John Parrington

Lysosomal two-pore channel subtype 2 (TPC2) regulates skeletal muscle autophagic signaling

Background: The endolysosomal TPC2 ion channel interacts with mTOR to regulate cellular energy utilization. Results: Mice lacking TPC2 display muscle atrophy phenotype with reduced muscle endurance, altered autophagy, and lysosomal enzymatic activities. Conclusion: TPC2 regulates autophagic signaling in skeletal muscle. Significance: TPC2 impacts protein turnover via regulating autophagy signaling in the process of tissue homeostasis and aging. Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal two-pore channel subtype 2 (TPC2) contributes to autophagy signaling and protein homeostasis in skeletal muscle. Muscles derived from Tpcn2−/− mice exhibit an atrophic phenotype with exacerbated autophagy under starvation. Compared with wild types, animals lacking TPC2 demonstrated an enhanced autophagy flux characterized by increased accumulation of autophagosomes upon combined stress induction by starvation and colchicine treatment. In addition, deletion of TPC2 in muscle caused aberrant lysosomal pH homeostasis and reduced lysosomal protease activity. Association between mammalian target of rapamycin and TPC2 was detected in skeletal muscle, allowing for appropriate adjustments to cellular metabolic states and subsequent execution of autophagy. TPC2 therefore impacts mammalian target of rapamycin reactivation during the process of autophagy and contributes to maintenance of muscle homeostasis.

PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

ABSTRACT Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1–/– male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1–/– males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility. Highlighted article: In mice, PLCζ-null sperm fail to trigger the Ca2+ oscillations in eggs that normally initiate embryogenesis in mammals, resulting in polyspermy and subfertility but not sterility.

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart.

Background: The Ca2+-releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) acts via lysosomal two-pore channels (TPC2). Results: Tpcn2−/− cardiac myocytes showed reduced acute responses to β-adrenoreceptor stimulation and chronically reduced cardiac hypertrophy and arrhythmogenesis. Conclusion: Acute and chronic effects of cardiac β-adrenoreceptor stimulation depend on NAADP acting via TPC2 in lysosomes. Significance: NAADP/TPC2 signaling pathways offer new strategies for cardiac therapeutics. Ca2+-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca2+ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca2+ responses in cardiac myocytes from Tpcn2−/− mice, unlike myocytes from wild-type (WT) mice. Ca2+/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca2+ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2−/− mice. Increases in amplitude of L-type Ca2+ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca2+-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.

Endolysosomal two‐pore channels regulate autophagy in cardiomyocytes

Two‐pore channels (TPCs) were identified as a novel family of endolysosome‐targeted calcium release channels gated by nicotinic acid adenine dinucleotide phosphate, as also as intracellular Na+ channels able to control endolysosomal fusion, a key process in autophagic flux. Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure. We report direct evidence indicating that TPCs are involved in regulating autophagy in cardiomyocytes, and that TPC knockout mice show alterations in the cardiac lysosomal system. TPC downregulation implies a decrease in the viability of cardiomyocytes under starvation conditions. In cardiac tissues from both humans and rats, TPC transcripts and protein levels were higher in females than in males, and correlated negatively with markers of autophagy. We conclude that the endolysosomal channels TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes, and also that they are differentially expressed in male and female hearts.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells

Background: TPCs are regulated by NAADP and other factors. Results: NAADP-induced Ca2+ release from acidic stores evokes depolarizing currents in pancreatic β cells. Inhibition of NAADP signaling or TPC knock out attenuates Ca2+ signaling and insulin secretion. Conclusion: NAADP-evoked Ca2+ release enhances β cell excitability and insulin secretion in response to glucose or sulfonylureas. Significance: NAADP signaling pathways offer novel therapeutic targets for diabetes treatment. Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells.

Absence of Intracellular Ion Channels TPC1 and TPC2 Leads to Mature-Onset Obesity in Male Mice, Due to Impaired Lipid Availability for Thermogenesis in Brown Adipose Tissue

Intracellular calcium-permeable channels have been implicated in thermogenic function of murine brown and brite/beige adipocytes, respectively transient receptor potential melastin-8 and transient receptor potential vanilloid-4. Because the endo-lysosomal two-pore channels (TPCs) have also been ascribed with metabolic functionality, we studied the effect of simultaneously knocking out TPC1 and TPC2 on body composition and energy balance in male mice fed a chow diet. Compared with wild-type mice, TPC1 and TPC2 double knockout (Tpcn1/2(-/-)) animals had a higher respiratory quotient and became obese between 6 and 9 months of age. Although food intake was unaltered, interscapular brown adipose tissue (BAT) maximal temperature and lean-mass adjusted oxygen consumption were lower in Tpcn1/2(-/-) than in wild type mice. Phosphorylated hormone-sensitive lipase expression, lipid density and expression of β-adrenergic receptors were also lower in Tpcn1/2(-/-) BAT, whereas mitochondrial respiratory chain function and uncoupling protein-1 expression remained intact. We conclude that Tpcn1/2(-/-) mice show mature-onset obesity due to reduced lipid availability and use, and a defect in β-adrenergic receptor signaling, leading to impaired thermogenic activity, in BAT.

The two pore channel TPC2 is dispensable in pancreatic β-cells for normal Ca²⁺ dynamics and insulin secretion.

Graphical abstract

Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca(2+) signaling during myogenesis in intact zebrafish.

We have recently characterized essential inositol 1,4,5-trisphosphate receptor (IP 3R) and ryanodine receptor (RyR)-mediated Ca(2+) signals generated during the differentiation of slow muscle cells (SMCs) in intact zebrafish embryos. Here, we show that the lysosomal two-pore channel 2 (TPC2) also plays a crucial role in generating, and perhaps triggering, these essential Ca(2+) signals, and thus contributes to the regulation of skeletal muscle myogenesis. We used a transgenic line of zebrafish that expresses the bioluminescent Ca(2+) reporter, aequorin, specifically in skeletal muscle, in conjunction with morpholino (MO)-based and pharmacological inhibition of TPC2, in both intact embryos and isolated SMCs. MO-based knock-down of TPC2 resulted in a dramatic attenuation of the Ca(2+) signals, whereas the introduction of TPCN2-MO and TPCN2 mRNA together partially rescued the Ca(2+) signaling signature. Embryos treated with trans-ned-19 or bafilomycin A1, a specific NAADP receptor inhibitor and vacuolar-type H(+)ATPase inhibitor, respectively, also displayed a similar disruption of SMC Ca(2+) signaling. TPC2 and lysosomes were shown via immunohistochemistry and confocal laser scanning microscopy to be localized in perinuclear and striated cytoplasmic domains of SMCs, coincident with patterns of IP 3R and RyR expression. These data together imply that TPC2-mediated Ca(2+) release from lysosomes acts upstream from RyR- and IP 3R-mediated Ca(2+) release, suggesting that the former might act as a sensitive trigger to initiate the SR-mediated Ca(2+)-induced-Ca(2+)-release essential for SMC myogenesis and function.

Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in β-adrenoceptor signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation–contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro. However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38−/− mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38−/− myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of β-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38−/− myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38−/− mice. Whole hearts isolated from CD38−/− mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of β-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Calcium signals regulated by NAADP and two-pore channels - their role in development, differentiation and cancer

Ca(2+) signals regulate a wide range of physiological processes. Intracellular Ca(2+) stores can be mobilized in response to extracellular stimuli via a range of signal transduction mechanisms, often involving recruitment of diffusible second messenger molecules. The Ca(2+) mobilizing messengers InsP 3 and cADPR release Ca(2+) from the endoplasmic reticulum via InsP 3 and ryanodine receptors, respectively, while a third messenger, NAADP, releases Ca(2+) from acidic endosomes and lysosomes. Bidirectional communication between the ER and acidic organelles has functional relevance for endolysosomal function as well as for the generation of Ca(2+) signals. The two-pore channels (TPCs) are currently strong candidates for being key components of NAADP-regulated Ca(2+) channels. Ca(2+) signals have been shown to play important roles in embryonic development and cell differentiation; however, much remains to be established about the exact signalling mechanisms involved. Investigation of the role of NAADP and TPCs in development and differentiation is still at an early stage, but recent studies have suggested that they play important roles at key developmental stages in vivo and are important mediators of differentiation of neurons, skeletal muscle cells and osteoclasts in vitro. NAADP signals and TPCs have also been implicated in autophagy, an important process in differentiation. Moreover, potential links between TPC2 and cancer have been recently identified. Further studies will be required to identify the precise mechanisms of action of TPCs and their link with NAADP signalling, and to relate these to their roles in differentiation and other key developmental processes in the cell and organism.