TRPML1 Research Articles

TRPML1 gating modulation by allosteric mutations and lipids

Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel whose loss-of-function mutations directly cause the lysosomal storage disorder mucolipidosis type IV (MLIV). TRPML1 can be allosterically regulated by various ligands including natural lipids and small synthetic molecules and the channel undergoes a global movement propagated from ligand-induced local conformational changes upon activation. In this study, we identified a functionally critical residue, Tyr404, at the C-terminus of the S4 helix, whose mutations to tryptophan and alanine yield gain- and loss-of-function channels, respectively. These allosteric mutations mimic the ligand activation or inhibition of the TRPML1 channel without interfering with ligand binding and both mutant channels are susceptible to agonist or antagonist modulation, making them better targets for screening potent TRPML1 activators and inhibitors. We also determined the high-resolution structure of TRPML1 in complex with the PI(4,5)P2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P2-bound TRPML1 structure at the same hotspot for agonists and antagonists, providing a plausible structural explanation for the inhibitory effect of sphingomyelin on agonist activation.

TRPML1 gating modulation by allosteric mutations and lipids.

Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel whose loss-of-function mutations directly cause the lysosomal storage disorder mucolipidosis type IV (MLIV). TRPML1 can be allosterically regulated by various ligands including natural lipids and small synthetic molecules and the channel undergoes a global movement propagated from ligand-induced local conformational changes upon activation. In this study, we identified a functionally critical residue, Tyr404, at the C-terminus of the S4 helix, whose mutations to tryptophan and alanine yield gain- and loss-of-function channels, respectively. These allosteric mutations mimic the ligand activation or inhibition of the TRPML1 channel without interfering with ligand binding and both mutant channels are susceptible to agonist or antagonist modulation, making them better targets for screening potent TRPML1 activators and inhibitors. We also determined the high-resolution structure of TRPML1 in complex with the PI(4,5)P2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P2-bound TRPML1 structure at the same hotspot for agonists and antagonists, providing a plausible structural explanation for the inhibitory effect of sphingomyelin on agonist activation.

Lysosomal cation channel TRPML1 suppression sensitizes acute myeloid leukemia cells to chemotherapeutics by inhibiting autophagy.

Despite the implementation of novel therapeutic regimens and extensive research efforts, chemoresistance remains a formidable challenge in the treatment of acute myeloid leukemia (AML). Notably, the involvement of lysosomes in chemoresistance has sparked interest in developing lysosome-targeted therapies to sensitize tumor cells to currently approved chemotherapy or as innovative pharmacological approaches. Moreover, as ion channels on the lysosomal membrane are critical regulators of lysosomal function, they present potential as novel targets for enhancing chemosensitivity. Here, we discovered that the expression of a lysosomal cation channel, namely transient receptor potential mucolipin 1 (TRPML1), was elevated in AML cells. Inhibiting TRPML1 individually does not impact the proliferation and apoptosis of AML cells. Importantly, inhibition of TRPML1 demonstrated the potential to modulate the sensitivity of AML cells to chemotherapeutic agents. Exploration of the underlying mechanisms revealed that suppression of TRPML1 impaired autophagy while concurrently increasing the production of reactive oxygen species (ROS) and ROS-mediated lipid peroxidation (Lipid-ROS) in AML cells. Finally, the knockdown of TRPML1 significantly reduced OCI-AML3 tumor growth following chemotherapy in a mouse model of human leukemia. In summary, targeting TRPML1 represents a promising approach for combination therapy aimed at enhancing chemosensitivity in treating AML.

Multimodal Imaging in Mucolipidosis Type IV: Siblings With Novel Genetic Variant.

Utilization of multimodal imaging techniques to diagnose cases of mucolipidosis type IV (ML-IV) and report a new genetic variant. This study is a case report. Case 1 involves a 4-year-old boy with corneal haziness and global developmental delay who showed an increased reflectivity of the corneal epithelium on anterior segment optical coherence tomography (AS-OCT). In addition, neurologic evaluation was suggestive of ML-IV. Further genetics evaluation confirmed ML-IV. Histology of the button revealed a thickened epithelial basement membrane. Case 2, the younger sibling, showed a milder corneal haze with similar changes on AS-OCT prompting us to further evaluate for ML-IV by genetics (positive MCOLN1 gene mutation). Both instances highlighted varied ML-IV presentations, but a persistent feature was hyperreflective epithelium. Our study emphasizes AS-OCT's role in screening ML-IV and advocates the role of genetic counseling of affected parents. We present 2 South-Asian siblings with ML-IV with a new genetic variant, emphasizing the utility of detailed ophthalmic and neurologic assessments using multimodal imaging.

A blood-brain barrier-penetrant AAV gene therapy improves neurological function in symptomatic mucolipidosis IV mice

Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits, and progressive vision loss. Using AAV9 and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the human MCOLN1 gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-oriented MCOLN1 gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1 in adult symptomatic Mcoln1-/- mice at a dose of 1e12 vg per mouse resulted in MCOLN1 expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression of MCOLN1 transcripts was also detected in the retina of the mouse, which had exhibited significant degeneration at the time of the treatment. However, no increase in retinal thickness was observed after the gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies.

Lysosomal Channels as New Molecular Targets in the Pharmacological Therapy of Neurodegenerative Diseases via Autophagy Regulation.

Besides controlling several organellar functions, lysosomal channels also guide the catabolic "self-eating" process named autophagy, which is mainly involved in protein and organelle quality control. Neuronal cells are particularly sensitive to the rate of autophagic flux either under physiological conditions or during the degenerative process. Accordingly, neurodegeneration occurring in Parkinson's (PD), Alzheimer's (AD), and Huntington's Diseases (HD), and Amyotrophic Lateral Sclerosis (ALS) as well as Lysosomal Storage Diseases (LSD) is partially due to defective autophagy and accumulation of toxic aggregates. In this regard, dysfunction of lysosomal ionic homeostasis has been identified as a putative cause of aberrant autophagy. From a therapeutic perspective, Transient Receptor Potential Channel Mucolipin 1 (TRPML1) and Two-Pore Channel isoform 2 (TPC2), regulating lysosomal homeostasis, are now considered promising druggable targets in neurodegenerative diseases. Compelling evidence suggests that pharmacological modulation of TRPML1 and TPC2 may rescue the pathological phenotype associated with autophagy dysfunction in AD, PD, HD, ALS, and LSD. Although pharmacological repurposing has identified several already used drugs with the ability to modulate TPC2, and several tools are already available for the modulation of TRPML1, many efforts are necessary to design and test new entities with much higher specificity in order to reduce dysfunctional autophagy during neurodegeneration.

Coxiella burnetii effector CvpE maintains biogenesis of Coxiella-containing vacuoles by suppressing lysosome tubulation through binding PI(3)P and perturbing PIKfyve activity on lysosomes

ABSTRACT Coxiella burnetii (C. burnetii) is the causative agent of Q fever, a zoonotic disease. Intracellular replication of C. burnetii requires the maturation of a phagolysosome-like compartment known as the replication permissive Coxiella-containing vacuole (CCV). Effector proteins secreted by the Dot/Icm secretion system are indispensable for maturation of a single large CCV by facilitating the fusion of promiscuous vesicles. However, the mechanisms of CCV maintenance and evasion of host cell clearance remain to be defined. Here, we show that C. burnetii secreted Coxiella vacuolar protein E (CvpE) contributes to CCV biogenesis by inducing lysosome-like vacuole (LLV) enlargement. LLV fission by tubulation and autolysosome degradation is impaired in CvpE-expressing cells. Subsequently, we found that CvpE suppresses lysosomal Ca2+ channel transient receptor potential channel mucolipin 1 (TRPML1) activity in an indirect manner, in which CvpE binds phosphatidylinositol 3-phosphate [PI(3)P] and perturbs PIKfyve activity in lysosomes. Finally, the agonist of TRPML1, ML-SA5, inhibits CCV biogenesis and C. burnetii replication. These results provide insight into the mechanisms of CCV maintenance by CvpE and suggest that the agonist of TRPML1 can be a novel potential treatment that does not rely on antibiotics for Q fever by enhancing Coxiella-containing vacuoles (CCVs) fission.

Intersection of chronic contractile activity and lysosomal defects in mediating lysosomal adaptations in skeletal muscle cells

Mitophagy is the cellular process that serves to degrade mitochondria when these organelles lose their potential for ATP production, generate excessive reactive oxygen species, and exhibit a reduced membrane potential. The terminal step of mitophagy is mediated by the lysosome, the organelle that degrades defective cellular cargos. Electron microscopy evidence shows that lysosomes become defective in aging muscle, as well as in lysosomal storage diseases, ultimately leading to cellular pathology. Recent research has shown that chronic contractile activity can induce rapid increases in lysosomal gene and protein expression in muscle. Our objective is to investigate whether this adaptation leads to greater lysosomal content and function, thus enhancing the capacity to degrade defective mitochondria. This result would suggest that reduced lysosomal function in muscle could be rescued by exercise. However, the underlying mechanisms mediating lysosomal turnover and function are not clear. To study this, we simulated lysosomal dysfunction in C2C12 myotubes using short-interfering RNA targeting the lysosomal calcium channel mucolipin-1 (MCOLN1) or the autophagosome-lysosome fusion protein, lysosomal-associated membrane protein 2 (LAMP2). We achieved a 60-70% knockdown, as well as a 40-50% knockdown, after a 24-hour transfection, in each lysosomal marker, respectively. The cells were then subjected to four days of electrical stimulation-induced chronic contractile activity. Changes in gene expression were measured via qPCR, while protein content was measured via western blotting techniques, in addition to lysosomal content and functional assessments via confocal microscopy and flow cytometry. Silencing of MCOLN1 or LAMP2 both resulted in a compensatory increase in the transcript and protein levels of lysosomal genes, including the lysosomal transcription factors TFEB, TFE3, as well as lysosomal cathepsins B and D. Interestingly, contractile activity superimposed on the MCOLN1 or LAMP2 knockdown did not further enhance these compensatory increases. This suggests that contractile activity and the cellular signaling that arises from lysosomal defects, operate similarly to initiate the biogenesis of lysosomes as a negative feedback mechanism to enhance lysosomal degradation capacity. The nature of these signaling pathways, potentially involving changes in lysosome-derived intracellular calcium driving organelle synthesis, recycling, or degradation represent novel avenues for upcoming research investigating skeletal muscle phenotypic adaptations. This work is supported by NSERC. This is the full abstract presented at the American Physiology Summit 2024 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.

A Novel Homozygous Variant in the MCOLN1 Gene Associated With Severe Oromandibular Dystonia and Parkinsonism.

Mucolipidosis type IV (MLIV) is a rare, progressive lysosomal storage disorder characterized by severe intellectual disability, delayed motor milestones and ophthalmologic abnormalities. MLIV is an autosomal recessive disease caused by mutations in the MCOLN1 gene, encoding mucolipin-1 which is responsible for maintaining lysosomal function. Here, we report a family of four Iranian siblings with cognitive decline, progressive visual and pyramidal disturbances, and abnormal movements manifested by severe oromandibular dystonia and parkinsonism. MRI scans of the brain demonstrated signal abnormalities in the white matter and thinning of the corpus callosum. Whole-exome sequencing identified a novel homozygous variant, c.362C > T:p. Thr121Met in the MCOLN1 gene consistent with a diagnosis of MLIV. The presentation of MLIV may overlap with a variety of other neurological diseases, and genetic analysis is an important strategy to clarify the diagnosis. This is an important point that clinicians should be familiar with. The novel variant c.362C > T:p. Thr121Met herein described may be related to a comparatively older age at onset. Our study also expands the clinical spectrum of MLIV associated with the MCOLN1 variants and introduces a novel likely pathogenic variant for testing in MLIV cases that remain unresolved.

TRPML1 as a potential therapeutic target for triple-negative breast cancer: a review.

Triple-negative breast cancer (TNBC) is the most refractory subtype of breast cancer, and effective treatments are urgently needed owing to its poor prognosis. Surgery, radiotherapy, and chemotherapy, alone or in combination, are the leading choices for TNBC therapy. Although promising approaches and procedures have emerged, several challenges, such as off-target effects, drug resistance, and severe side effects, remain to be addressed. Recently, transient receptor potential channel mucolipin 1 (TRPML1) has attracted the attention of researchers because its expression has been implicated in numerous diseases, including cancer. TRPML1 regulates biological events and signaling pathways, including autophagic flux, exocytosis, ionic homeostasis, and lysosomal biogenesis, all contributing to tumorigenesis and cancer progression. TRPML1 also functions as a building block for cancer cell growth, mitogenic signaling, priming tissues for metastasis, and activation of transcriptional programs, processes involved in several malignant tumors. This review provides an overview of breast cancer epidemiology and diagnostic techniques and then discusses the existing therapeutics. Additionally, we elaborate on the development of, and associated challenges to, TNBC diagnostics and treatment and the feasibility of TRPML1 as a therapeutic target for TNBC.

Neurodevelopmental delay, corneal clouding, hypomyelinating leukoencephalopathy: think mucolipidosis IV

Mucolipidosis type IV (MLIV) is an ultra-rare autosomal recessive lysosomal disorder characterized by typical neurological (early-onset developmental delay, spasticity), ocular (corneal clouding, retinopathy), and characteristic MRI findings (hypomyelinating leukoencephalopathy, thin corpus callosum, cerebellar atrophy). Although MLIV is predominantly reported in patients in the Ashkenazi Jewish community, it is a pan-ethnic disorder. The authors report on two siblings of Armenian origin with global neurodevelopmental delay, ophthalmological abnormalities and characteristic MRI features suggesting MLIV. Whole-Exome Sequencing confirmed two pathogenic variants in the MCOLN1 gene. This constellation of clinical and neuroimaging findings has been confirmed to provide a basis for the diagnosis of MLIV, and it is suggested that a focused diagnostic work-up should be considered for unexplained neurodevelopmental disorders.

Lisch Epithelial Corneal Dystrophy Is Caused by Heterozygous Loss-of-Function Variants in MCOLN1

To report the genetic etiology of Lisch epithelial corneal dystrophy (LECD). Multicenter cohort study. A discovery cohort of 27 individuals with LECD from 17 families, including 7 affected members from the original LECD family, 6 patients from 2 new families and 14 simplex cases, was recruited. A cohort of 6 individuals carrying a pathogenic MCOLN1 (mucolipin 1) variant was reviewed for signs of LECD. Next-generation sequencing or targeted Sanger sequencing were used in all patients to identify pathogenic or likely pathogenic variants and penetrance of variants. Nine rare heterozygous MCOLN1 variants were identified in 23 of 27 affected individuals from 13 families. The truncating nature of 7 variants and functional testing of 1 missense variant indicated that they result in MCOLN1 haploinsufficiency. Importantly, in the homozygous and compound-heterozygous state, 4 of 9 LECD-associated variants cause the rare lysosomal storage disorder mucolipidosis IV (MLIV). Autosomal recessive MLIV is a systemic disease and comprises neurodegeneration as well as corneal opacity of infantile-onset with epithelial autofluorescent lysosomal inclusions. However, the 6 parents of 3 patients with MLIV confirmed to carry pathogenic MCOLN1 variants did not have the LECD phenotype, suggesting MCOLN1 haploinsufficiency may be associated with reduced penetrance and variable expressivity. MCOLN1 haploinsufficiency is the major cause of LECD. Based on the overlapping clinical features of corneal epithelial cells with autofluorescent inclusions reported in both LECD and MLIV, it is concluded that some carriers of MCOLN1 haploinsufficiency-causing variants present with LECD.

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.

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.

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.

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.

Sulforaphane protects microvascular endothelial cells in lower limb ischemia/reperfusion injury mice.

Background: Microvascular damage is a key pathological factor in acute lower limb ischemia/reperfusion (I/R) injury. Current evidence suggests that sulforaphane (SFN) protects tissue from I/R injury. However, the role of SFN in acute lower limb I/R injury remains elusive. This study aimed to investigate the role and potential mechanism of SFN in I/R-related microvascular damage in the limb. Methods: Limb viability was evaluated by laser Doppler imaging, tissue edema analysis and histological analysis. Western blotting and immunofluorescence were applied to analyze the levels of apoptosis, oxidative stress, autophagy, transcription factor EB (TFEB) activity and mucolipin 1 (MCOLN1)-calcineurin signaling pathway. Results: SFN administration significantly ameliorated I/R-induced hypoperfusion, tissue edema, skeletal muscle fiber injury and endothelial cell (EC) damage in the limb. Pharmacological inhibition of NFE2L2 (nuclear factor, erythroid 2 like 2) reversed the anti-oxidation and anti-apoptosis effects of SFN on ECs. Additionally, silencing of TFEB by interfering RNA abolished the SFN-induced autophagy restoration, anti-oxidant response and anti-apoptosis effects on ECs. Furthermore, silencing of MCOLN1 by interfering RNA and pharmacological inhibition of calcineurin inhibited the activity of TFEB induced by SFN, demonstrating that SFN regulates the activity of TFEB through the MCOLN1-calcineurin signaling pathway. Conclusion: SFN protects microvascular ECs against I/R injury by TFEB-mediated autophagy restoration and anti-oxidant response.

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.

Autophagic Deficiency and Dedifferentiation in Podocytes of Mice Lacking Acid Ceramidase (Asah1) Gene

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain with a podocyte‐specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Asah1fl/fl/Podocre, Asah1 is a gene code of mouse acid ceramidase). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether autophagic deficiency and associated dedifferentiation of podocytes contribute to the pathogenesis of podocytopathy in Asah1fl/fl/Podocre mice. By Western blot analysis and immunofluorescent staining, we demonstrated the significant reduction of epithelial markers (ZO‐1 and P‐cadherin) and remarkable elevation of mesenchymal markers (α‐SMA and FSP‐1) in podocytes lack of Asah1 gene in vivo and in vitro. Also, we found that Asah1 gene knockout induced autophagic deficiency in podocytes, which were evidenced by autophagosome accumulation and decreased lysosome‐autophagosome interaction in these cells. Given the vital role of transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and interaction with other vehicles, we tested whether this lysosomal channel is implicated in autophagic deficiency in podocytes lacking Asah1 gene. As a selective TRPML1 channel agonist, ML‐SA5 enhanced lysosome‐autophagosome interaction in WT/WT podocytes, but only had partial effects on this interaction in Asah1fl/fl/Podocre podocytes. Autophagosome accumulation was also attenuated by ML‐SA5 in podocytes lacking Asah1 gene. On the contrary, ML‐SI1 as a TRPML1 channel inhibitor impaired autophagic flux in WT/WT podocytes and amplified autophagic deficiency in Asah1fl/fl/Podocre podocytes. The enhancements of lysosome trafficking and lysosome‐autophagosome interaction by ML‐SA5 were blocked by dynein inhibitors, EHNA and ciliobrevin D, indicating the contribution of dynein to lysosome movement in response to TRPML1 channel‐mediated Ca2+ release. Pathologically, Asah1 gene deletion‐induced podocyte dedifferentiation, reduction of slit diaphragm proteins, and proteinuria were inhibited by ML‐SA5 but enhanced by ML‐SI1. Based on these results, we conclude that Asah1 gene deletion induces autophagic deficiency and dedifferentiation in podocytes through blockade of lysosomal TRPML1 channel, thereby leading to the development of podocytopathy and nephrotic syndrome in Asah1fl/fl/Podocre mice.

Structural mechanism of allosteric activation of TRPML1 by PI(3,5)P2 and rapamycin

Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-permeable, nonselective cation channel ubiquitously expressed in the endolysosomes of mammalian cells and its loss-of-function mutations are the direct cause of type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease. TRPML1 is a ligand-gated channel that can be activated by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] as well as some synthetic small-molecule agonists. Recently, rapamycin has also been shown to directly bind and activate TRPML1. Interestingly, both PI(3,5)P2 and rapamycin have low efficacy in channel activation individually but together they work cooperatively and activate the channel with high potency. To reveal the structural basis underlying the synergistic activation of TRPML1 by PI(3,5)P2 and rapamycin, we determined the high-resolution cryoelectron microscopy (cryo-EM) structures of the mouse TRPML1 channel in various states, including apo closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus (a rapamycin analog)-bound open states. These structures, combined with electrophysiology, elucidate the molecular details of ligand binding and provide structural insight into how the TRPML1 channel integrates two distantly bound ligand stimuli and facilitates channel opening.