Expression Of TRPML1 Research Articles

Tissue expression of porcine transient receptor potential mucolipin protein channels and their differential responses to porcine reproductive and respiratory syndrome virus infection in vitro

Abstract Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious disease, posing a huge economic threat to the global swine industry. The transient receptor potential mucolipin proteins (TRPMLs) have been shown to be strongly associated with virus infection and other physiological processes in humans, but their tissue distribution and responses to PRRSV in pigs remain unknown. Material and Methods Quantitative reverse-transcription PCR analysis was undertaken to determine the optimal primer for TRPML expression detection and for quantifying that expression individually in different pig tissue samples. Meat Animal Research Center 145 (MARC-145) monkey kidney cells and the TRPML-specific activator mucolipin synthetic agonist 1 (ML-SA1) were used to reveal the relationship between TRPML and PRRSV-2 infection. Results The best primers for each TRPML gene used in a fluorescence-based quantitative method were identified and TRPML1 was found to be highly expressed in the kidneys and liver of pigs, while TRPML2 and TRPML3 were observed to be primarily expressed in the kidney and spleen tissues. The expression of TRPML2 was upregulated with the rise in PRRSV-2 infection titre but not the expression of TRPML1 or TRPML3, and ML-SA1 inhibited PRRSV-2 in a dose-dependent manner. Conclusion Our research revealed the gene expression of TRPMLs in pigs and identified that TRPML channels may act as key host factors against PRRSV infection, which could lead to new targets for the prevention and treatment of pig infectious diseases.

Gene expression of TRPMLs and its regulation by pathogen stimulation

The transient receptor potential mucolipin (TRPML) subfamily in mammalian has three members, namely TRPML1, TRPML2, and TRPML3, who play key roles in regulating intracellular Ca2+ homeostasis, endosomal pH, membrane trafficking and autophagy. Previous studies had shown that three TRPMLs are closely related to the occurrence of pathogen invasion and immune regulation in some immune tissues or cells, but the relationship between TRPMLs expression and pathogen invasion in lung tissue or cell remains elusive. Here, we investigated the expression distribution of three TRPML channels in mouse different tissues by qRT-PCR, and then found that all three TRPMLs were highly expressed in the mouse lung tissue, as well as mouse spleen and kidney tissues. The expression of TRPML1 or TRPML3 in all three mouse tissues had a significant down-regulation after the treatment of Salmonella or LPS, but TRPML2 expression showed a remarkable increase. Consistently, TRPML1 or TRPML3 but not TRPML2 in A549 cells also displayed a decreased expression induced by LPS stimulation, which shared a similar regulation pattern in the mouse lung tissue. Furthermore, the treatment of the TRPML1 or TRPML3 specific activator induced a dose-dependent up-regulation of inflammatory factors IL-1β, IL-6 and TNFα, suggesting that TRPML1 and TRPML3 are likely to play an important role in immune and inflammatory regulation. Together, our study identified the gene expression of TRPMLs induced by pathogen stimulation in vivo and in vitro, which may provide novel targets for innate immunity or pathogen regulation.

Coexpression of TRPML1 and TRPML2 Mucolipin Channels Affects the Survival of Glioblastoma Patients.

Among brain cancers, glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high cell heterogeneity, which can be linked to its high malignancy. We have previously demonstrated that TRPML1 channels affect the OS of GBM patients. Herein, by RT-PCR, FACS and Western blot, we demonstrated that TRPML1 and TRPML2 channels are differently expressed in GBM patients and cell lines. Moreover, these channels partially colocalized in ER and lysosomal compartments in GBM cell lines, as evaluated by confocal analysis. Interestingly, the silencing of TRPML1 or TRPML2 by RNA interference results in the decrease in the other receptor at protein level. Moreover, the double knockdown of TRPML1 and TRPML2 leads to increased GBM cell survival with respect to single-channel-silenced cells, and improves migration and invasion ability of U251 cells. Finally, the Kaplan–Meier survival analysis demonstrated that patients with high TRPML2 expression in absence of TRPML1 expression strongly correlates with short OS, whereas high TRPML1 associated with low TRPML2 mRNA expression correlates with longer OS in GBM patients. The worst OS in GBM patients is associated with the loss of both TRPML1 and TRPML2 channels.

Artemisia Leaf Extract protects against neuron toxicity by TRPML1 activation and promoting autophagy/mitophagy clearance in both in vitro and in vivo models of MPP+/MPTP-induced Parkinson's disease

BackgroundParkinson's disease (PD) is a neurodegenerative disorder involving the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Cellular clearance mechanisms, including the autophagy-lysosome pathway, are commonly affected in the pathogenesis of PD. The lysosomal Ca2+ channel mucolipin TRP channel 1 (TRPML1) is one of the most important proteins involved in the regulation of autophagy. Artemisia argyi Lev. et Vant., is a traditional Chinese herb, that has diverse therapeutic properties and is used to treat patients with skin diseases and oral ulcers. However, the neuroprotective effects of A. argyi are not explored yet. HypothesisThis study aims is to investigate the neuroprotective effects of A. argyi in promoting the TRPML1-mediated autophagy/mitophagy-enhancing effect MethodsIn this study, we used 1-methyl-4-phenyl-pyridinium (MPP+)-induced PD model established in an SH-SY5Y human neuroblastoma cell line as well as in a 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-induced PD model in C57BL/6 J mice. MTT assay was conducted to measure the cell viability and further MitoSoX and DCFDA assay were used to measure the ROS. Western blot analysis was used to access levels of TRPML1, p-DRP1 (ser616), p-AKT, PI3K, and β-catenin, Additionally, IF and IHC analysis to investigate the expression of TRPML1, LC3B, β-catenin, TH+, α-synuclein. Mitotracker stain was used to check mitophagy levels and a lysosomal intracellular activity kit was used to measure the lysosomal dysfunction. Behavioral studies were conducted by rotarod and grip strength experiments to check motor functions. ResultsIn our in vitro study, A. argyi rescued the MPP+-induced loss of cell viability and reduced the accumulation of mitochondrial and total reactive oxygen species (ROS). Subsequently, it increased the expression of TRPML1 protein, thereby inducing autophagy, which facilitated the clearance of toxic accumulation of α-synuclein. Furthermore, A. argyi played a neuroprotective role by activating the PI3K/AKT/β-catenin cell survival pathway. MPP+-mediated mitochondrial damage was overcome by upregulation of mitophagy and downregulation of the mitochondrial fission regulator p-DRP1 (ser616) in SH-SY5Y cells. In the in vivo study, A. argyi ameliorated impaired motor function and rescued TH+ neurons in the SNpc region. Similar to the results of the in vitro study, TRPML1, LC3B, and β-catenin expression was enhanced in the SNpc region in the A. argyi-treated mice brain. ConclusionThus, our results first demonstrate that A. argyi can exert neuroprotective effects by stimulating TRPML1 and rescuing neuronal cells by boosting autophagy/mitophagy and upregulating a survival pathway, suggesting that A. argyi can further be exploited to slow the progression of PD.

ML-SA1, a TRPML1 agonist, induces gastric secretion and gastrointestinal tract inflammation in vivo

Background: The effect of ML-SA1, a potent and specific TRPML1 channel agonist, on gastric secretion and subsequent impact to the gastrointestinal (GI) tract of mice was investigated.
 Methods: Twenty mice were divided into two groups, and respectively received either ML-SA1 or DMSO alone (as negative control), and then the intestinal propulsion rate were monitored. The stomach and intestinal tissues were sectioned for periodic acid Schiff (PAS), histopathological or immunohistochemical (IHC) analysis. TRPML1 expression level in AGS cells was assayed by western blot, and calcium imaging was performed in AGS cells after the GCaMP5G transfection.
 Results: Application of 150 μg/Kg ML-SA1 could result in significant decrease in intestinal propulsion rate and structural changes of the parietal cells in stomach and goblet cells in intestine of mice. Moreover, it induced inflammation to the duodenum section of intestine in mice. IHC staining also revealed that ML-SA1 could up-regulate TRPML1 expression in both the parietal cells of stomach pits and in the columnar epithelial cells of duodenum villi. Further study in AGS cells, a type of stomach cell line, demonstrated that ML-SA1 could enhance the expression of TRPML1 and induce the protrusion of micro-vesicles.
 Conclusions: All together, our results suggested that ML-SA1 was capable of activating TRPML1 in stomach cells and increasing gastric juice secretion. This provided a convincing attempt of applying ML-SA1 in animals and pointed out a new possible research direction that TRPML1 channel could be a potential new therapeutic target on hypochlorhydria or even in the field of gastrointestinal cancer research.

TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

SUMMARYWe screen ion channels and transporters throughout the genome to identify those required by human melanoma cells but not by normal human melanocytes. We discover that Mucolipin-1 (MCOLN1), which encodes the lysosomal cation channel TRPML1, is preferentially required for the survival and proliferation of melanoma cells. Loss of MCOLN1/TRPML1 function impairs the growth of patient-derived melanomas in culture and in xenografts but does not affect the growth of human melanocytes. TRPML1 expression and macropinocytosis are elevated in melanoma cells relative to melanocytes. TRPML1 is required in melanoma cells to negatively regulate MAPK pathway and mTORC1 signaling. TRPML1-deficient melanoma cells exhibit decreased survival, proliferation, tumor growth, and macropinocytosis, as well as serine depletion and proteotoxic stress. All of these phenotypes are partially or completely rescued by mTORC1 inhibition. Melanoma cells thus increase TRPML1 expression relative to melanocytes to attenuate MAPK and mTORC1 signaling, to sustain macropinocytosis, and to avoid proteotoxic stress.

Association of luteal cell degeneration and progesterone deficiency with lysosomal storage disorder mucolipidosis type IV in Mcoln1-/- mouse model†.

Transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1) (MCOLN1/Mcoln1) is a lysosomal counter ion channel. Mutations in MCOLN1 cause mucolipidosis type IV (MLIV), a progressive and severe lysosomal storage disorder with a slow onset. Mcoln1-/- mice recapitulate typical MLIV phenotypes but roles of TRPML1 in female reproduction are unknown. Despite normal mating activities, Mcoln1-/- female mice had reduced fertility at 2 months old and quickly became infertile at 5 months old. Progesterone deficiency was detected on 4.5 days post coitum/gestation day 4.5 (D4.5). Immunohistochemistry revealed TRPML1 expression in luteal cells of wild type corpus luteum (CL). Corpus luteum formation was not impaired in 5-6 months old Mcoln1-/- females indicated by comparable CL numbers in control and Mcoln1-/- ovaries on both D1.5 and D4.5. In the 5-6 months old Mcoln1-/- ovaries, histology revealed less defined corpus luteal cord formation, extensive luteal cell vacuolization and degeneration; immunofluorescence revealed disorganized staining of collagen IV, a basal lamina marker for endothelial cells; Nile Red staining detected lipid droplet accumulation, a typical phenotype of MLIV; immunofluorescence of heat shock protein 60 (HSP60, a mitochondrial marker) and in situ hybridization of steroidogenic acute regulatory protein (StAR, for the rate-limiting step of steroidogenesis) showed reduced expression of HSP60 and StAR, indicating impaired mitochondrial functions. Luteal cell degeneration and impaired mitochondrial functions can both contribute to progesterone deficiency in the Mcoln1-/- mice. This study demonstrates a novel function of TRPML1 in maintaining CL luteal cell integrity and function.

Transient Receptor Potential Mucolipin-1 Channels in Glioblastoma: Role in Patient's Survival.

A link between mucolipin channels and tumors has been recently suggested. Herein, we aim to investigate the transient receptor potential mucolipin (TRPML)-1 relevance in glioblastoma. The expression of this channel was evaluated via qRT-PCR and immunohistochemistry in biopsies from 66 glioblastoma patients and two human glioblastoma cell lines and compared to normal human brain, astrocytes, and epileptic tissues. The subcellular distribution of TRPML-1 was examined via confocal microscopy in the glioma cell lines. Then, to assess the role of TRPML-1, cell viability assays have been conducted in T98 and U251 cell lines treated with the specific TRPML-1 agonist, MK6-83. We found that MK6-83 reduced cell viability and induced caspase-3-dependent apoptosis. Indeed, the TRPML-1 silencing or the blockage of TRPML-1 dependent [Ca2+]i release abrogated these effects. In addition, exposure of glioma cells to the reactive oxygen species (ROS) inducer, carbonyl cyanide m-chlorophenylhydrazone (CCCP), stimulated a TRPML-1-dependent autophagic cell death, as demonstrated by the ability of the autophagic inhibitor bafilomycin A, the TRPML-1 inhibitor sphingomyelin, and the TRPML-1 silencing to completely inhibit the CCCP-mediated effects. To test a possible correlation with patient’s survival, Kaplan–Meier, univariate, and multivariate analysis have been performed. Data showed that the loss/reduction of TRPML-1 mRNA expression strongly correlates with short survival in glioblastoma (GBM) patients, suggesting that the reduction of TRPML-1 expression represents a negative prognostic factor in GBM patients.

Exosomal release through TRPML1-mediated lysosomal exocytosis is required for adipogenesis

The lysosomal Ca2+ permeable channel TRPML1 (MCOLN1) plays key roles in lysosomal membrane trafficking, including the fusion of late endosomes to lysosomes and lysosomal exocytosis, both of which are essential for release of exosomes into the extracellular milieu. Multiple lines of evidence indicate that the contents of adipocyte-derived exosomes mediate diverse cellular responses, including adipogenic differentiation. In this study, we aimed to define the potential roles of TRPML1 in lysosomal membrane trafficking during adipogenesis and in exosomal release. In response to adipogenic stimuli, the endogenous TRPML1 expression in OP9 pre-adipocytes was increased in a time-dependent manner, and the acute deletion of TRPML1 reduced lipid synthesis and expression of differentiation-related marker genes. Notably, mature adipocyte-derived exosomes were found to be necessary for adipogenesis and were dependent on TRPML1-mediated lysosomal exocytosis. Taken together, our findings indicate that TRPML1 mediates diverse roles in adipocyte differentiation and exosomal release. Further, we propose that TRPML1 should be considered as a regulator of obesity-related diseases.

PIKfyve accelerates phagosome acidification through activation of TRPML1 while arrests aberrant vacuolation independent of the Ca2+ channel.

PIKfyve phosphorylates PtdIns(3)P to PtdIns(3, 5)P2. One of the best characterized effector downstream of PtdIns(3, 5)P2 is a lysosomal Ca2+ channel, TRPML1. Although it has been reported that TRPML1 is involved in phagosome-lysosome fusion, the relevance of the Ca2+ channel in phagosome acidification has been denied. In this article, however, we demonstrated that the phagosome acidification was dependent on TRPML1. Based on the classical idea that Fluorescein isothiocyanate (FITC)-fluorescence is highly sensitive to acidic pH, we could estimate the phagosome acidification by time laps imaging. FITC-zymosan fluorescence that was engulfed by macrophages, decreased immediately after the uptake while the extinction of FITC-zymosan fluorescence was delayed in PIKfyve-deficient cells. The acidification arrest was completely rescued in the presence of Ca2+ ionophore A23187. Cells treated with a PIKfyve inhibitor, apilimod, also showed delayed phagosome acidification but were rescued by the overexpression of TRPML1. Additionally, TRPML1 agonist, ML-SA1 was effective to acidify the phagosome in PIKfyve-deficient cells. Another phenotype observed in PIKfyve-deficient cells is vacuole formation. Unexpectedly, enlarged vacuole formation in PIKfyve-deficient cells was not rescued by Ca2+ or over expression of TRPML1. It is likely that the acidification and vacuolation arrest is bifurcating downstream of PIKfyve.

Lysosome signaling controls the migration of dendritic cells

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.

TRPML2 and mucolipin evolution.

The TRPML2 protein, encoded by the Mcoln2 gene, is one of the three mucolipins (TRPML1-3), a subset of the TRP superfamily of ion channels. Although there are no thorough studies on the cellular distribution of TRPML2, its mRNA appears to be largely restricted to lymphocytes and other immune cells. This contrasts with the ubiquitous expression of TRPML1 and the limited but diverse expression of TRPML3 and clearly suggests a specialized role for TRPML2 in immunity. Localization studies indicate that TRPML2 is present in lysosomes (including the specialized lysosome-related organelle that B-lymphocytes use for processing of the antigen-bound B-cell receptor), late endosomes, recycling endosomes, and, at a much lower level, the plasma membrane. Heterologously expressed TRPML2, like TRPML1 and/or TRPML3, forms ion channels that can be activated by a gain-of-function mutation (alanine to proline in the fifth transmembrane domain, close to the pore) that favors the open state, by a transient reduction of extracellular sodium followed by sodium replenishment, by small chemicals related to sulfonamides, and by PI(3,5)P2, a rare phosphoinositide that naturally accumulates in the membranes of endosomes and lysosomes and thus could act as a physiologically relevant agonist. TRPML2 channels are inwardly rectifying and permeable to Ca(2+), Na(+), and Fe(2+). When heterologously co-expressed, TRPML2 can form heteromultimers with TRPML1 and TRPML3. In B-lymphocytes, TRPML2 and TRPML1 may play redundant roles in the function of their specialized lysosome. Although the specific subcellular function of TRPML2 is unknown, distribution and channel properties suggest roles in calcium release from endolysosomes, perhaps to regulate vesicle fusion and/or subsequent scission or to release calcium from intracellular acidic stores for signaling in the cytosol. Alternatively, TRPML2 could function in the plasma membrane, and its abundance in vesicles of the endocytic pathway could simply be due to regulation by endocytosis and exocytosis. The Mcoln2 gene is closely downstream from and in the same orientation as Mcoln3 in the genomes of most jawed vertebrates (from humans to sharks) with the exception of pigs, Xenopus tropicalis, and ray-finned fishes. The close homology of TRPML2 and 3 (closer to each other than to TRPML1) suggests that Mcoln2 and Mcoln3 arose from unequal crossing over that duplicated a common ancestor and placed both gene copies in tandem. These genes would have come apart subsequently in pigs, Xenopus, and the ancestor to ray-finned fishes. All jawed vertebrates for which we have thorough genomic knowledge have distinct Mcoln1, 2, and 3 genes (except ray-finned fishes which, probably due to the whole-genome duplication in their common ancestor, have two Mcoln1-like genes and two Mcoln3-like genes, although only one Mcoln2 gene). However, the available genomes of invertebrate deuterostomes (a sea urchin, lancelet, and two tunicates) contain a single mucolipin gene that is equally distant from the three vertebrate mucolipins. Hence, vertebrate mucolipins arose through two rounds of gene duplication (the first one likely producing Mcoln1 and the ancestor to Mcoln2 and 3) at some time between the onset of craniates and that of jawed vertebrates. This is also the evolutionary period during which adaptive immunity appeared. Given the restricted expression of TRPML2 in immune cells, this evolutionary history suggests a functional role in the adaptive immunity characteristic of vertebrates.

Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release

Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

A Role for the Ca 2+ Channel TRPML1 in Gastric Acid Secretion, Based on Analysis of Knockout Mice

Mutations in TRPML1, a lysosomal Ca(2+)-permeable TRP channel, lead to mucolipidosis type IV, a neurodegenerative lysosomal storage disease. An unusual feature of mucolipidosis type IV is constitutive achlorhydria. We produced Trpml1(-/-) (null) mice to investigate the requirement for this protein in gastric acid secretion. Trpml1-null mice were generated by gene targeting. The expression of Trpml1 and its role in acid secretion by gastric parietal cells were analyzed using biochemical, histologic, and ultrastructural approaches. Trpml1 is expressed by parietal cells and localizes predominantly to the lysosomes; it was dynamically palmitoylated and dephosphorylated in vivo following histamine stimulation of acid secretion. Trpml1-null mice had significant impairments in basal and histamine-stimulated gastric acid secretion and markedly reduced levels of the gastric proton pump. Histologic and ultrastructural analyses revealed that Trpml1(-/-) parietal cells were enlarged, had multivesicular and multi-lamellated lysosomes, and maintained an abnormal intracellular canalicular membrane. The intralysosomal Ca(2+) content and receptor-mediated Ca(2+) signaling were, however, unaffected in Trpml1(-/-) gastric glands, indicating that Trpml1 does not function in the regulation of lysosomal Ca(2+). Loss of Trpml1 causes reduced levels and mislocalization of the gastric proton pump and alters the secretory canaliculi, causing hypochlorhydria and hypergastrinemia. The lysosomal enlargement and defective intracellular canaliculi formation observed in Trpml1(-/-) parietal cells indicate that Trpml1 functions in the formation and trafficking of the tubulovesicles. This study provides direct evidence for the regulation of gastric acid secretion by a TRP channel; TRPML1 is an important protein in parietal cell apical membrane trafficking.

Expression of transient receptor potential channel mucolipin (TRPML) and polycystine (TRPP) in the mouse inner ear

Conclusions: TRPML3 may play distinct roles in the inner ear, such as stereociliar organization, sensory cell transduction, and inner ear fluid homeostasis, and TRPP3 may be important for fluid homeostasis in the inner ear. Objective: To study the expression of TRPML1–3 and TRPP2, 3, and 5 in the mouse inner ear. Materials and methods: Localization of TRPML1–3 and TRPP2, 3, and 5 in the inner ear of CBA/J mice was investigated by immunohistochemistry. Results: TRPML1–3 immunoreactivity was evident in the stria vascularis, spiral prominence, and spiral ligament. TRPML immunoreactivity was also observed in outer and inner hair cells, supporting cells, and spiral ganglion cells. The vestibular end organs, vestibular sensory cells, dark cells, and ganglion cells all showed immunoreactivity to TRPML. TRPP2 immunoreactivity was evident in the outer lining of the lateral wall of the cochlea, spiral ganglion cells, vestibular sensory cells, and ganglion cells. TRPP3 immunoreactivity was present in stria vascularis, spiral ganglion cells, vestibular sensory cells, dark cells, and ganglion cells. Faint TRPP5 immunoreactivity was observed in the spiral ganglion cells and vestibular ganglion cells.

PetroChina selects UOP technology for new integrated refining and petrochemicals complex

The lysosomal Ca2+ permeable channel TRPML1 (MCOLN1) plays key roles in lysosomal membrane trafficking, including the fusion of late endosomes to lysosomes and lysosomal exocytosis, both of which are essential for release of exosomes into the extracellular milieu. Multiple lines of evidence indicate that the contents of adipocyte-derived exosomes mediate diverse cellular responses, including adipogenic differentiation. In this study, we aimed to define the potential roles of TRPML1 in lysosomal membrane trafficking during adipogenesis and in exosomal release. In response to adipogenic stimuli, the endogenous TRPML1 expression in OP9 pre-adipocytes was increased in a time-dependent manner, and the acute deletion of TRPML1 reduced lipid synthesis and expression of differentiation-related marker genes. Notably, mature adipocyte-derived exosomes were found to be necessary for adipogenesis and were dependent on TRPML1-mediated lysosomal exocytosis. Taken together, our findings indicate that TRPML1 mediates diverse roles in adipocyte differentiation and exosomal release. Further, we propose that TRPML1 should be considered as a regulator of obesity-related diseases.

New zeotype for making amines

The lysosomal Ca2+ permeable channel TRPML1 (MCOLN1) plays key roles in lysosomal membrane trafficking, including the fusion of late endosomes to lysosomes and lysosomal exocytosis, both of which are essential for release of exosomes into the extracellular milieu. Multiple lines of evidence indicate that the contents of adipocyte-derived exosomes mediate diverse cellular responses, including adipogenic differentiation. In this study, we aimed to define the potential roles of TRPML1 in lysosomal membrane trafficking during adipogenesis and in exosomal release. In response to adipogenic stimuli, the endogenous TRPML1 expression in OP9 pre-adipocytes was increased in a time-dependent manner, and the acute deletion of TRPML1 reduced lipid synthesis and expression of differentiation-related marker genes. Notably, mature adipocyte-derived exosomes were found to be necessary for adipogenesis and were dependent on TRPML1-mediated lysosomal exocytosis. Taken together, our findings indicate that TRPML1 mediates diverse roles in adipocyte differentiation and exosomal release. Further, we propose that TRPML1 should be considered as a regulator of obesity-related diseases.

TRP-ML1 Regulates Lysosomal pH and Acidic Lysosomal Lipid Hydrolytic Activity

Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles, which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1(-/-) cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H(+) channel, and the increased lysosomal acidification in TRP-ML1(-/-) cells is likely caused by the loss of TRP-ML1-mediated H(+) leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1(-/-) cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1(-/-) cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1(-/-) cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.