The lysosomotrope GPN mobilises Ca2+ from acidic organelles.

Lysosomes are acidic Ca2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+ signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca2+ release. We conclude that Ca2+ signals evoked by GPN most likely derive from acidic organelles.

Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx.

Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca2+ release but their role in Ca2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca2+ release and Ca2+ entry. Ca2+ release required lysosomal and ER Ca2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca2+ channels, are capable of 'chatter' with ER Ca2+ channels. Our data identify new modalities for TRPML1 action.

Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.

Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease.

A computational model of lysosome-ER Ca2+ microdomains.

Acidic organelles form an important intracellular Ca(2+) pool that can drive global Ca(2+) signals through coupling with endoplasmic reticulum (ER) Ca(2+) stores. Recently identified lysosome-ER membrane contact sites might allow formation of Ca(2+) microdomains, although their size renders observation of Ca(2+) dynamics impractical. Here, we generated a computational model of lysosome-ER coupling that incorporated a previous model of the inositol trisphosphate (IP3) receptor as the ER Ca(2+) 'amplifier' and lysosomal leaks as the Ca(2+) 'trigger'. The model qualitatively described global Ca(2+) responses to the lysosomotropic agent GPN, which caused a controlled but substantial depletion of small solutes from the lysosome. Adapting this model to physiological lysosomal leaks induced by the Ca(2+) mobilising messenger NAADP demonstrated that lysosome-ER microdomains are capable of driving global Ca(2+) oscillations. Interestingly, our simulations suggest that the microdomain [Ca(2+)] need not be higher than that in the cytosol for responses to occur, thus matching the relatively high affinity of IP3 receptors for Ca(2+). The relative distribution and overall density of the lysosomal leaks dictated whether microdomains triggered or modulated global signals. Our data provide a computational framework for probing lysosome-ER Ca(2+) dynamics.

Direct mobilisation of lysosomal Ca2+ triggers complex Ca2+ signals.

Accumulating evidence implicates acidic organelles of the endolysosomal system as mobilisable stores of Ca(2+) but their relationship to the better-characterised endoplasmic reticulum (ER) Ca(2+) store remains unclear. Here we show that rapid osmotic permeabilisation of lysosomes evokes prolonged, spatiotemporally complex Ca(2+) signals in primary cultured human fibroblasts. These Ca(2+) signals comprised an initial response that correlated with lysosomal disruption and secondary long-lasting spatially heterogeneous Ca(2+) oscillations that required ER-localised inositol trisphosphate receptors. Electron microscopy identified extensive membrane contact sites between lysosomes and the ER. Mobilisation of lysosomal Ca(2+) stores is thus sufficient to evoke ER-dependent Ca(2+) release probably through lysosome-ER membrane contact sites, and akin to the proposed mechanism of action of the Ca(2+) mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Our data identify functional and physical association of discrete Ca(2+) stores important for the genesis of Ca(2+) signal complexity.

Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers.

Two-pore channels (TPCs) are Ca2+-permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P2-mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Two-pore channels and disease.

Two-pore channels (TPCs) are Ca2+-permeable endo-lysosomal ion channels subject to multi-modal regulation. They mediate their physiological effects through releasing Ca2+ from acidic organelles in response to cues such as the second messenger, NAADP. Here, we review emerging evidence linking TPCs to disease. We discuss how perturbing both local and global Ca2+ changes mediated by TPCs through chemical and/or molecular manipulations can induce or reverse disease phenotypes. We cover evidence from models of Parkinson's disease, non-alcoholic fatty liver disease, Ebola infection, cancer, cardiac dysfunction and diabetes. A need for more drugs targeting TPCs is identified.

An Endosomal NAADP-Sensitive Two-Pore Ca2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling.

Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.

Connecting Ca2+ and lysosomes to Parkinson disease.

The neurodegenerative movement disorder Parkinson disease (PD) is prevalent in the aged population. However, the underlying mechanisms that trigger disease are unclear. Increasing work implicates both impaired Ca2+ signalling and lysosomal dysfunction in neuronal demise. Here I aim to connect these distinct processes by exploring the evidence that lysosomal Ca2+ signalling is disrupted in PD. In particular, I highlight defects in lysosomal Ca2+ content and signalling through NAADP-regulated two-pore channels in patient fibroblasts harbouring mutations in the PD-linked genes, GBA1 and LRRK2. As an emerging contributor to PD pathogenesis, the lysosomal Ca2+ signalling apparatus could represent a novel therapeutic target.

Endoplasmic reticulum and lysosomal Ca²âº stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts.

Mutations in ß-glucocerebrosidase (encoded by GBA1) cause Gaucher disease (GD), a lysosomal storage disorder, and increase the risk of developing Parkinson disease (PD). The pathogenetic relationship between the two disorders is unclear. Here, we characterised Ca(2+) release in fibroblasts from type I GD and PD patients together with age-matched, asymptomatic carriers, all with the common N370S mutation in ß-glucocerebrosidase. We show that endoplasmic reticulum (ER) Ca(2+) release was potentiated in GD and PD patient fibroblasts but not in cells from asymptomatic carriers. ER Ca(2+) signalling was also potentiated in fibroblasts from aged healthy subjects relative to younger individuals but not further increased in aged PD patient cells. Chemical or molecular inhibition of ß-glucocerebrosidase in fibroblasts and a neuronal cell line did not affect ER Ca(2+) signalling suggesting defects are independent of enzymatic activity loss. Conversely, lysosomal Ca(2+) store content was reduced in PD fibroblasts and associated with age-dependent alterations in lysosomal morphology. Accelerated remodelling of Ca(2+) stores by pathogenic GBA1 mutations may therefore feature in PD.

Coupling acidic organelles with the ER through Ca²âº microdomains at membrane contact sites.

Acidic organelles such as lysosomes serve as non-canonical Ca(2+) stores. The Ca(2+) mobilising messenger NAADP is thought to trigger local Ca(2+) release from such stores. These events are then amplified by Ca(2+) channels on canonical ER Ca(2+) stores to generate physiologically relevant global Ca(2+) signals. Coupling likely occurs at microdomains formed at membrane contact sites between acidic organelles and the ER. Molecular analyses and computational modelling suggest heterogeneity in the composition of these contacts and predicted Ca(2+) microdomain behaviour. Conversely, acidic organelles might also locally amplify and temper ER-evoked Ca(2+) signals. Ca(2+) microdomains between distinct Ca(2+) stores are thus likely to be integral to the genesis of complex Ca(2+) signals.

Methods for monitoring lysosomal morphology.

Lysosomes are abundant organelles best known for their crucial role in macromolecule turnover. Lysosome dysfunction features in several diseases exemplified by the lysosomal storage disorders and is often associated with marked changes in lysosome structure. Lysosomal morphology may therefore serve as a sensitive readout of endocytic well-being. Here we describe methods for monitoring lysosome morphology in fixed and live cells using fluorescent probes and electron microscopy.

A "mix-and-match" approach to designing Ca(2+) microdomains at membrane-contact sites.

Ca(2+) microdomains are critical for regulating cellular activity and often form at membrane contact sites. Such sites between lysosomes and the ER potentially provide a platform for signaling by the Ca(2+) mobilizing messenger NAADP. However, at present we know little of how Ca(2+) release events are coordinated at these experimentally intractable junctions. We therefore developed a computational model of lysosome-ER microdomains, which suggested that small leaks of Ca(2+) from the lysosome couple to Ca(2+)-sensitive Ins(1,4,5)P 3 receptors on the ER to generate global, microdomain-dependent Ca(2+) signals. Here we discuss how the "mix-and-match" arrangement of different Ca(2+) signaling proteins on the "source" and "target" membranes might generate functionally heterogeneous Ca(2+) microdomains.