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Tight regulation of neuronal Zn2+is critical for physiological function. Multiple Zn2+transporters are expressed in the brain, yet their spatial distribution and distinct roles are largely unknown. Here, we show developmental regulation of the expression of Zn2+transporters ZIP1 and ZIP3 in mouse hippocampal neurons, corresponding to previously described increase in neuronal vesicular Zn2+during the first postnatal month. Rates of Zn2+uptake in cultured mouse hippocampal neurons, monitored using FluoZin-3 fluorescence, were higher in mature neurons, which express higher levels of ZIP1 and ZIP3. Zn2+uptake was attenuated by ∼50% following silencing of either ZIP1 or ZIP3. Expression of both ZIP1 and ZIP3 was ubiquitous on somas and most neuronal processes in the cultured neurons. In contrast, we observed distinct localization of the transporters in adult mouse hippocampal brain, with ZIP1 predominantly expressed in the CA3 stratum pyramidale, and ZIP3 primarily localized to the stratum lucidum. Consistent with their localization, silencing of ZIP1 expressionin vivoreduced Zn2+uptake in CA3 neurons while ZIP3 silencing reduced Zn2+influx into dentate gyrus (DG) granule cells in acute hippocampal slices. Strikingly,in vivosilencing of ZIP3, but not ZIP1, protected CA3 neurons from neurodegeneration following kainate-induced seizures. Our results indicate that distinct Zn2+transporters control Zn2+accumulation and toxicity in different neuronal populations in the hippocampus and suggest that selective regulation of Zn2+transporters can prevent seizure induced brain damage.SIGNIFICANCE STATEMENTZinc plays a major role in neuronal function and its dysregulation is associated with neurodegeneration. Multiple zinc transporters are expressed in neurons, yet little is known on their distinct roles. Here, we show that the plasma membrane ZIP1 and ZIP3 zinc transporters are expressed on distinct neuronal populations in the CA3 region of the hippocampus. We show that ZIP1 mediates zinc influx into postsynaptic cells, while ZIP3 is responsible for zinc re-uptake from this synapse into dentate granule cells. We further show that silencing of ZIP3, but not ZIP1, can rescue the postsynaptic cells from kainate-induced neurodegeneration. This suggests that neuronal zinc toxicity and degeneration can be modulated by regulation of specific zinc transporters function.

Impairments in neural lysosomal- and autophagic-mediated degradation of cellular debris contribute to neuritic dystrophy and synaptic loss. While these are well-characterized features of neurodegenerative disorders such as Alzheimer’s disease (AD), the upstream cellular processes driving deficits in pathogenic protein mishandling are less understood. Using a series of fluorescent biosensors and optical imaging in model cells, AD mouse models and human neurons derived from AD patients, we reveal a previously undescribed cellular signaling cascade underlying protein mishandling mediated by intracellular calcium dysregulation, an early component of AD pathogenesis. Increased Ca 2+ release via the endoplasmic reticulum (ER)-resident ryanodine receptor (RyR) is associated with reduced expression of the lysosome proton pump vacuolar-ATPase (vATPase) subunits (V1B2 and V0a1), resulting in lysosome deacidification and disrupted proteolytic activity in AD mouse models and human-induced neurons (HiN). As a result of impaired lysosome digestive capacity, mature autophagosomes with hyperphosphorylated tau accumulated in AD murine neurons and AD HiN, exacerbating proteinopathy. Normalizing AD-associated aberrant RyR-Ca 2+ signaling with the negative allosteric modulator, dantrolene (Ryanodex), restored vATPase levels, lysosomal acidification and proteolytic activity, and autophagic clearance of intracellular protein aggregates in AD neurons. These results highlight that prior to overt AD histopathology or cognitive deficits, aberrant upstream Ca 2+ signaling disrupts lysosomal acidification and contributes to pathological accumulation of intracellular protein aggregates. Importantly, this is demonstrated in animal models of AD, and in human iPSC-derived neurons from AD patients. Furthermore, pharmacological suppression of RyR-Ca 2+ release rescued proteolytic function, revealing a target for therapeutic intervention that has demonstrated effects in clinically-relevant assays.

Calcium dynamics control synaptic transmission. Calcium triggers synaptic vesicle fusion, determines release probability, modulates vesicle recycling, participates in long-term plasticity and regulates cellular metabolism. Mitochondria, the main source of cellular energy, serve as calcium signaling hubs. Mitochondrial calcium transients are primarily determined by the balance between calcium influx, mediated by the mitochondrial calcium uniporter (MCU), and calcium efflux through the sodium/lithium/calcium exchanger (NCLX). We identified a human recessive missense SLC8B1 variant that impairs NCLX activity and is associated with severe mental retardation. On this basis, we examined the effect of deleting NCLX in mice on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity. Neuronal mitochondria exhibited basal calcium overload, membrane depolarization, and a reduction in the amplitude and rate of calcium influx and efflux. We observed smaller cytoplasmic calcium transients in the presynaptic terminals of NCLX-KO neurons, leading to a lower probability of release and weaker transmission. In agreement, synaptic facilitation in NCLX-KO hippocampal slices was enhanced. Importantly, deletion of NCLX abolished long term potentiation of Schaffer collateral synapses. Our results show that NCLX controls presynaptic calcium transients that are crucial for defining synaptic strength as well as short- and long-term plasticity, key elements of learning and memory processes., Stavsky et al. examined the effects of deleting the mitochondrial sodium/lithium/calcium exchanger, NCLX, on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity in mice. Having identified a human mutation that impairs NCLX activity and is associated with mental retardation, they show that NCLX is crucial for defining synaptic strength and plasticity, which are pivotal elements of learning and memory.

Klotho is a transmembrane protein, which can be shed and act as a circulating hormone and is involved in regulating cellular calcium levels and inhibition of the PI3K/AKT pathway. As a longevity hormone, it protects normal cells from oxidative stress, and as a tumor suppressor it inhibits growth of cancer cells. Mechanisms governing these differential activities have not been addressed. Altered cellular metabolism is a hallmark of cancer and dysregulation of mitochondrial activity is a hallmark of aging. We hypothesized that klotho exerts its differential effects through regulation of these two hallmarks. Treatment with klotho inhibited glycolysis, reduced mitochondrial activity and membrane potential only in cancer cells. Accordingly, global metabolic screen revealed that klotho altered pivotal metabolic pathways, amongst them glycolysis and tricarboxylic acid cycle in breast cancer cells. Alteration of metabolic activity and increased AMP/ATP ratio lead to LKB1-dependent AMPK activation. Indeed, klotho induced AMPK phosphorylation; furthermore, inhibition of LKB1 partially abolished klotho’s tumor suppressor activity. By diminishing deltapsi (Δψ) klotho also inhibited mitochondria Ca2+ shuttling thereby impairing mitochondria communication with SOCE leading to reduced Ca2+ influx by SOCE channels. The reduced SOCE was followed by ER Ca2+ depletion and stress. These data delineate mechanisms mediating the differential effects of klotho toward cancer versus normal cells, and indicate klotho as a potent regulator of metabolic activity.

Acid sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~ pH 6, while its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na(+) and Ca(2+) sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na(+) and Ca(2+) signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na(+) and Ca(2+) signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na(+) and Ca(2+) waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 (PcTX1) or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na(+) and Ca(2+) signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na(+) signaling even in digitonin - permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.

Mitochondrial activity is crucial for the plasticity of central synapses, but how the firing pattern of pre- and postsynaptic neurons affects the mitochondria remains elusive. We recorded changes in the fluorescence of cytosolic and mitochondrial Ca2+ indicators in cell bodies, axons, and dendrites of cortical pyramidal neurons in mouse brain slices while evoking pre- and postsynaptic spikes. Postsynaptic spike firing elicited fast mitochondrial Ca2+ responses that were about threefold larger in the somas and apical dendrites than in basal dendrites and axons. The amplitude of these responses and metabolic activity were extremely sensitive to the firing frequency. Furthermore, while an EPSP alone caused no detectable Ca2+ elevation in the dendritic mitochondria, the coincidence of EPSP with a backpropagating spike produced prominent, highly localized mitochondrial Ca2+ hotspots. Our results indicate that mitochondria decode the spike firing frequency and the Hebbian temporal coincidences into the Ca2+ signals, which are further translated into the metabolic output and most probably lead to long-term changes in synaptic efficacy.

The mitochondrial solute carrier family 8 sodium/calcium/lithium exchanger, member B1 (NCLX) is an important mediator of calcium extrusion from mitochondria. In this study, we tested the hypothesis that physiological expression levels of NCLX are essential for maintaining neuronal resilience in the face of excitotoxic challenge. Using an shRNA-mediated approach, we showed that reduced NCLX expression exacerbates neuronal mitochondrial calcium dysregulation, mitochondrial membrane potential (ΔΨ

Mitochondrial activity is crucial for the plasticity of central synapses, but how the firing pattern of pre- and postsynaptic neurons affects the mitochondria remains elusive. We recorded changes in the fluorescence of cytosolic and mitochondrial Ca

Impaired phosphodiesterase (PDE) function and mitochondrial Ca

Mitochondrial Ca2+ transient is the earliest discovered organellar Ca2+ signaling pathway. It consist of a Ca2+ influx, mediated by mitochondrial Ca2+ uniporter (MCU), and mitochondrial Ca2+ efflux mediated by a Na+/Ca2+ exchanger (NCLX). Mitochondrial Ca2+ signaling machinery plays a fundamental role in linking metabolic activity to cellular Ca2+ signaling, and in controlling local Ca2+ concertation in distinct cellular compartments. Impaired balance between mitochondrial Ca2+ influx and efflux leads to mitochondrial Ca2+ overload, an early and key event in ischemic or neurodegenerative syndromes. Molecular identification of NCLX and MCU happened only recently. Surprisingly, MCU knockout yielded a relatively mild phenotype while conditional knockout of NCLX led to a rapid fatal heart failure. Here we will focus on recent functional and molecular studies on NCLX structure and its mode of regulation. We will describe the unique crosstalk of this exchanger with Na+ and Ca2+ signaling pathways in the cell membrane and the endoplasmic reticulum, and with protein kinases that posttranslationally modulate NCLX activity. We will critically compare selectivity of pharmacological blockers versus molecular control of NCLX expression and activity. Finally we will discuss why this exchanger is essential for survival and can serve as an attractive therapeutic target.

Manganese (Mn(2+)) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca(2+) inward gradient for active cellular exchange of Mn(2+). In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn(2+) and Ca(2+) fluxes. We found that (a) application of Mn(2+)-containing Ca(2+)-free solution to ZnT10-expressing cells triggers an influx of Mn(2+), (b) reintroduction of Ca(2+) leads to cellular Mn(2+) extrusion against an inward Mn(2+) gradient, and (c) the cellular transport of Mn(2+) by ZnT10 is coupled to a reciprocal movement of Ca(2+). Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn(2+)/Ca(2+) exchange to an uncoupled channel mode, permeable to both Ca(2+) and Mn(2+). The findings in our study identify the first ion transporter that uses the Ca(2+) gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins.

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn

Impaired phosphodiesterase (PDE) function and mitochondrial Ca2+ - [Ca2+]m signaling leads to cardiac failure, ischemic damage and dysfunctional learning and memory. Yet, a causative link between these pathways is unknown. Here, we fluorescently monitored [Ca2+]m transients in hippocampal neurons evoked by caffeine followed by depolarization. [Ca2+]m efflux was apparent in WT but diminished in neurons deficient in the mitochondrial Na+/Ca2+ exchanger NCLX. Surprisingly, neuronal depolarization-induced Ca2+ transients alone failed to evoke strong [Ca2+]m efflux in WT neurons. Caffeine is also a PDE inhibitor. Pretreatment with the PDE2 inhibitor Bay 60-7550 rescued [Ca2+]m efflux triggered by neuronal depolarization. Inhibition of PDE2 acted by diminishing the Ca2+ dependent reduction of mitochondrial cAMP, thereby promoting NCLX phosphorylation. Selective PDE2 inhibition also enhanced [Ca2+]m efflux triggered by neuromodulators. We found that protection of neurons against excitotoxic insults, conferred by PDE2 inhibition, was diminished in NCLX KO neurons, thus is NCLX dependent. Finally, administration of Bay 60-7550 enhanced new object recognition learning in WT but not in NCLX KO mice. Our results identify a long-sought link between PDE and [Ca2+]m signaling thereby providing new therapeutic targets.

SummaryMitochondrial metabolism is critical for brain function. However, the mechanisms linking mitochondrial energy production to neuronal activity are elusive. Using whole-cell electrical recordings from Layer 5 pyramidal neurons in cortical slices and fluorescence imaging of cytosolic, mitochondrial Ca2+ indicators and endogenous NAD(P)H, we revealed ultra-fast, spike-evoked mitochondrial Ca2+ transients temporally similar to cytosolic Ca2+ elevations. We demonstrate that, whereas single or few spikes elicit the mitochondrial Ca2+ transients throughout the cell, their amplitude is differentially regulated in distinct neuronal compartments. Thus, these signals were prominent in the soma and apical dendrites and ∼3 times smaller in basal dendrites and axons. The spike firing frequency had a subtle effect on the amplitude of the cytosolic Ca2+ elevations but dramatically affected mitochondrial Ca2+ transients and NAD(P)H oxidation and recovery rates. Moreover, while subthreshold EPSPs alone caused no detectable Ca2+ elevation in dendritic mitochondria, the Hebbian coincidence of unitary EPSP and postsynaptic spike produced a localized, single mitochondrial Ca2+ elevation. These findings suggest that neuronal mitochondria are uniquely capable of decoding firing frequency and EPSP-to-spike time intervals for tuning the metabolic rate and triggering changes in synaptic efficacy.

Modulation of neuronal K+/Cl- cotransporter 2 (KCC2) activity, which mediates Cl- export, is considered to be regulated by phosphorylation of its C-terminus and critical to neuronal function. Here, we show that KCC2 interacts with the SNARE protein Synaptosome Associated Protein 23, SNAP23, an essential component of membrane insertion machinery. Using KCC2 truncated mutants, we show that KCC2 C-terminal domain is essential for the Zn2+-sensitive receptor mZnR/GPR39 upregulation of KCC2 activity and membrane targeting in HEK293 cells. Surprisingly, we found that SNAP23 is essential for ZnR/GPR39-dependent upregulation of transport mediated by KCC2 and its epithelial isoform KCC3. Moreover, IκB kinase (IKK)-dependent phosphorylation of SNAP23 is essential for ZnR/GPR39 upregulation of KCC2 activity in mouse hippocampal neurons. Finally, we found that interaction of SNAP23 and Syntaxin 1A with KCC2 enhances its insertion into the neuronal membrane. Our results elucidate a G-protein coupled receptor dependent pathway for regulation of KCC activity that via interaction with SNARE proteins.

Consumption of coffee and caffeine enhances learning efficiency and reduces the risk of stroke and neurodegenerative syndromes by a molecular pathway that is poorly understood. Here we fluorescently monitored mitochondrial Ca2+ transients in hippocampal neurons evoked by caffeine. Surprisingly, caffeine also had a long-lasting potentiation effect on mitochondrial Ca2+ ([Ca2+]m) efflux evoked by depolarization, in WT but not in neurons deficient in the mitochondrial Na+/Ca2+ exchanger NCLX. We reasoned that the caffeine effect is linked to phosphodiesterase inhibition, and focused on PDE2 that has the highest affinity to caffeine and is targeted to the mitochondria. Pretreatment of neurons with the selective PDE2 inhibitor Bay 60-7550 mimicked caffeine and rescued [Ca2+]m efflux triggered by neuronal depolarization. PDE2 inhibition also enhanced [Ca2+]m efflux triggered by neuromodulators. Inhibition of PDE2 acted by increasing mitochondrial cAMP thereby promoting NCLX phosphorylation. We found that protection of neurons against excitotoxic insults, conferred by PDE2 inhibition was diminished in NCLX KO neurons, thus is NCLX dependent. Finally, administration of Bay 60-7550 enhanced new object recognition learning in WT but not in NCLX KO mice. Our results identify a pathway linking caffeine and PDE to [Ca2+]m signaling, thereby providing new therapeutic targets for treating cognitive impairment and excitotoxicity.

Despite the established role of mitochondria in cancer, the mechanisms by which mitochondrial Ca2+ (mtCa2+) regulates tumorigenesis remain incompletely understood. The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer. Here, we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX (SLC8B1) in human colorectal tumors and its association with advanced-stage disease in patients. Downregulation of NCLX causes mtCa2+ overload, mitochondrial depolarization, decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, and expression of epithelial-to-mesenchymal, hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to increased mitochondrial reactive oxygen species, which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells. Thus, loss of NCLX is a novel driver of metastasis, indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer., eLife digest Colorectal cancer is the second largest cause of cancer deaths worldwide. Even in cases where the cancer is diagnosed and treated early, cells can sometimes survive treatment and spread to other organs. Once the cancer has spread, the survival rate is less than 15%. Mitochondria are compartments in the cell that produce energy, and they play an important role in supporting the rapid growth of cancer cells. The levels of calcium ions in mitochondria control how they produce energy, a process that is altered in cancer cells. To better understand how calcium ions influence colorectal cancer growth, Pathak, Gueguinou et al. studied a protein called NCLX, which controls calcium levels by pumping them out of the mitochondria. Two mouse strains that were used to study what happens if NCLX is missing. The first strain was genetically modified to disable the gene for NCLX and then exposed to carcinogens. The second strain was injected with colorectal cancer cells from a human tumor that were lacking NCLX. In both strains, the tumors that formed were smaller than in mice with NCLX. However, the human cancer cells in the second model were more likely to spread to other organs. This is likely because the build-up of calcium ions in the mitochondria of mice lacking NCLX led to an increase in the production of hypoxia-inducible factor-1a, a protein that is a common driver of cancer spread. Pathak, Gueguinou et al. demonstrated how NCLX can affect colorectal cancer progression. It suggests that it may have opposing effects during early and late-stage colorectal cancer, encouraging tumor growth but also decreasing the spread to other organs. Further research could help refine treatments at different stages of the disease.

SummaryDespite the established role of mitochondria in tumorigenesis, the molecular mechanisms by which mitochondrial Ca2+ (mtCa2+) signaling regulates tumor growth and metastasis remain unknown. The crucial role of mtCa2+ in tumorigenesis is highlighted by the altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer cells. Here, we demonstrate that expression of the mitochondrial Na+/Ca2+ exchanger NCLX (SLC8B1) is decreased in colorectal tumors and is associated with advanced-stage disease in patients. We reveal that downregulation of NCLX leads to mtCa2+ overload, mitochondrial depolarization, mitophagy, and reduced tumor size. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, the expression of epithelial-to-mesenchymal transition (EMT), hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to an increase in mitochondrial reactive oxygen species (mtROS) which activates HIF1α signaling supporting the metastatic behavior of tumor cells lacking NCLX. Our results reveal that loss of NCLX expression is a novel driver of metastatic progression, indicating that control of mtCa2+ levels is a novel therapeutic approach in metastatic colorectal cancer.HighlightsThe expression of NCLX is decreased in colorectal tumors and is associated with advanced-stage disease in patients.NCLX plays a dichotomous role in colorectal tumor growth and metastasis.NCLX downregulation causes mitophagy and reduced colorectal cancer tumor growth.NCLX downregulation induces stemness, chemoresistance and metastasis through mtCa2+/ROS/HIF1α signaling axis.Graphical AbstractSignificanceMitochondrial Ca2+ (mtCa2+) homeostasis is essential for cellular metabolism and growth and plays a critical role in cancer progression. mtCa2+ uptake is mediated by an inner membrane protein complex containing the mitochondrial Ca2+ uniporter (MCU). mtCa2+ uptake by the MCU is followed by a ∼100-fold slower mtCa2+ extrusion mediated by the inner mitochondrial membrane ion transporter, the mitochondrial Na+/Ca2+ exchanger NCLX. Because NCLX is a slower transporter than the MCU, it is a crucial rate-limiting factor of mtCa2+ homeostasis that cannot easily be compensated by another Ca2+ transport mechanism. This represents the first study investigating the role of NCLX in tumorigenesis and metastasis. We demonstrate for the first time that colorectal cancers exhibit loss of NCLX expression and that this is associated with advanced-stage disease. Intriguingly, decreased NCLX function has a dichotomous role in colorectal cancer. Thus, we reveal that NCLX loss leads to reduced primary tumor growth and overall tumor burden in vivo. Yet, the consequential increases in mtCa2+ elicit pro-survival, hypoxic and gene transcription pathways that enhance metastatic progression. This dichotomy is a well-established feature of chemoresistant and recurrent tumor cells including cancer stem cells. Moreover, the downstream changes elicited by NCLX loss are reminiscent of mesenchymal colorectal cancer subtypes that display poor patient survival. Our data indicate that the demonstrated changes to the mtCa2+/mtROS/HIF1α signaling axis elicited through the loss of NCLX are a key adaptation and driver of metastatic colorectal cancer.

Mitochondrial Ca 2+ signaling is linking global cellular Ca 2+ signals to energy production and controls local Ca 2+ concentrations in the ER and cell membrane. On the other hand, aberrant change in mitochondrial Ca 2+ is a hallmark of ischemic and neurodegenerative syndromes. Mitochondrial Ca 2+ transients are mediated by a Ca 2+ channel, MCU (mitochondrial Ca 2+ uniporter), and by a much slower, rate-limiting, mitochondrial Na + /Ca 2+ exchanger, NCLX. This review describes recent studies on the essential role of NCLX in cardiac survival and in diverse physiological activities. It further focuses on the regulatory and transport sites that are controlling NCLX function.

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn2+ neurotoxicity. The mechanism mediating Zn2+ transport by ZnT1, however, has remained elusive. Here, we investigated ZnT1-dependent Zn2+ transport by measuring intracellular changes of this ion using the fluorescent indicator FluoZin-3. In primary mouse cortical neurons, which express ZnT1, transient addition of extracellular Zn2+ triggered a rise in cytosolic Zn2+, followed by its removal. Knockdown of ZnT1 by adeno associated viral (AAV)-short hairpin RNA (shZnT1) markedly increased rates of Zn2+ rise, and decreased rates of its removal, suggesting that ZnT1 is a primary route for Zn2+ efflux in neurons. Although Zn2+ transport by other members of the SLC30A family is dependent on pH gradients across cellular membranes, altered H+ gradients were not coupled to ZnT1-dependent transport. Removal of cytoplasmic Zn2+, against a large inward gradient during the initial loading phase, suggests that Zn2+ efflux requires a large driving force. We therefore asked if Ca2+ gradients across the membrane can facilitate Zn2+ efflux. Elimination of extracellular Ca2+ abolished Zn2+ efflux, while increased extracellular Ca2+ levels enhanced Zn2+ efflux. Intracellular Ca2+ rises, measured in GCaMP6 expressing neurons, closely paralleled cytoplasmic Zn2+ removal. Taken together, these results strongly suggest that ZnT1 functions as a Zn2+/Ca2+ exchanger, thereby regulating the transport of two ions of fundamental importance in neuronal signaling.

Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that accumulates in humans and affects the nervous system. To elucidate the mechanisms of HBCD neurotoxicity, we used transcriptomic profiling in brains of female mice exposed through their diet to HBCD (199 mg/kg body weight per day) for 28 days and compared with those of neuronal N2A and NSC-19 cell lines exposed to 1 or 2 µM HBCD. Similar pathways and functions were affected both in vivo and in vitro, including Ca2+ and Zn2+ signalling, glutamatergic neuron activity, apoptosis, and oxidative stress. Release of cytosolic free Zn2+ by HBCD was confirmed in N2A cells. This Zn2+ release was partially quenched by the antioxidant N-acetyl cysteine indicating that, in accordance with transcriptomic analysis, free radical formation is involved in HBCD toxicity. To investigate the effects of HBCD in excitable cells, we isolated mouse hippocampal neurons and monitored Ca2+ signalling triggered by extracellular glutamate or zinc, which are co-released pre-synaptically to trigger postsynaptic signalling. In control cells application of zinc or glutamate triggered a rapid rise of intracellular [Ca2+]. Treatment of the cultures with 1 µM of HBCD was sufficient to reduce the glutamate-dependent Ca2+ signal by 50%. The effect of HBCD on zinc-dependent Ca2+ signalling was even more pronounced, resulting in the reduction of the Ca2+ signal with 86% inhibition at 1 µM HBCD. Our results show that low concentrations of HBCD affect neural signalling in mouse brain acting through dysregulation of Ca2+ and Zn2+ homeostasis. Electronic supplementary material The online version of this article (doi:10.1007/s00204-017-2119-2) contains supplementary material, which is available to authorized users.

Background The Na+/Ca2 +/Li+ exchanger (NCLX) is a member of the Na+/Ca2 + exchanger family. NCLX is unique in its capacity to transport both Na+ and Li+, unlike other members, which are Na+ selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li+ or Na+ selective Ca2 + transport and map their putative location on NCLX cation transport site. Method We combined molecular modeling to map transport site of NCLX with euryarchaeal H+/Ca2 + exchanger, CAX_Af, and fluorescence analysis to monitor Li+ versus Na+ dependent mitochondrial Ca2 + efflux of transport site mutants of NCLX in permeabilized cells. Result Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca2 + exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li+/Ca2 + exchange activity but a reduced Na+/Ca2 + exchange activity. On the other hand, D471A showed dramatically reduced Li+/Ca2 + exchange, but Na+/Ca2 + exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca2 + binding residues was required to completely abolish both Na+/Ca2 + and Li+/Ca2 + exchange activities. Conclusions We identified distinct Na+ and Li+ selective residues in the NCLX transport site. We propose that functional segregation in Li+ and Na+ sites reflects the functional properties of NCLX required for Ca2 + exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane. General significance The results of this study provide functional insights into the unique Li+ and Na+ selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Administration of zinc, as a complement to oral rehydration solutions, effectively diminishes duration and severity of diarrhea, but it is not known whether it merely fulfills a nutritional deficiency, or if zinc has a direct role of regulating solute absorption. We show that Zn 2+ acts via a specific receptor, ZnR/GPR39, to reduce fluid loss. Intestinal fluid secretion triggered by cholera toxin (CTx) was lower in WT mice compared to ZnR/GPR39 KO. In the absence of dietary Zn 2+ we observed similar fluid accumulation in WT and ZnR/GPR39 KO mice, indicating that Zn 2+ and ZnR/GPR39 are both required for a beneficial effect of Zn 2+ in diarrhea. In primary colonocytes and in Caco-2 colonocytic cells, activation of ZnR/GPR39 enhanced Cl − transport, a critical factor in diarrhea, by upregulating K + /Cl − cotransporter (KCC1) activity. Importantly, we show basolateral expression of KCC1 in mouse and human colonocytes, thus identifying a novel Cl − absorption pathway. Finally, inhibition of KCC-dependent Cl − transport enhanced CTx-induced fluid loss. Altogether, our data indicate that Zn 2+ acting via ZnR/GPR39 has a direct role in controlling Cl − absorption via upregulation of basolateral KCC1 in the colon. Moreover, colonocytic ZnR/GPR39 and KCC1 reduce water loss during diarrhea and may therefore serve as effective drug targets.

Dendritic cells (DCs) mature upon an inflammatory trigger. However, an inflammatory trigger can lead to a semi-mature phenotype, allowing DCs to evoke tolerance and expedite the resolution of inflammation. This duality likely involves context-dependent modulation of inflammatory signaling. Human α1-antitrypsin (hAAT) promotes semimature DCs. We examined changes in a wide spectrum of signaling cascades in stimulated murine bone marrow-derived cells with hAAT. Upon stimulation by IL-1β+IFNγ, hAAT-treated cells depicted an attenuated calcium flux. Disrupting PKA or NF-κB pathways revoked only some hAAT-mediated outcomes. hAAT-treated cells exhibited a distict pattern of kinase phosphorylation. hAAT-mediated increase in Treg cells in-vitro required intact inflammatory signaling pathways. Taken together, hAAT appears to require a stimulated microenvironment to promote inflammatory resolution, setting it aside from classical anti-inflammatory agents. Further studies are required to identify the specific molecules targeted by hAAT that mediate these and other outcomes.

Klotho is a transmembrane protein, which can be shed and act as a circulating hormone and is involved in regulating cellular calcium levels and inhibition of the PI3K/AKT pathway. As a longevity hormone, it protects normal cells from oxidative stress, and as a tumor suppressor it inhibits growth of cancer cells. Mechanisms governing these differential activities have not been addressed. Altered cellular metabolism is a hallmark of cancer and dysregulation of mitochondrial activity is a hallmark of aging. We hypothesized that klotho exerts its differential effects through regulation of these two hallmarks. Treatment with klotho inhibited glycolysis, reduced mitochondrial activity and membrane potential only in cancer cells. Accordingly, global metabolic screen revealed that klotho altered pivotal metabolic pathways, amongst them glycolysis and tricarboxylic acid cycle in breast cancer cells. Alteration of metabolic activity and increased AMP/ATP ratio lead to LKB1-dependent AMPK activation. Indeed, klotho induced AMPK phosphorylation; furthermore, inhibition of LKB1 partially abolished klotho's tumor suppressor activity. By diminishing deltapsi (Δψ) klotho also inhibited mitochondria Ca

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca2+ signaling leading to tumor progression. The K+/Cl- co-transporters (KCC), activated by intracellular signaling, enhance breast cancer cell migration and invasion. We asked if ZnR/GPR39 enhances breast cancer cell malignancy by activating KCC. Activation of ZnR/GPR39 by Zn2+ upregulated K+/Cl- co-transport activity, measured using NH4+ as a surrogate to K+ while monitoring intracellular pH. Upregulation of NH4+ transport was monitored in tamoxifen resistant cells with functional ZnR/GPR39-dependent Ca2+ signaling but not in MCF-7 cells lacking this response. The NH4+ transport was Na+-independent, and we therefore focused on KCC family members. Silencing of KCC3, but not KCC4, expression abolished Zn2+-dependent K+/Cl- co-transport, suggesting that KCC3 is mediating upregulated NH4+ transport. The ZnR/GPR39-dependent KCC3 activation accelerated scratch closure rate, which was abolished by inhibiting KCC transport with [(DihydroIndenyl) Oxy] Alkanoic acid (DIOA). Importantly, silencing of either ZnR/GPR39 or KCC3 attenuated Zn2+-dependent scratch closure. Thus, a novel link between KCC3 and Zn2+, via ZnR/GPR39, promotes breast cancer cell migration and proliferation.

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca

NCLX, the mitochondrial Na+/Ca2+ transporter is a key player in Ca2+ signaling. However, its role in Na+ signaling is poorly understood. In this review we focus on Na+ signaling by NCLX, and discuss recent physiological and pathophysiological roles attributed to the Na+ influx into mitochondria.