A new life for an old pump: V-ATPase and neurotransmitter release.

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca(2+) ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca(2+)-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.

Pressing the right buttons: signaling in lymphangiogenesis.

Lymphatic vasculature is increasingly recognized as an important factor both in the regulation of normal tissue homeostasis and immune response and in many diseases, such as inflammation, cancer, obesity, and hypertension. In the last few years, in addition to the central role of vascular endothelial growth factor (VEGF)-C/VEGF receptor-3 signaling in lymphangiogenesis, significant new insights were obtained about Notch, transforming growth factor β/bone morphogenetic protein, Ras, mitogen-activated protein kinase, phosphatidylinositol 3 kinase, and Ca(2+)/calcineurin signaling pathways in the control of growth and remodeling of lymphatic vessels. An emerging picture of lymphangiogenic signaling is complex and in many ways distinct from the regulation of angiogenesis. This complexity provides new challenges, but also new opportunities for selective therapeutic targeting of lymphatic vasculature.

Ageing-related cardiomyocyte functional decline is sex and angiotensin II dependent.

Clinically, heart failure is an age-dependent pathological phenomenon and displays sex-specific characteristics. The renin-angiotensin system mediates cardiac pathology in heart failure. This study investigated the sexually dimorphic functional effects of ageing combined with angiotensin II (AngII) on cardiac muscle cell function, twitch and Ca(2+)-handling characteristics of isolated cardiomyocytes from young (~13 weeks) and aged (~87 weeks) adult wild type (WT) and AngII-transgenic (TG) mice. We hypothesised that AngII-induced contractile impairment would be exacerbated in aged female cardiomyocytes and linked to Ca(2+)-handling disturbances. AngII-induced cardiomyocyte hypertrophy was evident in young adult mice of both sexes and accentuated by age (aged adult ~21-23 % increases in cell length relative to WT). In female AngII-TG mice, ageing was associated with suppressed cardiomyocyte contractility (% shortening, maximum rate of shortening, maximum rate of relaxation). This was associated with delayed cytosolic Ca(2+) removal during twitch relaxation (Tau ~20 % increase relative to young adult female WT), and myofilament responsiveness to Ca(2+) was maintained. In contrast, aged AngII-TG male cardiomyocytes exhibited peak shortening equivalent to young TG; yet, myofilament Ca(2+) responsiveness was profoundly reduced with ageing. Increased pro-arrhythmogenic spontaneous activity was evident with age and cardiac AngII overexpression in male mice (42-55 % of myocytes) but relatively suppressed in female aged transgenic mice. Female myocytes with elevated AngII appear more susceptible to an age-related contractile deficit, whereas male AngII-TG myocytes preserve contractile function with age but exhibit desensitisation of myofilaments to Ca(2+) and a heightened vulnerability to arrhythmic activity. These findings support the contention that sex-specific therapies are required for the treatment of age-progressive heart failure.

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity.

Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors as Therapeutics: Rationales, Controversies, Clinical Experience.

Nicotinamide adenine dinucleotide (NAD+) biosynthesis from nicotinamide is used by mammalian cells to replenish their NAD+ stores and to avoid unwanted nicotinamide accumulation. Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in this biosynthetic pathway, almost invariably leads to intracellular NAD+ depletion and, when protracted, to ATP shortage and cell demise. Cancer cells and activated immune cells express high levels of NAMPT and are highly susceptible to NAMPT inhibitors, as shown by the activity of these agents in models of malignant and inflammatory disorders. As the spectrum of conditions which could benefit from pharmacological NAMPT inhibition becomes broader, the mechanisms accounting for their activity are also eventually becoming apparent, including the induction of autophagy and the impairment of Ca(2+) - and NF-κB-dependent signaling. Here, we discuss the rationales for exploiting NAMPT inhibitors in cancer and inflammatory diseases and provide an overview of the preclinical and clinical studies in which these agents have been evaluated.

Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.

Typically at dawn on a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to induce a timely heat shock response (HSR) and accumulate protective heat shock proteins in anticipation of harmful temperatures at mid-day. Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca(2+) channels that act as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to an HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild-type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused a hyper-thermoresponsive Ca(2+) influx and altered Ca(2+) signaling. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermoresponsive Ca(2+) channels in wild-type cells. Deletion of CNGCb led to a total absence of one and increased the open probability of the remaining two thermoresponsive Ca(2+) channels. Thus, CNGC2 and CNGCb are expected to form heteromeric Ca(2+) channels with other related CNGCs. These channels in the plasma membrane respond to increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance.

Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

Secretory IgA Induces Tolerogenic Dendritic Cells through SIGNR1 Dampening Autoimmunity in Mice.

IgA plays ambivalent roles in the immune system. The balance between inhibitory and activating responses relies on the multimerization status of IgA and interaction with their cognate receptors. In mucosal sites, secretory IgA (SIgA) protects the host through immune-exclusion mechanisms, but its function in the bloodstream remains unknown. Using bone marrow-derived dendritic cells, we found that both human and mouse SIgA induce tolerogenic dendritic cells (DCs) following binding to specific ICAM-3 grabbing nonintegrin receptor 1. This interaction was dependent on Ca(2+) and mannose residues. SIgA-primed DCs (SIgA-DCs) are resistant to TLR-dependent maturation. Although SIgA-DCs fail to induce efficient proliferation and Th1 differentiation of naive responder T cells, they generate the expansion of regulatory T cells through IL-10 production. SIgA-DCs are highly potent in inhibiting autoimmune responses in mouse models of type 1 diabetes and multiple sclerosis. This discovery may offer new insights about mucosal-derived DC immunoregulation through SIgA opening new therapeutic approaches to autoimmune diseases.

Protective role of amantadine in mitochondrial dysfunction and oxidative stress mediated by hepatitis C virus protein expression.

Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca(2+); (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15 min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization.

Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases.

Genetic analysis of fission yeast suggests a role for the spHop2-Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2-Mnd1 binds single-strand DNA ends of 3'-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca(2+) alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.

Duality of the murine CD8 compartment.

CD8αβ plays crucial roles in the thymic selection, differentiation, and activation of some, but not all, CD8(+) T cells, whereas CD8αα does not. To investigate these roles, we produced mice that expressed transgene P14 T-cell receptor β (TCRβ) chain and CD8β or did not (WT and KO mice, respectively). The primary CD8(+) T-cell response to acute lymphocytic choriomeningitis virus (LCMV) infection was predominantly D(b)/GP33 specific and CD8 independent in KO mice and was mostly CD8 dependent in WT mice. Cytotoxic T lymphocytes (CTL) from KO mice failed to mobilize intracellular Ca(2+) and to kill via perforin/granzyme. Their strong Fas/FasL-mediated cytotoxicity and IFN-γ response were signaled via a Ca(2+)-independent, PI3K-dependent pathway. This was also true for 15-20% of CD8-independent CTL found in WT mice. Conversely, the perforin/granzyme-mediated killing and IFN-γ response of CD8-dependent CTL were signaled via a Ca(2+), p56(lck), and nuclear factor of activated T cells-dependent pathway. Deep sequencing of millions of TCRα chain transcripts revealed that the TCR repertoires of preimmune CD8(+) T cells were highly diverse, but those of LCMV D(b)/GP33-specific CTL, especially from KO mice, were narrow. The immune repertoires exhibited biased use of Vα segments that encoded different complementary-determining region 1α (CDR1α) and CDR2α sequences. We suggest that TCR from WT CD8-independent T cells may engage MHC-peptide complexes in a manner unfavorable for efficient CD8 engagement and Ca(2+) signaling but permissive for Ca(2+)-independent, PI3K-dependent signaling. This duality of the CD8 compartment may provide organisms with broader protective immunity.

Astrocytic dysfunction: insights on the role in neurodegeneration.

For decades, astrocytes have been regarded as passive partners of neurons in central nervous system (CNS) function. Studies of the last 20 years, however, challenged this view by demonstrating that astrocytes possess functional receptors for neurotransmitters and respond to their stimulation via release of gliotransmitters, including glutamate. Notably, astrocytes react to synaptically released neurotransmitters with intracellular calcium ([Ca(2+)]) elevations, which result in the release of glutamate via regulated exocytosis and, possibly, other mechanisms. These findings have led to a new concept of neuron-glia intercommunication where astrocytes play an unsuspected dynamic role by integrating neuronal inputs and modulating synaptic activity. The additional observation that glutamate release from astrocytes is controlled by molecules linked to inflammatory reactions, such as the cytokine tumor necrosis factor alpha (TNFalpha) and prostaglandins (PGs), suggests that glia-to-neuron signalling may be sensitive to changes in the production of these mediators occurring in pathological conditions. Indeed, a local, parenchymal brain inflammatory reaction (neuroinflammation) characterized by astrocytic and microglial activation has been reported in several neurodegenerative disorders, including AIDS dementia complex, Alzheimer's disease and amyotrophic lateral sclerosis. This transition may be accompanied by functional de-regulation and even degeneration of the astrocytes with the consequent disruption of the cross-talk normally occurring between these cells and neurons. Incorrect neuron-astrocyte interactions may be involved in neuronal derangement and contribute to disease development. The findings reported in this review suggest that a better comprehension of the glutamatergic interplay between neurons and astrocytes may provide information about normal brain function and also highlight potential molecular targets for therapeutic interventions in pathology.

Spontaneous NA+ transients in individual mitochondria of intact astrocytes.

Mitochondria in intact cells maintain low Na(+) levels despite the large electrochemical gradient favoring cation influx into the matrix. In addition, they display individual spontaneous transient depolarizations. The authors report here that individual mitochondria in living astrocytes exhibit spontaneous increases in their Na(+) concentration (Na(mit)(+) spiking), as measured using the mitochondrial probe CoroNa Red. In a field of view with approximately 30 astrocytes, up to 1,400 transients per minute were typically detected under resting conditions. Na(mit)(+) spiking was also observed in neurons, but was scarce in two nonneural cell types tested. Astrocytic Na(mit)(+) spikes averaged 12.2 +/- 0.8 s in duration and 35.5 +/- 3.2 mM in amplitude and coincided with brief mitochondrial depolarizations; they were impaired by mitochondrial depolarization and ruthenium red pointing to the involvement of a cation uniporter. Na(mit)(+) spiking activity was significantly inhibited by mitochondrial Na(+)/H(+) exchanger inhibition and sensitive to cellular pH and Na(+) concentration. Ca(2+) played a permissive role on Na(mit)(+) spiking activity. Finally, the authors present evidence suggesting that Na(mit)(+) spiking frequency was correlated with cellular ATP levels. This study shows that, under physiological conditions, individual mitochondria in living astrocytes exhibit fast Na(+) exchange across their inner membrane, which reveals a new form of highly dynamic and localized functional regulation.

Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase.

The cellular response to an inflammatory stressor requires a proinflammatory cellular activation followed by a controlled resolution of the response to restore homeostasis. We hypothesized that biliverdin reductase (BVR) by binding biliverdin (BV) quells the cellular response to endotoxin-induced inflammation through phosphorylation of endothelial nitric oxide synthase (eNOS). The generated NO, in turn, nitrosylates BVR, leading to nuclear translocation where BVR binds to the Toll-like receptor-4 (TLR4) promoter at the Ap-1 sites to block transcription. We show in macrophages that BV-induced eNOS phosphorylation (Ser-1177) and NO production are mediated in part by Ca(2+)/calmodulin-dependent kinase kinase. Furthermore, we show that BVR is S-nitrosylated on one of three cysteines and that this posttranslational modification is required for BVR-mediated signaling. BV-induced nuclear translocation of BVR and inhibition of TLR4 expression is lost in macrophages derived from Enos(-/-) mice. In vivo in mice, BV provides protection from acute liver damage and is dependent on the availability of NO. Collectively, we elucidate a mechanism for BVR in regulating the inflammatory response to endotoxin that requires eNOS-derived NO and TLR4 signaling in macrophages.