adrenergic stimulation of BAT activates a PKA-dependent mitochondrial Ca2+ extrusion via the mitochondrial Na+/Ca2+ exchanger, NCLX. Adrenergic stimulation of NCLX-null brown adipocytes (BA) induces a profound mitochondrial Ca2+ overload and impaired uncoupled respiration. Core body temperature, PET imaging of glucose uptake and VO2 measurements confirm a thermogenic defect in NCLX-null mice. We show that Ca2+ overload induced by adrenergic stimulation of NCLX-null BAT, triggers the mitochondrial permeability transition pore (mPTP) opening, leading to a remarkable mitochondrial swelling and cell death. Treatment with mPTP inhibitors rescue mitochondrial function and thermogenesis in NCLX-null BAT, while calcium overload persists. Our findings identify a key pathway through which BA evade apoptosis during adrenergic stimulation of uncoupling. NCLX deletion transforms the adrenergic pathway responsible for thermogenesis activation into a death pathway.