Known and novel members of the endolysosomal transportome/channelome as candidates to rescue lysosomal storage diseases (LSDs)

The project presented herein addresses our limited understanding of organellar pharmacology. Specifically, the work was conceived to elucidate the biological relevance of the endolysosomal cation channels (mucolipins/TRPMLs and two-pore channels/TPCs) using novel, selective pharmacological modulators. On one hand, we developed a first-in-field selective TRPML2 agonist, ML2-SA1, which activates the TRPML2 ion channel on early endosomes, recycling endosomes, and lysosomes. We demonstrate how TRPML2 accelerates endosomal traffic, enhancing chemokine secretion and macrophage chemoattraction. TRPML2 activity is particularly important in the rapidly recycling pathway, where it mediates cargo transit directly from sorting endosomes to the plasma membrane. This function is largely conferred by its unqiue activation by membrane stretching, a feature we have shown to rely on a single amino acid in the TRPML2 phosphoinositide binding-pocket (L314). Mutation of L314 into its TRPML1/TRPML3 counterpart (L314R) abrogates TRPML2 osmosensitivity, and impedes the rapidly recycling pathway. These findings provide biological and structural information about TRPML2 function, laying the foundation for future endeavors modulating immune cell response and inflammation through the immune cell-restricted, druggable ion channel. Our primary motivation for investigating the endolysosomal ion channels is development of new treatments for diseases currently lacking therapies. The lysosomal storage diseases (LSDs) represent one such family of diseases, where endolysosomal protein defects result in lysosomal dysfunction and (often) neurodegeneration. Mucolipidosis type IV (MLIV) is caused by dysfunction of the lysosomal TRPML1 ion channel, causing blindness and early-onset neurodegeneration. Aiming to treat LSDs such as MLIV, we investigated the related lysosomal ion channel TPC2. We characterized various TPC2 polymorphisms that increase its activity, and developed agonists for TPC2 that either facilitate high Ca2+ fluxes arresting endosomal motility or Na+ fluxes facilitating lysosomal exocytosis and enhancing autophagy. We used CRISPR/Cas9 to develop new induced pluripotent stem cell (iPSC) models for Neuronal Ceroid Lipofuscinosis (colloquially termed “childhood dementia“) and MLIV, differentiating these into cortical neurons. We used the diseased human neurons to investigate treatments for LSDs, finding the autophagic enhancer tamoxifen and the two-pore channel 2 agonist TPC2-A1-P to counteract LSD phenotypes. TPC2-A1-P restored excessive lysosomal proteolysis, storage defects, and trafficking abnormalities in human MLIV neurons and patient fibroblasts. Similarly, TPC2-A1-P ameliorated LSD phenotypes in Niemann-Pick Disease type C1 fibroblasts (NPC1, also known as childhood Alzheimer’s Disease), another LSD marked by impaired activity of lysosomal cation channels. We finally performed a proof-of-concept in vivo investigation, treating MLIV mice with TPC2-A1-P. While DMSO-treated MLIV mice exhibited gliosis of the cerebellum and hippocampus, TPC2-A1-P-injected mouse brains featured much fewer glial cells, akin to the wild-type controls. These findings demonstrate that pharmacological modulation of the endolysosomal system can restore physiology in a variety of lysosomal storage diseases in vitro and in vivo

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

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

Summary: 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

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

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