Transient Receptor Potential Mucolipin 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.

Pseudorabies virus inhibits progesterone-induced inactivation of TRPML1 to facilitate viral entry.

Viral infection is a significant risk factor for fertility issues. Here, we demonstrated that infection by neurotropic alphaherpesviruses, such as pseudorabies virus (PRV), could impair female fertility by disrupting the hypothalamus-pituitary-ovary axis (HPOA), reducing progesterone (P4) levels, and consequently lowering pregnancy rates. Our study revealed that PRV exploited the transient receptor potential mucolipin 1 (TRPML1) and its lipid activator, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), to facilitate viral entry through lysosomal cholesterol and Ca2+. P4 antagonized this process by inducing lysosomal storage disorders and promoting the proteasomal degradation of TRPML1 via murine double minute 2 (MDM2)-mediated polyubiquitination. Overall, the study identifies a novel mechanism by which PRV hijacks the lysosomal pathway to evade P4-mediated antiviral defense and impair female fertility. This mechanism may be common among alphaherpesviruses and could contribute significantly to their impact on female reproductive health, providing new insights for the development of antiviral therapies.

Corynoxine promotes TFEB/TFE3-mediated autophagy and alleviates Aβ pathology in Alzheimer's disease models.

Autophagy impairment is a key factor in Alzheimer's disease (AD) pathogenesis. TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) are nuclear transcription factors that regulate autophagy and lysosomal biogenesis. We previously showed that corynoxine (Cory), a Chinese medicine compound, protects neurons from Parkinson's disease (PD) by activating autophagy. In this study, we investigated the effect of Cory on AD models in vivo and in vitro. We found that Cory improved learning and memory function, increased neuronal autophagy and lysosomal biogenesis, and reduced pathogenic APP-CTFs levels in 5xFAD mice model. Cory activated TFEB/TFE3 by inhibiting AKT/mTOR signaling and stimulating lysosomal calcium release via transient receptor potential mucolipin 1 (TRPML1). Moreover, we demonstrated that TFEB/TFE3 knockdown abolished Cory-induced APP-CTFs degradation in N2aSwedAPP cells. Our findings suggest that Cory promotes TFEB/TFE3-mediated autophagy and alleviates Aβ pathology in AD models.

Abstract 12784: Ischemic Cardiac Autophagy/Mitophagy is Regulated by TRPML1 and Mitochondrial Reactive Oxygen Species

Introduction: In myocardial infarction (MI), activated autophagy/mitophagy helps degrade cytoplasmic macromolecules/proteins/ organelles (especially damaged mitochondria), recycle breakdown products, and prevent oxidative damage and cell death. It had reported that reactive oxygen species (ROS) activates the transcription factor EB (TFEB) via a lysosomal transient receptor potential mucolipin 1 (TRPML1) Ca 2+ efflux. TFEB is thought to be the master regulator of lysosome biogenesis. Hypothesis: We investigated whether lysosomal TRPML1 was activated by ROS and contributed to ischemic cardiac autophagy/mitophagy. Methods: MI was induced in wild type C57BL/6J mice by ligated left anterior descending coronary artery. Mice were studied at three weeks after MI surgery. Results: The lysosomal-restricted TRPML1 Ca 2+ release channel was significantly upregulated by 84.8% in cardiac border zone (BZ) and 55.4% in a remote zone after MI. As expected, dephosphorylated TFEB (i.e., active TFEB), mitophagy marker Parkin, autophagy marker LC3II, and lysosome marker LAMP1 increased substantially after MI. Autophagy degradation was upregulated as measured by SQSTM1/p62. TRPML1 protein expression was reduced in a dose-dependent manner by TRPML1 shRNA AAV9 (AAV9 was injected 1 week after MI for two weeks). The protein levels of LC3II and LAMP1 significantly decreased in MI BZ after AAV9 transfection. Mitochondrial ROS scavenger (mitoTEMPO, 1 mg/kg/day, i.p.) treatment for two weeks (starting at 1 week after MI) significantly inhibited TRPML1, LAMP1, LC3II and Parkin expression in the MI BZ. Conversely, when TRPML1 was overexpressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the protein levels of LC3II/Parkin were increased. Speaking to human relevance, TRPML1 was significantly increased in human patients with ischemic cardiomyopathy. Conclusion: Autophagy and mitophagy after MI are regulated by TRPML1 and mitochondrial ROS.

Ferritin Is Secreted from Primary Cultured Astrocyte in Response to Iron Treatment via TRPML1-Mediated Exocytosis.

Impaired iron homeostasis has been proven to be one of the critical contributors to the pathology of Parkinson's disease (PD). Ferritin is considered an intracellular protein responsible for storing cytosolic iron. Recent studies have found that ferritin can be secreted from cells independent of the classical endoplasmic reticulum-Golgi system. However, the precise mechanisms underlying the secretion of ferritin in the brain were not elucidated. In the present study, we demonstrated that the primary cultured astrocytes do have the ability to secrete ferritin, which is enhanced by iron treatment. Increased ferritin secretion was accompanied by increased protein expression of ferritin response to iron stimulation. Further study showed that iron-induced expression and secretion of ferritin could be inhibited by CQ or 3-MA pretreatment. In addition, the knockdown of transient receptor potential mucolipin 1 (TRPML1) antagonized iron-induced ferritin secretion, accompanied by further increased intracellular protein levels of ferritin. Further study demonstrated that ferritin colocalized with LAMP1 in iron-treated astrocytes. On the contrary, ras-associated protein 27a (Rab27a) knockdown further enhanced iron-induced ferritin secretion and decreased intracellular protein levels of ferritin. Furthermore, we also showed that the secretory autophagy protein tripartite motif containing 16 (TRIM16) and sec22b decreased in iron-treated astrocytes. These results suggested that astrocytes might secrete ferritin via TRPML1-mediated exocytosis. This provides new evidence for the mechanisms underlying the secretion of ferritin in primary cultured astrocytes under a high iron environment.

FGF-18 Protects the Injured Spinal cord in mice by Suppressing Pyroptosis and Promoting Autophagy via the AKT-mTOR-TRPML1 axis.

Spinal cord injury (SCI) is a severe medical condition with lasting effects. The efficacy of numerous clinical treatments is hampered by the intricate pathophysiological mechanism of SCI. Fibroblast growth factor 18 (FGF-18) has been found to exert neuroprotective effects after brain ischaemia, but its effect after SCI has not been well explored. The aim of the present study was to explore the therapeutic effect of FGF-18 on SCI and the related mechanism. In the present study, a mouse model of SCI was used, and the results showed that FGF-18 may significantly affect functional recovery. The present findings demonstrated that FGF-18 directly promoted functional recovery by increasing autophagy and decreasing pyroptosis. In addition, FGF-18 increased autophagy, and the well-known autophagy inhibitor 3-methyladenine (3MA) reversed the therapeutic benefits of FGF-18 after SCI, suggesting that autophagy mediates the therapeutic effects of FGF-18 on SCI. A mechanistic study revealed that after stimulation of the protein kinase B (AKT)-transient receptor potential mucolipin 1 (TRPML1)-calcineurin signalling pathway, the FGF-18-induced increase in autophagy was mediated by the dephosphorylation and nuclear translocation of transcription factor E3 (TFE3). Together, these findings indicated that FGF-18 is a robust autophagy modulator capable of accelerating functional recovery after SCI, suggesting that it may be a promising treatment for SCI in the clinic.

Optineurin tunes outside-in signaling to regulate lysosome biogenesis and phagocytic clearance in the retina

Balancing the competing demands of phagolysosomal degradation and autophagy is a significant challenge for phagocytic tissues. Yet how this plasticity is accomplished in health and disease is poorly understood. In the retina, circadian phagocytosis and degradation of photoreceptor outer segments by the postmitotic retinal pigment epithelium (RPE) are essential for healthy vision. Disrupted autophagy due to mechanistic target of rapamycin (mTOR) overactivation in the RPE is associated with blinding macular degenerations; however, outer segment degradation is unaffected in these diseases, indicating that distinct mechanisms regulate these clearance mechanisms. Here, using advanced imaging and mouse models, we identify optineurin as a key regulator that tunes phagocytosis and lysosomal capacity to meet circadian demands and helps prioritize outer segment clearance by the RPE in macular degenerations. High-resolution live-cell imaging implicates optineurin in scissioning outer segment tips prior to engulfment, analogous to microglial trogocytosis of neuronal processes. Optineurin is essential for recruiting light chain 3 (LC3), which anchors outer segment phagosomes to microtubules and facilitates phagosome maturation and fusion with lysosomes. This dynamically activates transcription factor EB (TFEB) to induce lysosome biogenesis in an mTOR-independent, transient receptor potential-mucolipin 1 (TRPML1)-dependent manner. RNA-seq analyses show that expression of TFEB target genes temporally tracks with optineurin recruitment and that lysosomal and autophagy genes are controlled by distinct transcriptional programs in the RPE. The unconventional plasma membrane-to-nucleus signaling mediated by optineurin ensures outer segment degradation under conditions of impaired autophagy in macular degeneration models. Independent regulation of these criticalclearance mechanisms would help safeguard the metabolic fitness of the RPE throughout the organismal lifespan.

Intracellular Ca2+ signalling: unexpected new roles for the usual suspect.

Cytosolic Ca2+ signals are organized in complex spatial and temporal patterns that underlie their unique ability to regulate multiple cellular functions. Changes in intracellular Ca2+ concentration ([Ca2+]i) are finely tuned by the concerted interaction of membrane receptors and ion channels that introduce Ca2+ into the cytosol, Ca2+-dependent sensors and effectors that translate the elevation in [Ca2+]i into a biological output, and Ca2+-clearing mechanisms that return the [Ca2+]i to pre-stimulation levels and prevent cytotoxic Ca2+ overload. The assortment of the Ca2+ handling machinery varies among different cell types to generate intracellular Ca2+ signals that are selectively tailored to subserve specific functions. The advent of novel high-speed, 2D and 3D time-lapse imaging techniques, single-wavelength and genetic Ca2+ indicators, as well as the development of novel genetic engineering tools to manipulate single cells and whole animals, has shed novel light on the regulation of cellular activity by the Ca2+ handling machinery. A symposium organized within the framework of the 72nd Annual Meeting of the Italian Society of Physiology, held in Bari on 14-16th September 2022, has recently addressed many of the unexpected mechanisms whereby intracellular Ca2+ signalling regulates cellular fate in healthy and disease states. Herein, we present a report of this symposium, in which the following emerging topics were discussed: 1) Regulation of water reabsorption in the kidney by lysosomal Ca2+ release through Transient Receptor Potential Mucolipin 1 (TRPML1); 2) Endoplasmic reticulum-to-mitochondria Ca2+ transfer in Alzheimer's disease-related astroglial dysfunction; 3) The non-canonical role of TRP Melastatin 8 (TRPM8) as a Rap1A inhibitor in the definition of some cancer hallmarks; and 4) Non-genetic optical stimulation of Ca2+ signals in the cardiovascular system.

Increased expression or activation of TRPML1 reduces hepatic storage of toxic Z alpha-1 antitrypsin

Mutant Z alpha-1 antitrypsin (ATZ) accumulates in globules in the liver and is the prototype of proteotoxic hepatic disease. Therapeutic strategies aiming at clearance of polymeric ATZare needed. Transient receptor potential mucolipin-1 (TRPML1) is a lysosomal Ca2+ channel that maintains lysosomal homeostasis. In this study, we show that by increasing lysosomal exocytosis, TRPML1 gene transfer or small-molecule-mediated activation of TRPML1 reduces hepatic ATZ globules and fibrosis in PiZ transgenic mice that express the human ATZ. ATZ globule clearance induced by TRPML1 occurred without increase in autophagy or nuclear translocation of TFEB. Our results show that targeting TRPML1 and lysosomal exocytosis is a novel approach for treatment of the liver disease due to ATZ and potentially other diseases due to proteotoxic liver storage.

Lysosomal Ca2+ flux modulates automaticity in ventricular cardiomyocytes and correlates with arrhythmic risk.

Automaticity involves Ca2+ handling at the cell membrane and sarcoplasmic reticulum (SR). Abnormal or acquired automaticity is thought to initiate ventricular arrhythmias associated with myocardial ischemia. Ca2+ flux from mitochondria can influence automaticity, and lysosomes also release Ca2+. Therefore, we tested whether lysosomal Ca2+ flux could influence automaticity. We studied ventricular human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), hiPSC 3D engineered heart tissues (EHTs), and ventricular cardiomyocytes isolated from infarcted mice. Preventing lysosomal Ca2+ cycling reduced automaticity in hiPSC-CMs. Consistent with a lysosomal role in automaticity, activating the transient receptor potential mucolipin channel (TRPML1) enhanced automaticity, and two channel antagonists reduced spontaneous activity. Activation or inhibition of lysosomal transcription factor EB (TFEB) increased or decreased total lysosomes and automaticity, respectively. In adult ischemic cardiomyocytes and hiPSC 3D EHTs, reducing lysosomal Ca2+ release also inhibited automaticity. Finally, TRPML1 was up-regulated in cardiomyopathic patients with ventricular tachycardia (VT) compared with those without VT. In summary, lysosomal Ca2+ handling modulates abnormal automaticity, and reducing lysosomal Ca2+ release may be a clinical strategy for preventing ventricular arrhythmias.

A novel BRET-based assay to investigate binding and residence time of unmodified ligands to the human lysosomal ion channel TRPML1 in intact cells

Here, we report a bioluminescence resonance energy transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to the human transient receptor potential mucolipin 1 (hTRPML1), a lysosomal ion channel involved in several genetic diseases and cancer progression. This novel BRET assay can be used to determine equilibrium and kinetic binding parameters of unlabeled compounds to hTRPML1 using intact human-derived cells, thus complementing the information obtained using functional assays based on ion channel activation. We expect this new BRET assay to expedite the identification and optimization of cell-permeable ligands that interact with hTRPML1 within the physiologically relevant environment of lysosomes.

Visfatin-Induced Smooth Muscle Cell Stiffness Syndrome during Lysosomal Acid Ceramidase Deficiency

Recent studies have reported that intrinsic stiffness of smooth muscle cells (SMCs) plays an important role in contributing to arterial wall stiffness, which led to the concept of smooth muscle cell stiffness syndrome (SMCSS). However, the molecular mechanisms regulating the intrinsic stiffness of SMCs are still poorly understood. The present study tested whether lysosomal acid ceramidase (AC) determines intrinsic stiffness of mouse aortic SMCs via regulation of actin polymerization and integrin recycling. Atom force microscopy was used to study mechanical properties of SMCs. As an injurious adipokine, visfatin was found to reduce SMC height, but elevate cortical stiffness and focal adhesion in SMCs, indicating the augmentation of intrinsic stiffness. Moreover, visfatin-induced elevation of cortical stiffness was associated with increased actin polymerization in peripheral areas of SMCs. These pathological changes were much more remarkable in SMCs from smooth muscle-specific Asah1 gene (gene code of AC) knockout (Asah1fl/fl/SMcre) mice compared to SMCs from WT/WT mice. Super-resolution microscopy showed that multivesicular bodies (MVBs) as integrin degrading organelles were much less interacted or fused with lysosomes in SMCs from Asah1fl/fl/SMcre mice than WT/WT mice upon visfatin stimulation. In contrast, MVB-α5 integrin encounters increased more in SMCs from Asah1fl/fl/SMcre mice compared to SMCs from control littermates, suggesting that AC deficiency increases α5 integrin accumulation in MVB, which may enhance recycling endosome moving to cell membrane. Given the vital role of lysosomal transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and its interaction with MVB, we tested whether this lysosomal channel is disturbed by visfatin. By GCaMP3 Ca2+ imaging and whole-lysosome patch clamp recording, we found that ML-SA5 (TRPML1 channel agonist) induced remarkable lysosomal Ca2+ release in WT/WT SMCs, which was attenuated by visfatin. In SMCs lacking Asah1 gene, however, ML-SA5 induced much less Ca2+ release from lysosomes, which was almost abolished by visfatin. Lysosome trafficking analysis showed that ML-SA5 activation of lysosomal TRPML1 channels markedly increased lysosome movement in SMCs, which was significantly inhibited by visfatin and Asah1 gene deletion. Based on these results, we concluded that lysosomal AC deficiency leads to dysregulation of actin polymerization and integrin recycling in SMCs, which may be a novel molecular mechanism of SMCSS and ultimate arterial wall stiffening during obesity. This study was supported by NIH grants HL057244 and HL075316. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Blunting ROS/TRPML1 pathway protects AFB1-induced porcine intestinal epithelial cells apoptosis by restoring impaired autophagic flux

Aflatoxin B1 (AFB1) is a stable mycotoxin that contaminates animal feed on a large scale and causes severe damage to intestinal cells, induces inflammation and stimulates autophagy. Transient receptor potential mucolipin subfamily 1 (TRPML1) is a regulatory factor of autophagy, but the underlying mechanisms of TRPML1-mediated autophagy in AFB1 intestine toxicity remain elucidated. In the present study, AFB1 (0, 5, 10 μg/mL) was shown to reduce cell viability, increase reactive oxygen species (ROS) accumulation and apoptosis rate. Additionally, AFB1 caused structural damage to mitochondria and lysosomes and increased autophagosomes numbers. Furthermore, AFB1 promoted Ca2+ release by activating the TRPML1 channel, stimulated the expression of autophagy-related proteins, and induced autophagic flux blockade. Moreover, pharmacological inhibition of autophagosome formation by 3-methyladenine attenuated AFB1-induced apoptosis by downregulating the levels of TRPML1 and ROS, whereas blockade of autophagosome-lysosomal fusion by chloroquine alleviated AFB1-induced apoptosis by upregulating TRPML1 expression and exacerbating ROS accumulation. Intriguingly, blocking AFB1-induced autophagic flux generated ROS- and TRPML1-dependent cell death, as shown by the decreased apoptosis in the presence the free radical scavenger N-Acetyl-L-cysteine and the TRPML1 inhibitor ML-SI1. Overall, these results showed that AFB1 promoted apoptosis of IPEC-J2 cells by disrupting autophagic flux through activation of the ROS/TRPML1 pathway.

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.

Transient Receptor Potential Mucolipin-1 Participates in Intracerebral Hemorrhage-Induced Secondary Brain Injury by Inducing Neuroinflammation and Neuronal Cell Death.

Transient receptor potential mucolipin-1 (TRPML1) is the most abundantly and widely expressed channel protein in the TRP family. While numerous studies have been conducted involving many aspects of TRPML1, such as its role in cell biology, oncology, and neurodegenerative diseases, there are limited reports about what role it plays in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Here we examined the function of TRPML1 in ICH-induced SBI. The caudal arterial blood of rats was injected into the caudate nucleus of basal ganglia to establish an experimental ICH model. We observed that lentivirus downregulated the expression level of TRPML1 and chemical agonist promoted the enzyme activity of TRPML1. The results indicated that the protein levels of TRPML1 in brain tissues increased 24h after ICH. These results suggested that downregulated TRPML1 could significantly reduce inflammatory cytokines, and ICH induced the production of LDH and ROS. Furthermore, TRPML1 knockout relieved ICH-induced neuronal cell death and degeneration, and declines in learning and memory after ICH could be improved by downregulating the expression of TRPML1. In addition, chemical agonist-expressed TRPML1 showed the opposite effect and exacerbated SBI after ICH. In summary, this study demonstrated that TRPML1 contributed to brain injury after ICH, and downregulating TRPML1 could improve ICH-induced SBI, suggesting a potential target for ICH therapy.

Lysosomal Ca2+-flux contributes to abnormal automaticity-related arrhythmia in ventricular cardiomyopathy.

Abnormal automaticity contributes to ventricular arrhythmias during cardiac ischemia. This mechanism involves Ca2+ handling at the membrane and the sarcoplasmic reticulum (SR). We have described a contribution of mitochondrial Ca2+ flux to automaticity. Recent publications showed that lysosomal Ca2+ release influences SR Ca2+ release. Therefore, we determined if lysosomal Ca2+ flux could modify abnormal automaticity in ventricular cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiomyocytes (CMs) derived from infarcted mice were studied. The spontaneous beating rate of CMs was determined from action potentials or cytoplasmic Ca2+ transients. The hiPSC-CMs transfected with lysosomal transient receptor potential mucolipin Ca2+ release channel (TRPML1) siRNA had significantly reduced automaticity and a significantly increased SR Ca2+ release time constant. Moreover, the TRPML1 specific agonist, ML-SA1, accelerated the spontaneous beating while a TRPML1 specific antagonist, ML-SI1 substantially decreased this frequency. Proteasome inhibition by MG132 activated lysosomal transcription factor EB (TFEB). TFEB activation increased lysosomes and resulted in increased automaticity. Proteasome inhibition had the opposite effect. Blocking the lysosomal two-pore Ca2+ channel (TPC2) or inhibiting the V-type ATPase responsible for lysosomal Ca2+ loading had similar effects on automaticity. Furthermore, inhibition lysosomal Ca2+ release reduced the abnormal automaticity in adult cardiomyocytes isolated after myocardial infarction. As expected based upon hiPSC-CMs and mouse cardiomyocytes data, the protein level of TRPML1 in heart failure (HF) patients with tachycardia (VT) was significantly enhanced when compared with that in HF patients without VT. Lysosomes Ca2+-cycling modulated automaticity in ventricular hiPSC-CMs or cardiomyocytes from infarcted mouse hearts and contributed to HF patient VT events, implying that changes in lysosomal Ca2+ handling may play an important role in abnormal automaticity related arrhythmia.

The inhibition of TRPML1/TFEB leads to lysosomal biogenesis disorder, contributes to developmental fluoride neurotoxicity

Fluoride is capable of inducing developmental neurotoxicity; regrettably, the mechanism is obscure. We aimed to probe the role of lysosomal biogenesis disorder in developmental fluoride neurotoxicity—specifically, the regulating effect of the transient receptor potential mucolipin 1 (TRPML1)/transcription factor EB (TFEB) signaling pathway on lysosomal biogenesis. Sprague-Dawley rats were given fluoridated water freely, during pregnancy to the parental rats to 2 months after delivery to the offspring. In addition, neuroblastoma SH-SY5Y cells were treated with sodium fluoride (NaF), with or without mucolipin synthetic agonist 1 (ML-SA1) or adenovirus TFEB (Ad-TFEB) intervention. Our findings revealed that NaF impaired learning and memory as well as memory retention capacities in rat offspring, induced lysosomal biogenesis disorder, and decreased lysosomal degradation capacity, autophagosome accumulation, autophagic flux blockade, apoptosis, and pyroptosis. These changes were evidenced by the decreased expression of TRPML1, nuclear TFEB, LAMP2, CTSB, and CTSD, as well as increased expression of LC3-II, p62, cleaved PARP, NLRP3, Caspase1, and IL-1β. Furthermore, TRPML1 activation and TFEB overexpression both restored TFEB nuclear protein expression and promoted lysosomal biogenesis while enhancing lysosomal degradation capacity, recovering autophagic flux, and attenuating NaF-induced apoptosis and pyroptosis. Taken together, these results show that NaF promotes the progression of developmental fluoride neurotoxicity by inhibiting TRPML1/TFEB expression and impeding lysosomal biogenesis. Notably, the activation of TRPML1/TFEB alleviated NaF-induced developmental neurotoxicity. Therefore, TRPML1/TFEB may be promising markers of developmental fluoride neurotoxicity.

Characterization of Endo-Lysosomal Cation Channels Using Calcium Imaging.

Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca2+-permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca2+ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca2+ measurements, (2) peri-endo-lysosomal Ca2+ imaging using genetically encoded Ca2+ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca2+ imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca2+ imaging by directing Ca2+ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca2+ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca2+ imaging.

Abstract 9834: Lysosomal Ca 2+ Release Contribute to Arrhythmia After Myocardial Infarction

Introduction: In heart failure (HF), mitochondria Ca 2+ handling is involved in arrhythmogenesis by modulating diastolic sarcoplasmic reticulum (SR) Ca 2+ release. Recently, it has been reported that lysosomal Ca 2+ release can trigger a SR Ca 2+ release. Hypothesis: We investigated whether lysosomal Ca 2+ flux could contribute to ischemic HF arrhythmia in a similar manner to mitochondria. Methods: Ischemic HF was induced in wild type FVB/NJ mice by ligated left anterior descending coronary artery. Mice were studied at three weeks after myocardial infarction (MI). Results: The lysosomal-restricted transient receptor potential mucolipin 1 (TRPML1) Ca 2+ release channel, but not the two-pore channel 2, was significantly upregulated by 84.8% in cardiac border zone and 55.4% in a remote zone after MI, and cardiomyocyte TRPML1 was significantly increased in human patients with ischemic or nonischemic cardiomyopathy. As expected, TRPML1 co-localized with the lysosome marker, LAMP1. The master transcriptional lysosomal regulator, dephosphorylated TFEB (i.e., active TFEB) and LAMP1 labelled lysosomes increased after MI. Lysosomal Ca 2+ release directly as indicated by GCaMP7-TRPML1, a fluorescent Ca 2+ sensor coupled to TRPML1, was upregulated in MI mouse CMs. TRPML1 specific agonist, ML-SA1, could evoke Ca 2+ sparks in the CMs isolated from MI mice, and these sparks were inhibited by a specific antagonist, ML-SI1. Furthermore, thapsigargin or high concentration ryanodine, inhibited Ca 2+ loading and release from the SR respectively and prevented TRPML1-dependent Ca 2+ sparks in the MI CMs. TRPML1-dependent sparks were not affected by S107, a drug which specifically inhibits Ca 2+ leak, however. Blocking TRPML1depressed early and delayed afterdepolarizations (EADs and DADs) in CMs isolated from MI mice while increasing TRPML1 activity enhanced triggered activity. The inhibition of TRPML1 had no effect on the APs recorded from sham mouse CMs, however. ML-SA1 could increase induced ventricular fibrillation episodes in MI mice while ML-SI1 could reduce arrhythmia by optical mapping and pacing. Conclusions: Lysosomes contribute to arrhythmic risk in MI by releasing Ca 2+ during diastole causing SR diastolic Ca 2+ release and triggered activity.

Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.