Grant C. Churchill

Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium.

Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the acidic compartment (which we define as the late endosome and the lysosome) protein, NPC1. The function of NPC1 is unknown, but when it is dysfunctional, sphingosine, glycosphingolipids, sphingomyelin and cholesterol accumulate. We have found that NPC1-mutant cells have a large reduction in the acidic compartment calcium store compared to wild-type cells. Chelating luminal endocytic calcium in normal cells with high-affinity Rhod-dextran induced an NPC disease cellular phenotype. In a drug-induced NPC disease cellular model, sphingosine storage in the acidic compartment led to calcium depletion in these organelles, which then resulted in cholesterol, sphingomyelin and glycosphingolipid storage in these compartments. Sphingosine storage is therefore an initiating factor in NPC1 disease pathogenesis that causes altered calcium homeostasis, leading to the secondary storage of sphingolipids and cholesterol. This unique calcium phenotype represents a new target for therapeutic intervention, as elevation of cytosolic calcium with curcumin normalized NPC1 disease cellular phenotypes and prolonged survival of the NPC1 mouse.

NAADP Mobilizes Ca2+ from Reserve Granules, Lysosome-Related Organelles, in Sea Urchin Eggs

Abstract Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca 2+ in many cells and species. Unlike other Ca 2+ -mobilizing messengers, NAADP mobilizes Ca 2+ from an unknown store that is not the endoplasmic reticulum, the store traditionally associated with messenger-mediated Ca 2+ signaling. Here, we demonstrate the presence of a Ca 2+ store in sea urchin eggs mobilized by NAADP that is dependent on a proton gradient maintained by an ATP-dependent vacuolar-type proton pump. Moreover, we provide pharmacological and biochemical evidence that this Ca 2+ store is the reserve granule, the functional equivalent of a lysosome in the sea urchin egg. These findings represent an unsuspected mechanism for messenger-mediated Ca 2+ release from lysosome-related organelles.

Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells

Many hormones and neurotransmitters evoke Ca2+ release fromintracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3)1 and cyclic adenosine 5′-diphosphate-ribose (cADPR), are established Ca2+-mobilizing messengers that activate Ca2+ release through intracellular InsP3 and ryanodine receptors, respectively. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca2+ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca2+-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP),, an endogenous metabolite of β-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1–2 s) and long-lasting (0.2–1 min) Ca2+ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP3, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca2+ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.

Identification of a chemical probe for NAADP by virtual screening

Research into the biological role of the Ca2+-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with 3D-shape and electrostatic similarity. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca2+ increases in mouse pancreatic beta cells.

Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson’s disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-β (CaMKK-β)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets.

NAADP induces Ca2+ oscillations via a two‐pool mechanism by priming IP3‐ and cADPR‐sensitive Ca2+ stores

In sea urchin eggs, Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) potently self‐inactivates but paradoxically induces long‐term Ca2+ oscillations. We investigated whether NAADP‐induced Ca2+ oscillations arise from the recruitment of other Ca2+ release pathways. NAADP, inositol trisphosphate (IP3) and cyclic ADP‐ribose (cADPR) all mobilized Ca2+ from internal stores but only NAADP consistently induced Ca2+ oscillations. NAADP‐induced Ca2+ oscillations were partially inhibited by heparin or 8‐amino‐cADPR alone, but eliminated by the presence of both, indicating a requirement for both IP3‐ and cADPR‐dependent Ca2+ release. Thapsigargin completely blocked IP3 and cADPR responses as well as NAADP‐induced Ca2+ oscillations, but only reduced the NAADP‐mediated Ca2+ transient. Following NAADP‐mediated release from this Ca2+ pool, the amount of Ca2+ in the Ca2+‐induced Ca2+ release stores was increased. These results support a mechanism in which Ca2+ oscillations are initiated by Ca2+ release from NAADP‐sensitive Ca2+ stores (pool 1) and perpetuated through cycles of Ca2+ uptake into and release from Ca2+‐induced Ca2+ release stores (pool 2). These results provide the first direct evidence in support of a two‐pool model for Ca2+ oscillations.

NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.

Important questions remain concerning how elevated blood glucose levels are coupled to insulin secretion from pancreatic beta cells and how this process is impaired in type 2 diabetes. Glucose uptake and metabolism in beta cells cause the intracellular Ca(2+) concentration ([Ca(2+)](i)) to increase to a degree necessary and sufficient for triggering insulin release. Although both Ca(2+) influx and Ca(2+) release from internal stores are critical, the roles of inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine dinucleotide phosphate ribose (cADPR) in regulating the latter have proven equivocal. Here we show that glucose also increases [Ca(2+)](i) via the novel Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP) in the insulin-secreting beta-cell line MIN6. NAADP binds to specific, high-affinity membrane binding sites and at low concentrations elicits robust Ca(2+) responses in intact cells. Higher concentrations of NAADP inactivate NAADP receptors and attenuate the glucose-induced Ca(2+) increases. Importantly, glucose stimulation increases endogenous NAADP levels, providing strong evidence for recruitment of this pathway. In conclusion, our results support a model in which NAADP mediates glucose-induced Ca(2+) signaling in pancreatic beta cells and are the first demonstration in mammalian cells of the presence of endogenous NAADP levels that can be regulated by a physiological stimulus.

Purified TPC Isoforms Form NAADP Receptors with Distinct Roles for Ca2+ Signaling and Endolysosomal Trafficking

Summary Intracellular Ca2+ signals constitute key elements in signal transduction. Of the three major Ca2+ mobilizing messengers described, the most potent, nicotinic acid adenine dinucleotide phosphate (NAADP) is the least well understood in terms of its molecular targets [1]. Recently, we showed that heterologous expression of two-pore channel (TPC) proteins enhances NAADP-induced Ca2+ release, whereas the NAADP response was abolished in pancreatic beta cells from Tpcn2 gene knockout mice [2]. However, whether TPCs constitute native NAADP receptors is unclear. Here we show that immunopurified endogenous TPC complexes possess the hallmark properties ascribed to NAADP receptors, including nanomolar ligand affinity [3–5]. Our study also reveals important functional differences between the three TPC isoforms. Thus, TPC1 and TPC2 both mediate NAADP-induced Ca2+ release, but the subsequent amplification of this trigger Ca2+ by IP3Rs is more tightly coupled for TPC2. In contrast, TPC3 expression suppressed NAADP-induced Ca2+ release. Finally, increased TPC expression has dramatic and contrasting effects on endolysosomal structures and dynamics, implicating a role for NAADP in the regulation of vesicular trafficking. We propose that NAADP regulates endolysosomal Ca2+ storage and release via TPCs and coordinates endoplasmic reticulum Ca2+ release in a role that impacts on Ca2+ signaling in health and disease [6].

Sperm Deliver a New Second Messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.

Interactions between calcium release pathways: multiple messengers and multiple stores.

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles. In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.