TRPML1: Role In Autophagy And Potential Target To Treat Lysosomal Storage Disorders

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signaling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release via mucolipin 1 (TRPML1) activates calcineurin, which binds and de-phosphorylates TFEB, thus promoting its nuclear translocation. Induction of autophagy and lysosomal biogenesis via TFEB required TRPML1-mediated calcineurin activation, linking lysosomal calcium signaling to both calcineurin regulation and autophagy induction. In addition to the role of TRPML1 on sustaining transcriptional autophagy program, through the activation of TFEB, we also found that TRPML1-activation induces the recruitment of PtdIns(3)P-binding proteins to the nascent autophagosome, whereas genetic or pharmacological inhibition of TRPML1 channel inhibits autophagy initiation. Importantly, alteration of this function has pathological consequences, and thus we found that autophagosome formation is impaired in human fibroblasts from patients affected of mucolipidosis IV (MLIV; a severe lysosomal storage disorder caused by mutations in TRPML1). By using specific compound inhibitors during starvation, we found that TRPML1-mediated induction of autophagosome biogenesis requires calmodulin, CaMKKβ, and the PtdIns(3)-generating enzyme VPS34. Therefore, we hypothezise that during starvation, TRPML1 activation releases lysosomal calcium that activates a calcium-depedent pathway involving CaMKKβ and the induction of two essential protein complexes involved in autophagy initiation such as ULK1 and PIK3C3 complexes. In parallel studies, we used high content (HC) screening approaches to identify small molecules able to ameliorate the MLIV phenotype. In one of them, we tested whether previously identified drugs (150 FDA compounds) inducing TFEB translocation and cellular clearance might be active in MLIV patient cells. This screening resulted in the identification of 3 drugs able to induce TFEB nuclear translocation confirming the importance of the TRPML1-mediated signalling to promote TFEB activity. In addition, only one of these hits were able to reduce the pathological accumulation of autophagic substrates such as p62 and NBR1 in MLIV human fibroblasts. In a second independent HC-screening, we developed a cell-based assay to identify FDA-drugs able to reduce cholesterol accumulation in MLIV cells. We identify 8 small molecules able to reduce cholesterol accumulation in MLIV human fibroblasts that will need further characterization to define their ability to ameliorate the phenotype of this devastating disease. In summary, we found two novel signaling pathways triggered by TRPML1-dependent lysosomal calcium release that regulate cellular homeostasis by both promoting autophagy initiation and sustaining transcriptional programs inducing autophagic and lysosomal genes during starvation. Finally, we use part of this knowledge to develop cell-based high content screening assays that identified 9 FDA-approved compounds able to ameliorate the autophagic impairment and reduce lipid storage in MLIV disease cells

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)P-2, 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

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.This work was funded by the European Union’s Horizon 2020 research and innovation programme (BATCure, grant No. 666918 to DLM, JPB, SEM, TB and SS). JPB is funded by the Agencia Estatal de Investigación (PID2019-105699RB-I00/ AEI / 10.13039/501100011033 and RED2018-102576-T), Plan Nacional sobre Drogas (2020I028), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA and Fundación Ramón Areces. SS was funded by a grant from the Mila’s Miracle Foundation. TB was supported by German Research Council (DFG) grant FOR2625. SM benefits from MRC funding to the MRC Laboratory for Molecular Cell Biology University Unit at UCL (award code MC_U12266B) towards laboratory and office space. We acknowledge Marcella Cesana for providing the TFEB virus. Graphical abstract was created using BioRender.com

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease

TRPML1: The Ca(2+)retaker of the lysosome

Efficient functioning of lysosome is necessary to ensure the correct performance of a variety of intracellular processes such as degradation of cargoes coming from the endocytic and autophagic pathways, recycling of organelles, and signaling mechanisms involved in cellular adaptation to nutrient availability. Mutations in lysosomal genes lead to more than 50 lysosomal storage disorders (LSDs). Among them, mutations in the gene encoding TRPML1 (MCOLN1) cause Mucolipidosis type IV (MLIV), a recessive LSD characterized by neurodegeneration, psychomotor retardation, ophthalmologic defects and achlorhydria. At the cellular level, MLIV patient fibroblasts show enlargement and engulfment of the late endo-lysosomal compartment, autophagy impairment, and accumulation of lipids and glycosaminoglycans. TRPML1 is the most extensively studied member of a small family of genes that also includes TRPML2 and TRPML3, and it has been found to participate in vesicular trafficking, lipid and ion homeostasis, and autophagy. In this review we will provide an update on the latest and more novel findings related to the functions of TRPMLs, with particular focus on the emerging role of TRPML1 and lysosomal calcium signaling in autophagy. Moreover, we will also discuss new potential therapeutic approaches for MLIV and LSDs based on the modulation of TRPML1-mediated signaling

Neurodegenerative Lysosomal Storage Disorders: TPC2 Comes to the Rescue!

Lysosomal storage diseases (LSDs) resulting from inherited gene mutations constitute a family of disorders that disturb lysosomal degradative function leading to abnormal storage of macromolecular substrates. In most LSDs, central nervous system (CNS) involvement is common and leads to the progressive appearance of neurodegeneration and early death. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of neurodegenerative LSDs. One of the main basic mechanisms through which the endolysosomal ion channels regulate the function of the endolysosomal system is Ca2+ release, which is thought to be essential for intracellular compartment fusion, fission, trafficking and lysosomal exocytosis. The intracellular TRPML (transient receptor potential mucolipin) and TPC (two-pore channel) ion channel families constitute the main essential Ca2+-permeable channels expressed on endolysosomal membranes, and they are considered potential drug targets for the prevention and treatment of LSDs. Although TRPML1 activation has shown rescue effects on LSD phenotypes, its activity is pH dependent, and it is blocked by sphingomyelin accumulation, which is characteristic of some LSDs. In contrast, TPC2 activation is pH-independent and not blocked by sphingomyelin, potentially representing an advantage over TRPML1. Here, we discuss the rescue of cellular phenotypes associated with LSDs such as cholesterol and lactosylceramide (LacCer) accumulation or ultrastructural changes seen by electron microscopy, mediated by the small molecule agonist of TPC2, TPC2-A1-P, which promotes lysosomal exocytosis and autophagy. In summary, new data suggest that TPC2 is a promising target for the treatment of different types of LSDs such as MLIV, NPC1, and Batten disease, both in vitro and in vivo

Flavonoids increase melanin production and reduce proliferation, migration and invasion of melanoma cells by blocking endolysosomal/melanosomal TPC2

Two-pore channel 2 (TPC2) resides in endolysosomal membranes but also in lysosome-related organelles such as the melanin producing melanosomes. Gain-of-function polymorphisms in hTPC2 are associated with decreased melanin production and blond hair color. Vice versa genetic ablation of TPC2 increases melanin production. We show here an inverse correlation between melanin production and melanoma proliferation, migration, and invasion due to the dual activity of TPC2 in endolysosomes and melanosomes. Our results are supported by both genetic ablation and pharmacological inhibition of TPC2. Mechanistically, our data show that loss/block of TPC2 results in reduced protein levels of MITF, a major regulator of melanoma progression, but an increased activity of the melanin-generating enzyme tyrosinase. TPC2 inhibition thus provides a twofold benefit in melanoma prevention and treatment by increasing, through interference with tyrosinase activity, the synthesis of UV blocking melanin in melanosomes and by decreasing MITF-driven melanoma progression by increased GSK3β-mediated MITF degradation

Estradiol analogs attenuate autophagy, cell migration and invasion by direct and selective inhibition of TRPML1, independent of estrogen receptors

The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17β-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion

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