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Mitochondria and lysosomes are critical for cellular homeostasis, and dysfunction of both organelles has been implicated in numerous diseases. Recently, interorganelle contacts between mitochondria and lysosomes were identified and found to regulate mitochondrial dynamics. However, whether mitochondria–lysosome contacts serve additional functions by facilitating the direct transfer of metabolites or ions between the two organelles has not been elucidated. Here, using high spatial and temporal resolution live-cell microscopy, we identified a role for mitochondria–lysosome contacts in regulating mitochondrial calcium dynamics through the lysosomal calcium efflux channel, transient receptor potential mucolipin 1 (TRPML1). Lysosomal calcium release by TRPML1 promotes calcium transfer to mitochondria, which was mediated by tethering of mitochondria–lysosome contact sites. Moreover, mitochondrial calcium uptake at mitochondria–lysosome contact sites was modulated by the outer and inner mitochondrial membrane channels, voltage-dependent anion channel 1 and the mitochondrial calcium uniporter, respectively. Since loss of TRPML1 function results in the lysosomal storage disorder mucolipidosis type IV (MLIV), we examined MLIV patient fibroblasts and found both altered mitochondria–lysosome contact dynamics and defective contact-dependent mitochondrial calcium uptake. Thus, our work highlights mitochondria–lysosome contacts as key contributors to interorganelle calcium dynamics and their potential role in the pathophysiology of disorders characterized by dysfunctional mitochondria or lysosomes.

Abstract Introduction/Objective Mucolipidosis type IV (MLIV) is a lysosomal storage disorder with a gastric phenotype. Patients develop achlorhydia and compensatory hypergastrinemia, with pathology showing vacuolated parietal cells with lysosomal inclusions. MLIV is caused by mutation of TRPML1, a lysosomal membrane calcium channel required for trafficking proton pumps to the apical surface in parietal cells. We report the first case of MLIV predisposing to a neuroendocrine tumor (NET). This novel finding could impact management of patients with this disease, who may require frequent monitoring for NETs. We also show that immunohistochemistry for TRPML1 and H+/K+ ATPase can be used in conjunction to identify gastric MLIV. We hope that our updated immunohistochemical tools can aid in diagnosis of this disease. Methods/Case Report 34-year-old woman with MLIV requiring endoscopic resection for an incidentally discovered gastric polyp. Pathology showed Grade 2 NET invasion into submucosa (pT1). Background mucosa showed markedly vacuolated parietal cells, ranging from unilocular to multilocular with up to 20 vacuoles per cell. We stained the patient’s and a control stomach with antibodies against the TRPML1 and H+/K+ ATPase proteins. The H+/K+ ATPase antibody highlighted healthy parietal cells in the control stomach with strong cytoplasmic signal, but strikingly outlined lysosomal inclusions in MLIV parietal cells. TRPML-1 antibody showed cytoplasmic staining in parietal cells in the control. However, TRPML-1 staining showed markedly decreased signal in the patient and nuclear staining, possibly representing mislocalization. Results (if a Case Study enter NA) NA Conclusion We present the first case of MLIV predisposing to a NET in the stomach. NETs have potential for metastatic spread with subsequent mortality risk. Clinicians should maintain suspicion for NET development in this population, and protocols for frequent surveillance should be considered. This is also the first report using immunohistochemistry for TRPML1 and H+/K+ ATPase in combination to identify affected parietal cells in MLIV. Possibly, these tools can be used in the future by pathologists to diagnose MLIV.

Transient receptor potential mucolipin 1 (TRPML1) is a Ca 2+ -releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis. Two structures of human transient receptor potential mucolipin 1 (TRPML1), in the closed and agonist-bound open states, have been resolved by electron cryo-microscopy. Closing in on ion channels Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca( II )-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation.

A cryo-electron microscopy structure shows that the mucolipin domain of the lysosomal calcium channel TRPML3 binds phosphatidylinositol-3,5-bisphosphate and gates the channel. Closing in on ion channels Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca( II )-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation. The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily 1 , 2 and is composed of three members: TRPML1–TRPML3. TRPMLs are the major Ca 2+ -permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca 2+ from organelles, which is important for various physiological processes, including organelle trafficking and fusion 3 . Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint–waddler ( Va ) mouse phenotype 4 , 5 , 6 . Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P 2 ), whereas other phosphoinositides such as PtdIns(4,5)P 2 , which is enriched in plasma membranes, inhibit TRPMLs 7 , 8 . Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P 2 and inhibition by PtdIns(4,5)P 2 8 . However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P 2 and increase their Ca 2+ conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset ( Callithrix jacchus ) at an overall resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P 2 binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.

Background: Protein aggregates are degraded via the autophagy-lysosome pathway and alterations in the lysosomal system lead to the accumulation of pathogenic proteins, including aggregates of α-synuclein in Parkinson’s disease (PD). The importance of the endolysosomal transient receptor potential cation channel, mucolipin subfamily 1 (TRPML1) for lysosomal function is highlighted by the fact that TRPML1 mutations cause the lysosomal storage disease Mucolipidosis type IV. In this study, we investigated the mechanism by which activation of TRPML1 affects degradation of α-synuclein. Methods: As a model of α-synuclein pathology, we expressed the pathogenic A53T a-synuclein mutant in HEK293T cells. These cells were treated with the synthetic TRPML1 agonist ML-SA1. The amount of α-synuclein protein was determined by immunoblots. The abundance of aggregates and autolysosomal vesicles was determined by fluorescence microscopy and immunocytochemistry. Findings were confirmed by life-cell imaging and by application of ML-SA1 and the TRPML1 antagonist ML-SI3 to human dopaminergic neurons and human stem cell-derived neurons. Results: ML-SA1 reduced the percentage of HEK293T cells with a-synuclein aggregates and the amount of a-synuclein protein. The effect of ML-SA1 was blocked by pharmacological and genetic inhibition of autophagy. Consistent with TRPML function, it required the membrane lipid PI(3,5)P2 and cytosolic calcium. ML-SA1 shifted the composition of autophagosomes towards a higher fraction of mature autolysosomes, also in presence of α-synuclein. In neurons, inhibition of TRPML1 by its antagonist ML-SI3 blocked autophagosomal clearance, whereas the agonist ML-SA1 shifted the composition of α-synuclein particles towards a higher fraction of acidified particles. ML-SA1 was able to override the effect of Bafilomycin A1, which blocks the fusion of the autophagosome and lysosome and its acidification. Conclusion: These findings suggest, that activating TRPML1 with ML-SA1 facilitates clearance of α-synuclein aggregates primarily by affecting the late steps of the autophagy, i.e. by promoting autophagosome maturation. In consent with recent work by others, our findings indicate that TRPML1 might constitute a plausible therapeutic target for PD, that warrants further validation in rodent models of α-synuclein pathology.

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)P 2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P 2 -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.

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 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 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 variants and introduces a novel likely pathogenic variant for testing in MLIV cases that remain unresolved.

Efficient lysosomal Ca 2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca 2+ -activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca 2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca 2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca 2+ release was compromised in these LSDs. BK activation corrected the impaired Ca 2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs.

Mucolipidosis IV (MLIV) is an ultra-rare, recessively inherited lysosomal disorder resulting from inactivating mutations in MCOLN1 , the gene encoding the lysosomal cation channel TRPML1. The disease primarily affects the central nervous system (CNS) and manifests in the first year with cognitive and motor developmental delay, followed by a gradual decline in neurological function across the second decade of life, blindness, and premature death in third or fourth decades. Brain pathology manifestations in MLIV are consistent with hypomyelinating leukodystrophy with brain iron accumulation. Presently, there are no approved or investigational therapies for MLIV, and pathogenic mechanisms remain largely unknown. The MLIV mouse model, Mcoln1 −/− mice, recapitulates all major manifestations of the human disease. Here, to better understand the pathological mechanisms in the MLIV brain, we performed cell type specific LC–MS/MS proteomics analysis in the MLIV mouse model and reconstituted molecular signatures of the disease in either freshly isolated populations of neurons, astrocytes, oligodendrocytes, and neural stem cells, or whole tissue cortical homogenates from young adult symptomatic Mcoln1 −/− mice. Our analysis confirmed on the molecular level major histopathological hallmarks of MLIV universally present in Mcoln1 −/− tissue and brain cells, such as hypomyelination, lysosomal dysregulation, and impaired metabolism of lipids and polysaccharides. Importantly, pathway analysis in brain cells revealed mitochondria-related alterations in all Mcoln1 −/− brain cells, except oligodendrocytes, that was not possible to resolve in whole tissue. We also report unique proteome signatures and dysregulated pathways for each brain cell population used in this study. These data shed new light on cell-intrinsic mechanisms of MLIV and provide new insights for biomarker discovery and validation to advance translational studies for this disease.

Background Mucolipidosis type IV (MLIV) is a rare autosomal recessive lysosomal storage disorder due to biallelic pathogenic variants in the MCOLN1 gene. Its main impact is on the central nervous system, leading to severe psychomotor delays, progressive visual impairment, and characteristic brain abnormalities. Methods A 12-year-old male from a consanguineous Iranian family underwent clinical and imaging evaluations for suspected MLIV. Exome sequencing identified the causative variant, confirmed by Sanger co-segregation analysis, in silico tools assessed pathogenicity, protein stability, and structural impact, followed by 3D modeling (I-TASSER) and protein interaction analysis (STRING). Molecular dynamics simulations were performed with GROMACS 2020.4 employing the GROMOS96 43a1 force field to compare wild-type and mutant structures, evaluating key parameters, including root mean square deviation (RMSD), radius of gyration (Rg), hydrogen bond profiles, and solvent-accessible surface area (SASA), were analyzed, and results which were visualized using GraphPad Prism. Results Exome sequencing revealed a previously unreported homozygous nonsense variant in MCOLN1 (NM_020533.3: c.1384G > T; p.Glu462). This variant introduces a premature termination codon predicted to yield a truncated protein if translated; however, it is likely subject to nonsense-mediated mRNA decay, leading to transcript degradation and consequent loss of functional protein. Sanger sequencing confirmed the variant and its co-segregation within the family, with both parents heterozygous carriers and the patient homozygous. Bioinformatic analysis classified the variant as likely pathogenic, with high deleteriousness scores. Structural modeling indicated disruption of a helical domain. STRING analysis demonstrated strong functional associations between MCOLN1 and its paralogs MCOLN2 and MCOLN3, supporting its biological relevance. This variant expands the known spectrum of genetic causes of MLIV. Conclusion We report the first Iranian case of MLIV due to a novel homozygous nonsense variant in MCOLN1 (c.1384G > T; p.Glu462*). These findings expand the spectrum of MLIV, underscore phenotypic variability and the value of population-specific genetic data in rare disease diagnostics, and support the inclusion of this variant in targeted diagnostic panels for Iranian patients.