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.

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion.

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca2+ influx, but is also dependent on Ca2+ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca2+-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca2+ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

Whole-body analysis of TRPML3 (MCOLN3) expression using a GFP-reporter mouse model reveals widespread expression in secretory cells and endocrine glands.

TRPML3 (mucolipin 3, MCOLN3) is an endolysosomal cation channel belonging to the TRPML subfamily of transient receptor potential channels. Gain-of-function mutations in the Trpml3 gene cause deafness, circling behavior and coat color dilution in mice due to cell death of TRPML3-expressing hair cells of the inner ear or skin melanocytes, respectively. Furthermore, TRPML3 was found to play a role in the long term survival of cochlear hair cells (its absence contributing to presbycusis), in specialized giant lysosomes that neonatal (birth to weaning) enterocytes used for the uptake and digestion of maternal milk nutrients, and in the expulsion of exosome-encased bacteria such as uropathogenic E. coli, infecting bladder epithelial cells. Recently, TRPML3 was found to be expressed at high levels in alveolar macrophages and loss of TRPML3 results in a lung emphysema phenotype, confirmed in two independently engineered Trpml3 knockout lines. TRPML3 is not ubiquitously expressed like its relative TRPML1 and thus cellular expression of TRPML3 on a whole-tissue level remains, with the exceptions mentioned above, largely elusive. To overcome this problem, we generated a τGFP reporter mouse model for TRPML3 and compared expression data obtained from this model by immunofluorescence on tissue sections with immunohistochemistry using TRPML3 antibodies and in situ hybridization. We thus uncovered expression in several organs and distinct cell types. We confirmed TRPML3 expression in both neonatal and adult alveolar macrophages, in melanocytes of hair follicles and glabrous skin, in principle cells of the collecting duct of the neonatal and adult kidney, and in olfactory sensory neurons of the olfactory epithelium, including its fibres protruding to the glomeruli of the olfactory bulb. Additionally, we localized TRPML3 in several glands including parathyroid, thyroid, salivary, adrenal, and pituitary gland, testes and ovaries, suggestive of potential roles for the channel in secretion or uptake of different hormones.

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 and Lung Disease.

Endolysosomal cation channels are emerging as key players of endolysosomal function such as endolysosomal trafficking, fusion/fission, lysosomal pH regulation, autophagy, lysosomal exocytosis, and endocytosis. Diseases comprise lysosomal storage disorders (LSDs) and neurodegenerative diseases, metabolic diseases, pigmentation defects, cancer, immune disorders, autophagy related diseases, infectious diseases and many more. Involvement in lung diseases has not been a focus of attention so far but recent developments in the field suggest critical functions in lung physiology and pathophysiology. Thus, loss of TRPML3 was discovered to exacerbate emphysema formation and cigarette smoke induced COPD due to dysregulated matrix metalloproteinase 12 (MMP-12) levels in the extracellular matrix of the lung, a known risk factor for emphysema/COPD. While direct lung function measurements with the exception of TRPML3 are missing for other endolysosomal cation channels or channels expressed in lysosome related organelles (LRO) in the lung, links between those channels and important roles in lung physiology have been established such as the role of P2X4 in surfactant release from alveolar epithelial Type II cells. Other channels with demonstrated functions and disease relevance in the lung such as TRPM2, TRPV2, or TRPA1 may mediate their effects due to plasma membrane expression but evidence accumulates that these channels might also be expressed in endolysosomes, suggesting additional and/or dual roles of these channels in cell and intracellular membranes. We will discuss here the current knowledge on cation channels residing in endolysosomes or LROs with respect to their emerging roles in lung disease.

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.

TRPML Cation Channels in Inflammation and Immunity.

Background: In 1883, Ilya Mechnikov discovered phagocytes and established the concept of phagocytosis by macrophages. In 1908, he was awarded the Nobel Prize in Physiology/Medicine for his findings, which laid the foundations for today's understanding of the innate immune response. Only in the 1960s, Max Cooper and Robert Good significantly advanced our understanding of the immune system by demonstrating that B- and T-cells cooperate to regulate the adaptive immune response. Both, innate and adaptive immune response are essential to effectively protect the individual against infectious agents, such as viruses, bacterial or insect toxins, or allergens. Innate immune responses occur rapidly upon exposure to noxious or infectious agents or organisms, in contrast to the adaptive immune system that needs days rather than hours to develop and acts primarily on the basis of antigen-specific receptors expressed on the surface of B- and T-lymphocytes. In recent years, it has become evident that endosomes and lysosomes are involved in many aspects of immune cell function, such as phagocytosis, antigen presentation and processing by antigen-presenting cells, release of proinflammatory mediators, e.g., by mast cells, or secretion of the pore-forming protein perforin by cytotoxic T lymphocytes. Several lysosomal storage disorders (LSDs) have been associated with defects in immune system function or immune system hyperactivity, such as Gaucher, Fabry, or Niemann-Pick type C1 disease, mucopolysaccharidoses (MPS), gangliosidosis, or juvenile neuronal ceroid lipofuscinosis (JNCL). Beside accumulating evidence on the importance of endolysosomes in immune cell function, recent results suggest direct roles of endolysosomal ion channels, such as the TRPML channels (mucolipins), which are members of the transient receptor potential (TRP) superfamily of non-selective cation channels, for different aspects of immune cell function. The aim of this review is to discuss the current knowledge about the roles of TRPML channels in inflammation and immunity, and to assess their potential as drug targets to influence immune cell functions. Advances: Examples of recently established roles of TRPML channels in immune system function and immune response include the TRPML1-mediated modulation of secretory lysosomes, granzyme B content, and tuning of effector function in NK cells, TRPML1-dependent directional dendritic cell (DC) migration and DC chemotaxis, and the role of TRPML2 in chemokine release from LPS-stimulated macrophages. Outlook: Although our understanding of the functional roles of TRPML channels in inflammation and immunity is still in its infancy, a few interesting findings have been made in the past years, encouraging further and more detailed work on the role of TRPMLs, e.g., in intracellular trafficking and release of chemokines, cytokines, or granzyme B, or in phagocytosis and bacterial toxin and virus trafficking through the endolysosomal machinery.