Structural basis for inhibition of the lysosomal two-pore channel TPC2 by a small molecule antagonist.

Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.

P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2.

Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Lysosomal proteomics reveals mechanisms of neuronal apoE4associated lysosomal dysfunction.

ApoE4 is the primary risk factor for Alzheimer's Disease. While apoE is primarily expressed by astrocytes, AD pathology including endosomal abnormalities and mitochondrial dysfunction first occurs in neurons. Lysosomes are poised at the convergence point between these features. We find that apoE4-expressing cells exhibit lysosomal alkalinization, reduced lysosomal proteolysis, and impaired mitophagy. To identify driving factors for this lysosomal dysfunction, we performed quantitative lysosomal proteome profiling. This revealed that apoE4 expression results in lysosomal depletion of Lgals3bp and accumulation of Tmed5 in both Neuro-2a cells and postmitotic human neurons. Modulating the expression of both proteins affected lysosomal function, with Tmed5 knockdown rescuing lysosomal alkalinization in apoE4 cells, and Lgals3bp knockdown causing lysosomal alkalinization and reduced lysosomal density in apoE3 cells. Taken together, our work reveals that apoE4 exerts gain-of-toxicity by alkalinizing the lysosomal lumen, pinpointing lysosomal Tmed5 accumulation and Lgals3bp depletion as apoE4-associated drivers for this phenotype.

TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1, and Batten disease.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.

Endolysosomal cation channels point the way towards precision medicine of cancer and infectious diseases.

Infectious diseases and cancer are among the key medical challenges that humankind is facing today. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of both groups of diseases. The development of advanced patch-clamp technologies has allowed us to directly characterize ion fluxes through endolysosomal ion channels in their native environments. Endolysosomes are essential organelles for intracellular transport, digestion and metabolism, and maintenance of homeostasis. The endolysosomal ion channels regulate the function of the endolysosomal system through four basic mechanisms: calcium release, control of membrane potential, pH change, and osmolarity regulation. In this review, we put particular emphasis on the endolysosomal cation channels, including TPC2 and TRPML2, which are particularly important in monocyte function. We discuss existing endogenous and synthetic ligands of these channels and summarize current knowledge of their impact on channel activity and function in different cell types. Moreover, we summarize recent findings on the importance of TPC2 and TRPML2 channels as potential drug targets for the prevention and treatment of the emerging infectious diseases and cancer.

TRPMLs and TPCs: Targets for lysosomal storage and neurodegenerative disease therapy?

Neurodegenerative diseases (ND) pose a serious health burden to society and healthcare systems alike, with increasing incidence rates especially within aging populations. Alzheimer's disease (AD) is the most prevalent type of ND or dementia, followed by Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. Progressive neurological dysfunction and regional neuronal loss constitute the common characteristics of ND. Many ND are accompanied by accumulation of protein aggregates such as extracellular amyloid-ß (in AD), intraneuronal hyper-phosphorylated tau (in AD), or α-synuclein (in PD). Two main systems are responsible for the clearance of damaged, dysfunctional or senescent proteins inside cells: the autophagy-lysosomal pathway and the ubiquitin-proteasome system. The importance of lysosomes in neurodegenerative processes is further highlighted by clinical phenotypes of lysosomal storage disorders (LSDs), comprising more than 70 inheritable diseases caused by mutations in lysosomal enzymes or lysosomal membrane proteins, often resulting in severe neurodegeneration. Dysfunctional lysosomal proteins and enzymes result in the lysosomal accumulation of undigested macromolecules, e.g. lipids, glycoproteins, glycosaminoglycans, or gangliosides. Defects in intracellular transport pathways involving endosomes and lysosomes are increasingly recognized as drivers of neurodegenerative disease pathology including AD and PD. Thus, accumulation of damaged proteins and organelles (e.g. mitochondria) in neurons and glial cells overwhelms the capacity of intracellular recycling and degradation mechanisms, exacerbating disease pathology. Endolysosomal ion channels have recently been established as important regulators of lysosomal exocytosis, ion homeostasis/pH, endolysosomal trafficking, fusion and fission, and autophagy. In particular two non-selective endolysosomal cation channel families, the mucolipin/TRPML/MCOLN channels and the two-pore channels/TPCs will be discussed here as potential pharmacological targets for LSD/ND treatment.

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.

JPT2: The missing link between intracellular Ca2+ release channels and NAADP?

NAADP (nicotinic acid adenine dinucleotide phosphate) is a potent second messenger releasing Ca2+ from endolysosomes through two-pore channels (TPCs) and from the endoplasmic reticulum (ER) through type 1 ryanodine receptors (RyR1). How NAADP activates these channels, whether directly or indirectly, has been a matter of debate for more than a decade. Now a protein has emerged possibly providing the missing link between TPCs/RyR1 and NAADP.

Human genome diversity data reveal that L564P is the predominant TPC2 variant and a prerequisite for the blond hair associated M484L gain-of-function effect.

The endo-lysosomal two-pore channel (TPC2) has been established as an intracellular cation channel of significant physiological and pathophysiological relevance in recent years. For example, TPC2-/- mice show defects in cholesterol degradation, leading to hypercholesterinemia; TPC2 absence also results in mature-onset obesity, and a role in glucagon secretion and diabetes has been proposed. Infections with bacterial toxins or viruses e.g., cholera toxin or Ebola virus result in reduced infectivity rates in the absence of TPC2 or after pharmacological blockage, and TPC2-/- cancer cells lose their ability to migrate and metastasize efficiently. Finally, melanin production is affected by changes in hTPC2 activity, resulting in pigmentation defects and hair color variation. Here, we analyzed several publicly available genome variation data sets and identified multiple variations in the TPC2 protein in distinct human populations. Surprisingly, one variation, L564P, was found to be the predominant TPC2 isoform on a global scale. By applying endo-lysosomal patch-clamp electrophysiology, we found that L564P is a prerequisite for the previously described M484L gain-of-function effect that is associated with blond hair. Additionally, other gain-of-function variants with distinct geographical and ethnic distribution were discovered and functionally characterized. A meta-analysis of genome-wide association studies was performed, finding the polymorphisms to be associated with both distinct and overlapping traits. In sum, we present the first systematic analysis of variations in TPC2. We functionally characterized the most common variations and assessed their association with various disease traits. With TPC2 emerging as a novel drug target for the treatment of various diseases, this study provides valuable insights into ethnic and geographical distribution of TPC2 polymorphisms and their effects on channel activity.

Two-pore and TRP cation channels in endolysosomal osmo-/mechanosensation and volume regulation.

Two pore channels (TPCs) and mucolipins (TRPML) are the most prominent cation channels expressed in endolysosomes. Recently, roles of TPCs and TRPML2 have been revealed in regulating and detecting osmotically-driven changes in the surface-to-volume ratio of endolysosomes to promote endocytic and recycling traffic. TPCs and TRPML2 are highly expressed in macrophages and contribute to immune cell function. Here, we provide an overview of the emerging roles of these channels in innate immune cells, in particular macrophages, and highlight two models for osmo-mechanical regulation of intracellular organelle volume, trafficking, and cell homeostasis involving either TPCs or TRPML2.

TRPML2 is an osmo/mechanosensitive cation channel in endolysosomal organelles.

Endolysosomes are dynamic, intracellular compartments, regulating their surface-to-volume ratios to counteract membrane swelling or shrinkage caused by osmotic challenges upon tubulation and vesiculation events. While osmosensitivity has been extensively described on the plasma membrane, the mechanisms underlying endolysosomal surface-to-volume ratio changes and identities of involved ion channels remain elusive. Endolysosomes mediate endocytosis, exocytosis, cargo transport, and sorting of material for recycling or degradation. We demonstrate the endolysosomal cation channel TRPML2 to be hypotonicity/mechanosensitive, a feature crucial to its involvement in fast-recycling processes of immune cells. We demonstrate that the phosphoinositide binding pocket is required for TRPML2 hypotonicity-sensitivity, as substitution of L314 completely abrogates hypotonicity-sensitivity. Last, the hypotonicity-insensitive TRPML2 mutant L314R slows down the fast recycling pathway, corroborating the functional importance of hypotonicity-sensitive TRPML2. Our results highlight TRPML2 as an accelerator of endolysosomal trafficking by virtue of its hypotonicity-sensitivity, with implications in immune cell surveillance and viral trafficking.

Discovery of lipophilic two-pore channel agonists.

Two-pore channels (TPCs) have been a hot topic in recent literature. Their involvement in various diseases such as viral infections and cancer is of great interest for drug research. Due to their localization in the endolysosomal system and the lack of cell-permeable activators, complex techniques were required for studying channel functions. Here, we review the first published lipophilic small-molecule activators of TPCs. In independent high-throughput screens, several new agonists were discovered, which now allow simple and fast investigation of TPCs in more detail in intact cells and in vivo. Zhang et al. identified tricyclic and phenothiazine antidepressants as TPC1 and TPC2 activators by screening a library of approved drugs. In contrast, Gerndt et al. screened an extensive compound library with mostly new chemotypes and drug structures. The latter resulted in two structurally distinct high-affinity agonists, which are able to selectively activate TPC2 in either an NAADP- or PI(3,5)P2 -like manner. Here, we discuss the advantages and drawbacks of the identified molecules and their structural features. The versatility by which TPCs can be activated indicates many opportunities for future studies.

Evolutionary Aspects of TRPMLs and TPCs.

Transient receptor potential (TRP) or transient receptor potential channels are a highly diverse family of mostly non-selective cation channels. In the mammalian genome, 28 members can be identified, most of them being expressed predominantly in the plasma membrane with the exception of the mucolipins or TRPMLs which are expressed in the endo-lysosomal system. In mammalian organisms, TRPMLs have been associated with a number of critical endo-lysosomal functions such as autophagy, endo-lysosomal fusion/fission and trafficking, lysosomal exocytosis, pH regulation, or lysosomal motility and positioning. The related non-selective two-pore cation channels (TPCs), likewise expressed in endosomes and lysosomes, have also been found to be associated with endo-lysosomal trafficking, autophagy, pH regulation, or lysosomal exocytosis, raising the question why these two channel families have evolved independently. We followed TRP/TRPML channels and TPCs through evolution and describe here in which species TRP/TRPMLs and/or TPCs are found, which functions they have in different species, and how this compares to the functions of mammalian orthologs.

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

The protein interaction networks of mucolipins and two-pore channels.

BACKGROUND: The endolysosomal, non-selective cation channels, two-pore channels (TPCs) and mucolipins (TRPMLs), regulate intracellular membrane dynamics and autophagy. While partially compensatory for each other, isoform-specific intracellular distribution, cell-type expression patterns, and regulatory mechanisms suggest different channel isoforms confer distinct properties to the cell. SCOPE OF REVIEW: Briefly, established TPC/TRPML functions and interaction partners ('interactomes') are discussed. Novel TRPML3 interactors are shown, and a meta-analysis of experimentally obtained channel interactomes conducted. Accordingly, interactomes are compared and contrasted, and subsequently described in detail for TPC1, TPC2, TRPML1, and TRPML3. MAJOR CONCLUSIONS: TPC interactomes are well-defined, encompassing intracellular membrane organisation proteins. TRPML interactomes are varied, encompassing cardiac contractility- and chaperone-mediated autophagy proteins, alongside regulators of intercellular signalling. GENERAL SIGNIFICANCE: Comprising recently proposed targets to treat cancers, infections, metabolic disease and neurodegeneration, the advancement of TPC/TRPML understanding is of considerable importance. This review proposes novel directions elucidating TPC/TRPML relevance in health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Selective agonist of TRPML2 reveals direct role in chemokine release from innate immune cells.

Cytokines and chemokines are produced and secreted by a broad range of immune cells including macrophages. Remarkably, little is known about how these inflammatory mediators are released from the various immune cells. Here, the endolysosomal cation channel TRPML2 is shown to play a direct role in chemokine trafficking and secretion from murine macrophages. To demonstrate acute and direct involvement of TRPML2 in these processes, the first isoform-selective TRPML2 channel agonist was generated, ML2-SA1. ML2-SA1 was not only found to directly stimulate release of the chemokine CCL2 from macrophages but also to stimulate macrophage migration, thus mimicking CCL2 function. Endogenous TRPML2 is expressed in early/recycling endosomes as demonstrated by endolysosomal patch-clamp experimentation and ML2-SA1 promotes trafficking through early/recycling endosomes, suggesting CCL2 being transported and secreted via this pathway. These data provide a direct link between TRPML2 activation, CCL2 release and stimulation of macrophage migration in the innate immune response.