Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling.

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation "two-pore channel" (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2-/-) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2-/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Low Temperature Delays the Effects of Ischemia in Bergmann Glia and in Cerebellar Tissue Swelling.

Cerebral ischemia results in oxygen and glucose deprivation that most commonly occurs after a reduction or interruption in the blood supply to the brain. The consequences of cerebral ischemia are complex and involve the loss of metabolic ATP, excessive K+ and glutamate accumulation in the extracellular space, electrolyte imbalance, and brain edema formation. So far, several treatments have been proposed to alleviate ischemic damage, yet few are effective. Here, we focused on the neuroprotective role of lowering the temperature in ischemia mimicked by an episode of oxygen and glucose deprivation (OGD) in mouse cerebellar slices. Our results suggest that lowering the temperature of the extracellular 'milieu' delays both the increases in [K+]e and tissue swelling, two dreaded consequences of cerebellar ischemia. Moreover, radial glial cells (Bergmann glia) display morphological changes and membrane depolarizations that are markedly impeded by lowering the temperature. Overall, in this model of cerebellar ischemia, hypothermia reduces the deleterious homeostatic changes regulated by Bergmann glia.

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion.

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca2+ influx, but is also dependent on Ca2+ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca2+-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca2+ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

Unexpected Motherhood-Triggered Hearing Loss in the Two-Pore Channel (TPC) Mutant Mouse.

Calcium signaling is crucial for many physiological processes and can mobilize intracellular calcium stores in response to environmental sensory stimuli. The endolysosomal two-pore channel (TPC), regulated by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), is one of the key components in calcium signaling. However, its role in neuronal physiology remains largely unknown. Here, we investigated to what extent the acoustic thresholds differed between the WT mice and the TPC KO mice. We determined the thresholds based on the auditory brainstem responses (ABRs) at five frequencies (between 4 and 32 kHz) and found no threshold difference between the WT and KO in virgin female mice. Surprisingly, in lactating mothers (at P9-P10), the thresholds were higher from 8 to 32 kHz in the TPC KO mice compared to the WT mice. This result indicates that in the TPC KO mice, physiological events occurring during parturition altered the detection of sounds already at the brainstem level, or even earlier.

Neurophysiological functions and pharmacological tools of acidic and non-acidic Ca2+ stores.

Ca2+ signalling is of prime importance in controlling numerous cell functions in the brain. Endolysosomes are acidic organelles currently emerging as important Ca2+ stores in astrocytes, microglia, endothelial cells, and neurons. In neurons, these acidic Ca2+ stores are found in axons, soma, dendrites, and axon endings and could provide local sources of Ca2+ to control synaptic transmission, neuronal plasticity, and autophagy to name a few. This review will address how acidic Ca2+ stores are recruited in response to cell stimulation. We will focus on the role of the endolysosomal two-pore channels (TPCs) and their physiological agonist nicotinic acid adenine dinucleotide phosphate (NAADP) and how they interact with cyclic ADP-ribose and ryanodine receptors from the endoplasmic reticulum. Finally, this review will describe new pharmacological tools and animal mutant models now available to explore acidic Ca2+ stores as key elements in brain function and dysfunction.

CD38-NADase is a new major contributor to Duchenne muscular dystrophic phenotype.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration. Two important deleterious features are a Ca2+ dysregulation linked to Ca2+ influxes associated with ryanodine receptor hyperactivation, and a muscular nicotinamide adenine dinucleotide (NAD+ ) deficit. Here, we identified that deletion in mdx mice of CD38, a NAD+ glycohydrolase-producing modulators of Ca2+ signaling, led to a fully restored heart function and structure, with skeletal muscle performance improvements, associated with a reduction in inflammation and senescence markers. Muscle NAD+ levels were also fully restored, while the levels of the two main products of CD38, nicotinamide and ADP-ribose, were reduced, in heart, diaphragm, and limb. In cardiomyocytes from mdx/CD38-/- mice, the pathological spontaneous Ca2+ activity was reduced, as well as in myotubes from DMD patients treated with isatuximab (SARCLISA® ) a monoclonal anti-CD38 antibody. Finally, treatment of mdx and utrophin-dystrophin-deficient (mdx/utr-/- ) mice with CD38 inhibitors resulted in improved skeletal muscle performances. Thus, we demonstrate that CD38 actively contributes to DMD physiopathology. We propose that a selective anti-CD38 therapeutic intervention could be highly relevant to develop for DMD patients.

Enhanced Discriminative Abilities of Auditory Cortex Neurons for Pup Calls Despite Reduced Evoked Responses in C57BL/6 Mother Mice.

A fundamental task for the auditory system is to process communication sounds according to their behavioral significance. In many mammalian species, pup calls became more significant for mothers than other conspecific and heterospecific communication sounds. To study the cortical consequences of motherhood on the processing of communication sounds, we recorded neuronal responses in the primary auditory cortex of virgin and mother C57BL/6 mice which had similar ABR thresholds. In mothers, the evoked firing rate in response to pure tones was decreased and the frequency receptive fields were narrower. The responses to pup and adult calls were also reduced but the amount of mutual information (MI) per spike about the pup call's identity was increased in mother mice. The response latency to pup and adult calls was significantly shorter in mothers. Despite similarly decreased responses to guinea pig whistles, the response latency, and the MI per spike did not differ between virgins and mothers for these heterospecific vocalizations. Noise correlations between cortical recordings were decreased in mothers, suggesting that the firing rate of distant neurons was more independent from each other. Together, these results indicate that in the most commonly used mouse strain for behavioral studies, the discrimination of pup calls by auditory cortex neurons is more efficient during motherhood.

A multiscale analysis in CD38-/- mice unveils major prefrontal cortex dysfunctions.

Autism spectrum disorder (ASD) is characterized by early onset of behavioral and cognitive alterations. Low plasma levels of oxytocin (OT) have also been found in ASD patients; recently, a critical role for the enzyme CD38 in the regulation of OT release was demonstrated. CD38 is important in regulating several Ca2+-dependent pathways, but beyond its role in regulating OT secretion, it is not known whether a deficit in CD38 expression leads to functional modifications of the prefrontal cortex (PFC), a structure involved in social behavior. Here, we report that CD38-/- male mice show an abnormal cortex development, an excitation-inhibition balance shifted toward a higher excitation, and impaired synaptic plasticity in the PFC such as those observed in various mouse models of ASD. We also show that a lack of CD38 alters social behavior and emotional responses. Finally, examining neuromodulators known to control behavioral flexibility, we found elevated monoamine levels in the PFC of CD38-/- adult mice. Overall, our study unveiled major changes in PFC physiologic mechanisms and provides new evidence that the CD38-/- mouse could be a relevant model to study pathophysiological brain mechanisms of mental disorders such as ASD.-Martucci, L. L., Amar, M., Chaussenot, R., Benet, G., Bauer, O., de Zélicourt, A., Nosjean, A., Launay, J.-M., Callebert, J., Sebrié, C., Galione, A., Edeline, J.-M., de la Porte, S., Fossier, P., Granon, S., Vaillend, C., Cancela, J.-M., A multiscale analysis in CD38-/- mice unveils major prefrontal cortex dysfunctions.

Effect of dopaminergic D1 receptors on plasticity is dependent of serotoninergic 5-HT1A receptors in L5-pyramidal neurons of the prefrontal cortex.

Major depression and schizophrenia are associated with dysfunctions of serotoninergic and dopaminergic systems mainly in the prefrontal cortex (PFC). Both serotonin and dopamine are known to modulate synaptic plasticity. 5-HT1A receptors (5-HT1ARs) and dopaminergic type D1 receptors are highly represented on dendritic spines of layer 5 pyramidal neurons (L5PyNs) in PFC. How these receptors interact to tune plasticity is poorly understood. Here we show that D1-like receptors (D1Rs) activation requires functional 5HT1ARs to facilitate LTP induction at the expense of LTD. Using 129/Sv and 5-HT1AR-KO mice, we recorded post-synaptic currents evoked by electrical stimulation in layer 2/3 after activation or inhibition of D1Rs. High frequency stimulation resulted in the induction of LTP, LTD or no plasticity. The D1 agonist markedly enhanced the NMDA current in 129/Sv mice and the percentage of L5PyNs displaying LTP was enhanced whereas LTD was reduced. In 5-HT1AR-KO mice, the D1 agonist failed to increase the NMDA current and orientated the plasticity towards L5PyNs displaying LTD, thus revealing a prominent role of 5-HT1ARs in dopamine-induced modulation of plasticity. Our data suggest that in pathological situation where 5-HT1ARs expression varies, dopaminergic treatment used for its ability to increase LTP could turn to be less and less effective.

Low doses of arginine butyrate derivatives improve dystrophic phenotype and restore membrane integrity in DMD models.

A new approach to treating Duchenne muscular dystrophy was investigated by using the ester or amide covalent association of arginine [nitric oxide (NO) pathway] and butyrate [histone deacetylase (HDAC) inhibition] in mdx mice and patient myotubes. Two prodrugs were synthesized, and the beneficial effects on dystrophic phenotype were studied. Nerve excitability abnormalities detected in saline-treated mice were almost totally rescued in animals treated at low doses (50-100 mg/kg/d). Force and fatigue resistance were improved ≈60% and 3.5-fold, respectively, and the percentage of necrosis in heart sections was reduced ≈90% in the treated mice. A decrease of >50% in serum creatine kinase indicated an overall improvement in the muscles. Restoration of membrane integrity was studied directly by measuring the reduction (≈74%) of Evans blue incorporation in the limb muscles of the treated animals, the increase in utrophin level, and the normalization of lipid composition of the heart. In cultures of human myotubes (primary cells and cell line), both prodrugs and HDAC inhibitors increased by 2- to 4-fold the utrophin level, which was correctly localized at the membrane. ß-Dystroglycan and embryonic myosin protein levels were also increased. Finally, a 50% reduction in the number of spontaneous Ca(2+) spikes was observed after treatment with NO synthase substrate and HDAC inhibitors. Overall, the beneficial effects were obtained with doses 10 (in vivo) and 5 (in vitro) times lower than those of the salt formulation. Altogether, these data constitute proof of principle of the beneficial effects of low doses of arginine butyrate derivatives on muscular dystrophy, enhancing the NO pathway and inhibiting HDAC.

The ecto-enzyme CD38 is a nicotinic acid adenine dinucleotide phosphate (NAADP) synthase that couples receptor activation to Ca2+ mobilization from lysosomes in pancreatic acinar cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ecto-enzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca(2+) signals by discharging acidic Ca(2+) stores and leading to digestive enzyme secretion. From cells derived from CD38(-/-) mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca(2+)-signaling pathways in these cells by restoring Ca(2+) mobilization from lysosomes during CCK-induced Ca(2+) signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not co-localized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca(2+) release from lysosomal stores in pancreatic acinar cells.

Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization.

In neurons, voltage-gated Ca(2+) channels and nuclear Ca(2+) signaling play important roles, such as in the regulation of gene expression. However, the link between electrical activity and biochemical cascade activation involved in the generation of the nuclear Ca(2+) signaling is poorly understood. Here we show that depolarization of Aplysia neurons induces the translocation of ADP-ribosyl cyclase, a Ca(2+) messenger synthesizing enzyme, from the cytosol into the nucleus. The translocation is dependent on Ca(2+) influx mainly through the voltage-dependent L-type Ca(2+) channels. We report also that specific nucleoplasmic Ca(2+) signals can be induced by three different calcium messengers, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), both produced by the ADP-ribosyl cyclase, and inositol 1,4,5-trisphosphate (IP(3)). Moreover, our pharmacological data show that NAADP acts on its own receptor, which cooperates with the IP(3) and the ryanodine receptors to generate nucleoplasmic Ca(2+) oscillations. We propose a new model where voltage-dependent L-type Ca(2+) channel-induced nuclear translocation of the cytosolic cyclase is a crucial step in the fine tuning of nuclear Ca(2+) signals in neurons.

Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers.

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.

Brain mitochondrial defects amplify intracellular [Ca2+] rise and neurodegeneration but not Ca2+ entry during NMDA receptor activation.

According to the "indirect" excitotoxicity hypothesis, mitochondrial defects increase Ca2+ entry into neurons by rendering NMDA-R hypersensitive to glutamate. We tested this hypothesis by investigating in the rat striatum and cultured striatal cells how partial mitochondrial complex II inhibition produced by 3-nitropropionic acid (3NP) modifies the toxicity of the NMDA-R agonist quinolinate (QA). We showed that nontoxic 3NP treatment, leading to partial inhibition of complex II activity, greatly exacerbated striatal degeneration produced by slightly toxic QA treatment through an "all-or-nothing" process. The potentiation of QA-induced cell death by 3NP was associated with increased calpain activity and massive calpain-mediated cleavage of several postsynaptic proteins, suggesting major neuronal Ca2+ deregulation in the striatum. However, Ca2+ anomalies probably do not result from NMDA-R hypersensitivity. Indeed, brain imaging experiments using [(18)F]fluorodeoxyglucose indirectly showed that 3NP did not increase QA-induced ionic perturbations at the striatal glutamatergic synapses in vivo. Consistent with this, the exacerbation of QA toxicity by 3NP was not related to an increase in the QA-induced entry of 45Ca2+ into striatal neurons. The present results demonstrate that the potentiation of NMDA-R-mediated excitotoxicity by mitochondrial defects involves primarily intracellular Ca2+ deregulation, in the absence of NMDA-R hypersensitivity.

The Ca2+-releasing messenger NAADP, a new player in the nervous system.

Many physiological processes are controlled by a great diversity of Ca2+ signals. Within cell, Ca2+ signals depend upon Ca2+ entry and/or Ca2+ release from internal Ca2+ stores. The control of Ca2+-store mobilization is ensured by a family of messengers comprising inositol 1,4,5 trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). From recent works, new concepts have emerged where activation of the cells by outside stimuli, acting at the plasma membrane, results in the synthesis of multiple Ca2+-releasing messengers which may interact and shape complex Ca2+ signals in the cytosol as well as in the nucleus. This contribution will cover the most recent advances on NAADP signalling with some emphasis on neurons.

Role of NAADP and cADPR in the induction and maintenance of agonist-evoked Ca2+ spiking in mouse pancreatic acinar cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.