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Heat shock can be a lethal stressor. Previously, we described a CUL-6/cullin-ring ubiquitin ligase complex in the nematode Caenorhabditis elegans that is induced by intracellular intestinal infection and proteotoxic stress and that promotes improved survival upon heat shock (thermotolerance). Here, we show that CUL-6 promotes thermotolerance by targeting the heat shock protein HSP-90 for degradation. We show that CUL-6-mediated lowering of HSP-90 protein levels, specifically in the intestine, improves thermotolerance. Furthermore, we show that lysosomal function is required for CUL-6-mediated promotion of thermotolerance and that CUL-6 directs HSP-90 to lysosome-related organelles upon heat shock. Altogether, these results indicate that a CUL-6 ubiquitin ligase promotes organismal survival upon heat shock by promoting HSP-90 degradation in intestinal lysosomes. Thus, HSP-90, a protein commonly associated with protection against heat shock and promoting degradation of other proteins, is itself degraded to protect against heat shock.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimers disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

The yeast vacuole membrane can phase separate into ordered and disordered domains, a phenomenon that is required for micro-lipophagy under nutrient limitation. Despite its importance as a biophysical model and physiological significance, it is not yet resolved if specific lipidome changes drive vacuole phase separation. Here we report that the metabolism of sphingolipids (SLs) and their sorting into the vacuole membrane can control this process. We first developed a vacuole isolation method to identify lipidome changes during the onset of phase separation in early stationary stage cells. We found that early stationary stage vacuoles are defined by an increased abundance of putative raft components, including 40% higher ergosterol content and a nearly 3-fold enrichment in complex SLs (CSLs). These changes were not found in the corresponding whole cell lipidomes, indicating that lipid sorting is associated with domain formation. Several facets of SL composition-headgroup stoichiometry, longer chain lengths, and increased hydroxylations-were also markers of phase-separated vacuole lipidomes. To test SL function in vacuole phase separation, we carried out a systematic genetic dissection of their biosynthetic pathway. The abundance of CSLs controlled the extent of domain formation and associated micro-lipophagy processes, while their headgroup composition altered domain morphology. These results suggest that lipid trafficking can drive membrane phase separation in vivo and identify SLs as key mediators of this process in yeast.

Macrophages and autophagy are intricately linked, both playing vital roles in maintaining homeostasis and responding to disease. Macrophages, known for their ‘eating’ function, rely on a sophisticated digestion system to process a variety of targets, from apoptotic cells to pathogens. The connection between macrophages and autophagy is established early in their development, influencing both differentiation and mature functions. Autophagy regulates essential immune functions, such as inflammation control, pathogen clearance, and antigen presentation, linking innate and adaptive immunity. Moreover, it modulates cytokine production, ensuring a balanced inflammatory response that prevents excessive tissue damage. Autophagy also plays a critical role in macrophage polarization, influencing their shift between pro‐inflammatory and anti‐inflammatory states. This review explores the role of autophagy in macrophages, emphasizing its impact across various tissues and pathological conditions, and detailing the cellular and molecular mechanisms by which autophagy shapes macrophage function. [ABSTRACT FROM AUTHOR]

Acquired drug resistance is a major challenge in the management of cancer, which underscores the need for discovery and development of novel therapeutic strategies. We report here the mechanism of the anti-cancer activity of a small coordinate complex composed of the rare earth metal praseodymium (Pr) and mercaptopyridine oxide (MPO; pyrithione). Exposure of cancer cells to relatively low concentrations of the conjugate Pr-MPO (5 µM) significantly impairs cell survival in a p53-independent manner and irrespective of the drug resistant phenotype. Mechanistically, Pr-MPO-induced cell death is caspase-independent, not inhibitable by necrostatin, but associated with the appearance of autophagy markers. However, further analysis revealed incomplete autophagic flux, thus suggesting altered integrity of lysosomal machinery. Supporting the lysosomal targeting activity are data demonstrating increased lysosomal Ca2+ accumulation and alkalinization, which coincides with cytosolic acidification (drop in pHc from 7.75 to 7.00). In parallel, an increase in lysosomal activity of glycosidase alpha acid (GAA), involved in passive glycogen breakdown, correlates with rapid depletion of glucose stores upon Pr-MPO treatment. This is associated with swift cataplerosis of TCA cycle intermediates, loss of NAD+/NADH and increase in pyruvate dehydrogenase (PDH) activity to compensate for pyruvate loss. Addition of exogenous pyruvate rescued cell survival. Notably, lysosomal impairment and metabolic catastrophe triggered by Pr-MPO are suggestive of Zn2+-mediated cytotoxicity, which is confirmed by the ability of Zn2+ chelator TPEN to block Pr-MPO-mediated anti-tumor activity. Together, these results highlight the ability of the small molecule lanthanide conjugate to target the cells' waste clearing machinery as well as mitochondrial metabolism for Zn2+-mediated execution of cancer cells, which could have therapeutic potential against cancers with high metabolic activity. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is activated in many pathophysiological conditions, leading to TBK1-dependent interferon production in higher organisms. However, primordial functions of STING independent of TBK1 are poorly understood. Here, through proteomics and bioinformatics approaches, we identify lysosomal biogenesis as an unexpected function of STING. Transcription factor EB (TFEB), an evolutionarily conserved regulator of lysosomal biogenesis and host defense, is activated by STING from multiple species, including humans, mice, and frogs. STING-mediated TFEB activation is independent of TBK1, but it requires STING trafficking and its conserved proton channel. GABARAP lipidation, stimulated by the channel of STING, is key for STING-dependent TFEB activation. STING stimulates global upregulation of TFEB-target genes, mediating lysosomal biogenesis and autophagy. TFEB supports cell survival during chronic sterile STING activation, a common condition in aging and age-related diseases. These results reveal a primordial function of STING in the biogenesis of lysosomes, essential organelles in immunity and cellular stress resistance. [Display omitted] • TFEB and TFE3 are conserved effectors of STING, predating type I interferon signaling • Noncanonical GABARAP lipidation, triggered by the channel of STING, activates TFEB/TFE3 • STING stimulates cytoprotective upregulation of lysosomal biogenesis and autophagy • The cGAS-STING-TFEB axis is a primordial form of the antiviral DNA-sensing pathway Lv et al. discover TFEB/TFE3 as evolutionarily ancient effectors of the cGAS-STING DNA-sensing innate immune pathway. TFEB/TFE3 activation via the channel of STING integrates cytosolic DNA sensing, a general cellular stress-sensing mechanism, with lysosomal biogenesis and autophagy upregulation, conserved and general strategies for host defense and stress clearance. [ABSTRACT FROM AUTHOR]

Autophagosome (AP)–lysosome/vacuole fusion is one of the hallmarks of macroautophagy. Membrane features and changes during the fusion process have mostly been described using two-dimensional (2D) models with one AP and one lysosome/vacuole. The outer membrane (OM) of a closed mature AP has been suggested to fuse with the lysosomal/vacuolar membrane. However, the descriptions in some studies for fusion-related issues are questionable or incomplete. The correct membrane features of APs and lysosomes/vacuoles are the prerequisite for describing the fusion process. We searched the literature for representative membrane features of AP-related structures based on electron microscopy (EM) graphs of both animal and yeast cells and re-evaluated the findings. We also summarized the main 2D models describing the membrane changes during AP–lysosome/vacuole fusion in the literature. We used three-dimensional (3D) models to characterize the known and unknown membrane changes during and after fusion of the most plausible 2D models. The actual situation is more complex, since multiple lysosomes may fuse with the same AP in mammalian cells, multiple APs may fuse with the same vacuole in yeast cells, and in some mutant cells, phagophores (unclosed APs) fuse with lysosomes/vacuoles. This review discusses the membrane features and highly dynamic changes during AP (phagophore)–lysosome/vacuole fusion. The resulting information will improve the understanding of AP–lysosome/vacuole fusion and direct the future research on AP–lysosome/vacuole fusion and regeneration. [ABSTRACT FROM AUTHOR]

The mTOR inhibitor everolimus has been approved as a sequential or second‐line therapy for renal cell carcinoma (RCC). However, the development of drug resistance limits its clinical applications. This study aims to address the challenge of everolimus resistance and provide new insights into the treatment of advanced RCC. Here, the cytotoxicity of the DNA methyltransferase 1 (DNMT1) inhibitor SGI‐1027 in inducing cell vacuolation and methuosis is discovered and demonstrated for the first time. Additionally, SGI‐1027 exerts synergistic effects with everolimus, as their combination suppresses the growth, migration, and invasion of renal cancer cells. Mechanistically, apoptosis and GSDME‐dependent pyroptosis triggered by lysosomal membrane permeability (LMP) are observed. The upregulation of GSDME expression and increased lysosomal activity in renal cancer cells provide a therapeutic window for the combination of these two drugs to treat renal cancer. The combination treatment exhibits effective anti‐tumor activity and is well tolerated in a subcutaneous tumor model. Overall, this study validates and reveals the specific cytotoxicity property of SGI‐1027 and its potent synergistic effect with everolimus, offering new insights into advanced RCC therapy and everolimus‐resistance overcoming. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder with early autophagy deficits. Our study probed the role of lysosomal-related genes (LRGs) in AD. Using the Gene Expression Omnibus (GEO) database, we analyzed differentially expressed genes (DEGs) in AD. AD-related genes and lysosomal-related genes (LRGs) were extracted from public databases. Leveraging the UpSetR package, we identified differentially expressed LRGs (DE-LRGs). Subsequently, consensus cluster analysis was used to stratify AD patients into distinct molecular subtypes based on DE-LRGs. Immune cell patterns were studied via Single-Sample Gene Set Enrichment Analysis (ssGSEA). Molecular pathways were assessed through Gene Set Variation Analysis (GSVA), while Mendelian Randomization (MR) discerned potential gene-AD causations. To reinforce our bioinformatics findings, we conducted in vitro experiments. In total, 52 DE-LRGs were identified, with LAMP1, VAMP2, and CTSB as standout hub genes. Leveraging the 52 DE-LRGs, AD patients were categorized into three distinct molecular subtypes. Interestingly, the three aforementioned hub genes exhibited significant predictive accuracy for AD differentiation across the subtypes. The ssGSEA further illuminated correlations between LAMP1, VAMP2, and CTSB with plasma cells, fibroblasts, eosinophils, and endothelial cells. GSVA analysis underscored significant associations of LAMP1, VAMP2, and CTSB with NOTCH, TGFβ, and P53 pathways. Compellingly, MR findings indicated a potential causative relationship between LAMP1, CTSB, and AD. Augmenting our bioinformatics conclusions, in vitro tests revealed that LAMP1 potentially alleviates AD progression by amplifying autophagic processes. LAMP1 and CTSB emerge as potential AD biomarkers, paving the way for innovative therapeutic interventions [ABSTRACT FROM AUTHOR]

Danon disease, an X-linked dominant vacuolar cardiomyopathy and skeletal myopathy, is caused by a primary deficiency of lysosome-associated membrane protein-2 (LAMP-2). This disease is one of the autophagy-related muscle diseases. Male patients present with the triad of cardiomyopathy, myopathy, and intellectual disability, while female patients present with cardiomyopathy. The disease's leading cause of death is heart failure, and its prognostic factor is cardiomyopathy. Pathologically, the disease is characterized by the appearance of unique autophagic vacuoles with sarcolemmal features (AVSFs). Twenty-six families have been found to have this disease in Japan. It has been over 40 years since the first report of this disease by Danon et al. and over 20 years since the identification of the causative gene, LAMP2, by Nishino et al. Although the pathogenetic mechanism of Danon disease remains unestablished, the first clinical trials using AAV vectors have finally begun in recent years. The development of novel therapies is expected in the future. [ABSTRACT FROM AUTHOR]

Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome–lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS. [Display omitted] • MBP interacts with SARS-CoV-2 proteins N, E, nsp9 and nsp16 in mammalian cells. • MBP interacts and colocalizes with SARS-CoV-2 protein ORF3a in mammalian cells. • MBP counteracts ORF3a-mediated failure of the autophagy machinery. • Basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition. [ABSTRACT FROM AUTHOR]

Chlorosis in azaleas is characterized by an interveinal yellowing of leaves that is typically caused by a deficiency of iron. This condition is usually due to the inability of cells to properly acquire iron as a consequence of unfavorable conditions, such as an elevated pH, rather than insufficient iron levels. The causes and effects of chlorosis were found to have similarities with those pertaining to a recently presented hypothesis that describes a pathogenic process in Alzheimer disease. This hypothesis states that iron becomes sequestered (e.g., by amyloid β and tau), causing a functional deficiency of iron that disrupts biochemical processes leading to neurodegeneration. Additional mechanisms that contribute to iron becoming unavailable include iron-containing structures not undergoing proper recycling (e.g., disrupted mitophagy and altered ferritinophagy) and failure to successfully translocate iron from one compartment to another (e.g., due to impaired lysosomal acidification). Other contributors to a functional deficiency of iron in patients with Alzheimer disease include altered metabolism of heme or altered production of iron-containing proteins and their partners (e.g., subunits, upstream proteins). A review of the evidence supporting this hypothesis is presented. Also, parallels between the mechanisms underlying a functional iron-deficient state in Alzheimer disease and those occurring for chlorosis in plants are discussed. Finally, a model describing the generation of a functional iron deficiency in Alzheimer disease is put forward. [ABSTRACT FROM AUTHOR]

The autophagy-lysosome pathway plays an essential role in promoting lipid catabolism and preventing hepatic steatosis in non-alcoholic fatty liver disease (NAFLD). Transcription factor EB (TFEB) enhances the autophagy-lysosome pathway by regulating the expression of genes related to autophagy and lysosome biogenesis. Therefore, targeting TFEB provides a novel strategy for the treatment of lipid metabolic diseases. In this study, the antiallergic drug desloratadine was screened and identified as a novel TFEB agonist. Desloratadine effectively induced translocation of TFEB to the nucleus and promoted autophagy and lysosome biogenesis. Desloratadine-induced TFEB activation was dependent on AMPK rather than mTORC1. Moreover, desloratadine treatment enhanced clearance of lipid droplets in cells induced by fatty acids oleate and palmitate. Furthermore, high-fat diet (HFD) induced obesity mouse model experiments indicated treatment with desloratadine markedly reduced the body weight of HFD-fed mice, as well as the levels of hepatic triglycerides and total cholesterol, serum glutamic pyruvic transaminase and glutamic-oxaloacetic transaminase. Oil red O staining showed the liver fat was significantly reduced after desloratadine treatment, and H&E staining analysis demonstrated hepatocellular ballooning was improved. In addition, autophagy and lysosomal biogenesis was stimulated in the liver of desloratadine treated mice. Altogether, these findings demonstrate desloratadine ameliorates hepatic steatosis through activating the TFEB-mediated autophagy-lysosome pathway, thus desloratadine has an exciting potential to be used to treat fatty liver disease. [ABSTRACT FROM AUTHOR]

Our previous study showed that pyridoxine 5'-phosphate oxidase (PNPO) is a tissue biomarker of ovarian cancer (OC) and has a prognostic implication but detailed mechanisms remain unclear. The current study focused on PNPO-regulated lysosome/autophagy-mediated cellular processes and the potential role of PNPO in chemoresistance. We found that PNPO was overexpressed in OC cells and was a prognostic factor in OC patients. PNPO significantly promoted cell proliferation via the regulation of cyclin B1 and phosphorylated CDK1 and shortened the G2M phase in a cell cycle. Overexpressed PNPO enhanced the biogenesis and perinuclear distribution of lysosomes, promoting the degradation of autophagosomes and boosting the autophagic flux. Further, an autolysosome marker LAMP2 was upregulated in OC cells. Silencing LAMP2 suppressed cell growth and induced cell apoptosis. LAMP2-siRNA blocked PNPO action in OC cells, indicating that the function of PNPO on cellular processes was mediated by LAMP2. These data suggest the existence of the PNPO-LAMP2 axis. Moreover, silencing PNPO suppressed xenographic tumor formation. Chloroquine counteracted the promotion effect of PNPO on autophagic flux and inhibited OC cell survival, facilitating the inhibitory effect of PNPO-shRNA on tumor growth in vivo. Finally, PNPO was overexpressed in paclitaxel-resistant OC cells. PNPO-siRNA enhanced paclitaxel sensitivity in vitro and in vivo. In conclusion, PNPO has a regulatory effect on lysosomal biogenesis that in turn promotes autophagic flux, leading to OC cell proliferation, and tumor formation, and is a paclitaxel-resistant factor. These data imply a potential application by targeting PNPO to suppress tumor growth and reverse PTX resistance in OC. [ABSTRACT FROM AUTHOR]

In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41, core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases. Abbreviations: CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex. [ABSTRACT FROM AUTHOR]

The renoprotective effects of SLC5A2/SGLT2 (solute carrier 5 (sodium/glucose cotransporter), member 2) inhibitors have recently been demonstrated in non-diabetic chronic kidney disease (CKD), even without overt albuminuria. However, the mechanism underlying this renoprotection is largely unclear. We investigated the renoprotective mechanisms of the SLC5A2 inhibitor empagliflozin with a focus on ALB (albumin) reabsorption and macroautophagy/autophagy in proximal tubules using wild-type or drug-inducible lrp2/Megalin or atg5 knockout mice with high-fat diet (HFD)-induced obesity or 5/6 nephrectomy that elevated intraglomerular pressure without overt albuminuria. Empagliflozin treatment of HFD-fed mice reduced several hallmarks of lipotoxicity in the proximal tubules, such as phospholipid accumulation in the lysosome, inflammation and fibrosis. Empagliflozin, which decreases intraglomerular pressure, not only reduced the HFD-induced increase in ALB reabsorption via LRP2 in the proximal tubules (i.e. total nephron ALB filtration), as assessed by urinary ALB excretion caused by genetic ablation of Lrp2, but also ameliorated the HFD-induced imbalance in circulating ALB-bound fatty acids. Empagliflozin alleviated the HFD-induced increase in autophagic demand and successfully prevented autophagic stagnation in the proximal tubules. Similarly, empagliflozin decreased ALB exposure and autophagic demand in 5/6 nephrectomized mice. Finally, empagliflozin reduced HFD-induced vulnerability to ischemia–reperfusion injury, whereas LRP2 blockade and atg5 ablation separately diminished this effect. Our findings indicate that empagliflozin reduces ALB exposure and prevents autophagic stagnation in the proximal tubules even without overt albuminuria. Autophagy improvement may be critical for the renoprotection mediated by SLC5A2 inhibition. [ABSTRACT FROM AUTHOR]

Ageing is defined as the progressive loss of tissue function and regenerative capacity and is caused by both intrinsic factors i.e. the natural accumulation of damage, and extrinsic factors i.e. damage from environmental stressors. Cellular senescence, in brief, is an irreversible exit from the cell cycle that occurs primarily in response to excessive cellular damage, such as from ultraviolet (UV) exposure and oxidative stress, and it has been comprehensively demonstrated to contribute to tissue and organismal ageing. In this review, we will focus on the skin, an organ which acts as an essential protective barrier against injury, insults, and infection. We will explore the evidence for the existence and contribution of cellular senescence to skin ageing. We discuss the known molecular mechanisms driving senescence in the skin, with a focus on the dysregulation of the master growth regulator, mechanistic Target of Rapamycin Complex 1 (mTORC1). We explore the interplay of dysregulated mTORC1 with lysosomes and how they contribute to senescence phenotypes. [ABSTRACT FROM AUTHOR]

It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

DABMA is a chemical molecule optimized from the parent compound ABMA and exhibits broad‐spectrum antipathogenic activity by modulating the host's endolysosomal and autophagic pathways. Both DABMA and ABMA inhibit severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in a cellular assay, which further expands their anti‐pathogen spectrum in vitro. However, their precise mechanism of action has not yet been resolved. TMEM175 is a newly characterized endolysosomal channel which plays an essential role in the homeostasis of endosomes and lysosomes as well as organelle fusion. Here, we show that DABMA increases the endosomal TMEM175 current through organelle patch clamping with an EC50 of 17.9 μm. Depletion of TMEM175 protein significantly decreases the antitoxin activity of DABMA and affects its action on acidic‐ and Rab7‐positive endosomes as well as on endolysosomal trafficking. Thus, TMEM175 is necessary for DABMA's activity and may represent a druggable target for the development of anti‐infective drugs. Moreover, DABMA, as an activator of the TMEM175 channel, may be useful for the in‐depth characterization of the physiological and pathological roles of this endolysosomal channel. [ABSTRACT FROM AUTHOR]

Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. This lysosomal TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD. Synopsis: In addition to their degradative functions, lysosomes play an important role in coordinating cellular responses to changes in nutrient availability. This study reveals that TBK1 is recruited to lysosomes when amino acids are abundant where it phosphorylates Rab7 and thus relieves Rab7-dependent suppression of mTORC1 signaling from lysosomes. TBK1 is recruited to lysosomes when amino acids are abundant. Rab7 (serine 72) is a substrate of TBK1 at lysosomes. Rab7 suppresses mTORC1 activity and this is relieved by TBK1-dependent phosphorylation of Rab7 on serine 72. The ALS-FTD associated E696K TBK1 mutant accumulates at lysosomes resulting in increased Rab7 phosphorylation and mTORC1 activity. TBK1 promotes mTORC1 activation by amino acids at the lysosome surface. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy-lysosome function promotes growth and survival of cancer cells, making them attractive targets for cancer therapy. One intriguing lysosomal target is PPT1 (palmitoyl-protein thioesterase 1). PPT1 inhibitors derived from chloroquine block autophagy, have significant antitumor activity in preclinical models and are being developed for clinical trials. However, the role of PPT1 in tumorigenesis remains poorly understood. Here we report that in melanoma cells, acute siRNA or pharmacological PPT1 inhibition led to increased ferroptosis sensitivity and significant loss of viability, whereas chronic PPT1 knockout using CRISPR-Cas9 produced blunted ferroptosis that led to sustained viability and growth. Each mode of PPT1 inhibition produced lysosome-autophagy inhibition but distinct proteomic changes, demonstrating the complexity of cellular adaptation mechanisms. To determine whether total genetic loss of Ppt1 would affect tumorigenesis in vivo, we developed a Ppt1 conditional knockout mouse model. We then crossed it into the BrafCA, PtenloxP, Tyr:CreERT2 melanoma mouse model to investigate the impact of Ppt1 loss on tumorigenesis. Loss of Ppt1 had no impact on melanoma histology, time to tumor initiation, or survival of tumor-bearing mice. These results suggest that chemical PPT1 inhibitors produce different adaptations than genetic PPT1 inhibition, and additional studies are warranted to fully understand the mechanism of chloroquine derivatives that target PPT1 in cancer.Abbreviations: 4-HT: 4-hydroxytamoxifen; BRAF: B-Raf proto-oncogene, serine/threonine kinase; cKO: conditional knockout; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9; DC661: A specific PPT1 inhibitor; DMSO: dimethyl sulfoxide; Dox; doxycycline hyclate; Easi-CRISPR: efficient additions with ssDNA inserts-CRISPR; GNS561/ezurpimtrostat: A PPT1 inhibitor; Hug: human guide; iCas: inducible CRISPR-Cas9; KO: knockout; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LDLR: low density lipoprotein receptor; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NT: non-target; PTEN: phosphatase and tensin homolog; PPT1: palmitoyl-protein thioesterase 1; RSL3: RAS-selective lethal small molecule 3; SCRIB/SCRB1: scribble planar cell polarity protein; Tyr:CreERT2: tyrosinase-driven Cre recombinase fused with the tamoxifen-inducible mutant ligand binding domain of the human estrogen receptor; UGCG: UDP-glucose ceramide glucosyltransferase; WT: wild-type. [ABSTRACT FROM AUTHOR]

Lysosomes are acidic organelles involved in crucial intracellular functions, including the degradation of organelles and protein, membrane repair, phagocytosis, endocytosis, and nutrient sensing. Given these key roles of lysosomes, maintaining their homeostasis is essential for cell viability. Thus, to preserve lysosome integrity and functionality, cells have developed a complex intracellular system, called lysosome quality control (LQC). Several stressors may affect the integrity of lysosomes, causing Lysosomal membrane permeabilization (LMP), in which membrane rupture results in the leakage of luminal hydrolase enzymes into the cytosol. After sensing the damage, LQC either activates lysosome repair, or induces the degradation of the ruptured lysosomes through autophagy. In addition, LQC stimulates the de novo biogenesis of functional lysosomes and lysosome exocytosis. Alterations in LQC give rise to deleterious consequences for cellular homeostasis. Specifically, the persistence of impaired lysosomes or the malfunctioning of lysosomal processes leads to cellular toxicity and death, thereby contributing to the pathogenesis of different disorders, including neurodegenerative diseases (NDs). Recently, several pieces of evidence have underlined the importance of the role of lysosomes in NDs. In this review, we describe the elements of the LQC system, how they cooperate to maintain lysosome homeostasis, and their implication in the pathogenesis of different NDs. [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is a multifaceted inflammatory disorder stemming from the aberrant activation of trypsin within the pancreas. Despite the contribution of various factors to the pathogenesis of AP, such as trypsin activation, dysregulated increases in cytosolic Ca2+ levels, inflammatory cascade activation, and mitochondrial dysfunction, the precise molecular mechanisms underlying the disease are still not fully understood. Mitophagy, a cellular process that preserves mitochondrial homeostasis under stress, has emerged as a pivotal player in the context of AP. Research suggests that augmenting mitophagy can mitigate pancreatic injury by clearing away malfunctioning mitochondria. Elucidating the role of mitophagy in AP may pave the way for novel therapeutic strategies. This review article aims to synthesize the current research findings on mitophagy in AP and underscore its significance in the clinical management of the disorder. [ABSTRACT FROM AUTHOR]

As a self‐degrading and highly conserved survival mechanism, autophagy plays an important role in maintaining cell survival and recycling. The discovery of autophagy‐related (ATG) genes has revolutionized our understanding of autophagy. Lysosomal membrane proteins (LMPs) are important executors of lysosomal function, and increasing evidence has demonstrated their role in the induction and regulation of autophagy. In addition, the functional dysregulation of the process mediated by LMPs at all stages of autophagy is closely related to neurodegenerative diseases and cancer. Here, we review the role of LMPs in autophagy, focusing on their roles in vesicle nucleation, vesicle elongation and completion, the fusion of autophagosomes and lysosomes, and degradation, as well as their broad association with related diseases. [ABSTRACT FROM AUTHOR]

Endocytosis is a quintessential feature of eukaryotes, and the emergence of endocytosis played a major role in the origin and evolution of eukaryotes. During the early evolution of eukaryotes, phagocytosis and the digestion of prey (i.e., bacteria) combined with the endocytosis of macromolecules opened a new source of nutrients beyond osmotrophy. Pathogenic and commensal protozoa have retained endocytosis as a major mechanism of nutrient acquisition even though, in theory, nutrients could be obtained from the host through osmotrophy. Nearly all pathogenic protozoa exhibit endocytosis and have lysosomal-like compartments that function as digestive vacuoles, and endocytosis appears to play a major role in the acquisition of nutrients. Cryptosporidium is a possible exception that may not exhibit endocytosis. Phagotrophy, however, is only observed in parasites of the intestinal lumen and appears to have been lost in blood and tissue parasites. Overall, the basic features of endocytosis and lysosomes are similar to other eukaryotes. Nonetheless, adaptation to the host has generated some novel features that are specific to certain protozoan lineages. [ABSTRACT FROM AUTHOR]

The productive replication of human immunodeficiency virus type 1 (HIV-1) involves intricate interactions between viral proteins and host cell machinery. However, the contributions of the lysosomal pathways for HIV-1 replication are not fully understood. The goal of this study was to determine the impact of lysosome-targeting compounds on HIV-1 replication and identify the cellular changes that are linked to HIV-1 inhibition using cell culture models of HIV-1 infection. Here, we demonstrate that the treatment of cells with various pharmacological agents known to inhibit lysosomal functions interfere with HIV-1 replication. The vacuolar ATPase (V-ATPase) inhibitor bafilomycin A1 exerted a potent inhibition of HIV-1 replication. Bafilomycin A1 inhibition of HIV-1 was independent of coreceptor tropism of HIV-1. Our data suggest that bafilomycin A1 inhibits HIV-1 at the post-integration steps of the virus life cycle, which include viral gene expression, virus assembly, and/or egress. Analysis of the cellular alterations following bafilomycin A1 treatment indicates that bafilomycin A1 causes a disruption in lysosome structure and functions. Treatment of cells with bafilomycin A1 caused an accumulation of unesterified cholesterol in lysosomes along with the expansion of the lysosomal compartments. Interestingly, the overexpression of the lysosomal cholesterol transporter Niemann–Pick type C 1 (NPC1) partially relieved bafilomycin A1 inhibition of HIV-1. Collectively, our data suggest that bafilomycin A1 inhibits HIV-1 replication in part by disrupting the lysosomal cholesterol trafficking pathway. [ABSTRACT FROM AUTHOR]

Limited nutrient availability in the tumor microenvironment can cause the rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We show here that the limitation of glucose, glutamine and serum from the culture medium resulted in the survival of a population of cancer cells with high viability and capacity to form tumors in vivo. These cells also displayed a remarkable increase in the abundance and size of lysosomes. Moreover, lysosomes were located mainly in the perinuclear region in nutrient‐limited cells; this translocation was mediated by a rapid post‐transcriptional increase in the key endolysosomal trafficking protein Rab7a. The acidic lysosomes in nutrient‐limited cells could trap weakly basic drugs such as doxorubicin, mediating resistance of the cells to the drug, which could be partially reversed with the lysosomal inhibitor bafilomycin A1. An in vivo chorioallantoic membrane (CAM) assay indicated a remarkable decrease in microtumor volume when nutrient‐limited cells were treated with 5‐Fluorouracil (5‐FU) and bafilomycin A1 compared to cells treated with either agent alone. Overall, our data indicate the activation of complementary pathways with nutrient limitation that can enable cancer cells to survive, proliferate and acquire drug resistance. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a common inflammatory skin disease, in particular among infants, and is characterized, among other things, by a modification in fatty acid and ceramide composition of the skin's stratum corneum. Palmitic acid and stearic acid, along with C16-ceramide and 2-hydroxy C16-ceramide, occur strikingly in AD. They coincide with a simultaneous decrease in very long-chain ceramides and ultra-long-chain ceramides, which form the outermost lipid barrier. Ceramides originate from cellular sphingolipid/ceramide metabolism, comprising a well-orchestrated network of enzymes involving various ELOVLs and CerSs in the de novo ceramide synthesis and neutral and acid CERase in degradation. Contrasting changes in long-chain ceramides and very long-chain ceramides in AD can be more clearly explained by the compartmentalization of ceramide synthesis. According to our hypothesis, the origin of increased C16-ceramide and 2-hydroxy C16-ceramide is located in the lysosome. Conversely, the decreased ultra-long-chain and very long-chain ceramides are the result of impaired ELOVL fatty acid elongation. The suggested model's key elements include the lysosomal aCERase, which has pH-dependent long-chain C16-ceramide synthase activity (revaCERase); the NADPH-activated step-in enzyme ELOVL6 for fatty acid elongation; and the coincidence of impaired ELOVL fatty acid elongation and an elevated lysosomal pH, which is considered to be the trigger for the altered ceramide biosynthesis in the lysosome. To maintain the ELOVL6 fatty acid elongation and the supply of NADPH and ATP to the cell, the polyunsaturated PPARG activator linoleic acid is considered to be one of the most suitable compounds. In the event that the increase in lysosomal pH is triggered by lysosomotropic compounds, compounds that disrupt the transmembrane proton gradient or force the breakdown of lysosomal proton pumps, non-HLA-classified AGEP may result. [ABSTRACT FROM AUTHOR]

Mitophagy is a mechanism that maintains mitochondrial integrity and homeostasis and is thought to promote longevity and reduce the risk of age-related neurodegenerative diseases, including Alzheimer's disease (AD). Here, we investigate the abundance of mitochondrial reactive oxygen species (ROS), mitochondrial function, and mitophagy in primary fibroblasts from patients with sporadic AD (sAD) and normal healthy controls. The results show increased levels of mitochondrial ROS, changes in mitochondrial morphology, altered bioenergetic properties, and defects in autophagy, mitophagy, and lysosome-mediated degradation pathways in sAD fibroblasts relative to control fibroblasts. Interestingly, lysosome abundance and the staining of lysosomal markers remained high, while the capacity of lysosome-dependent degradation was lower in sAD fibroblasts than in controls fibroblasts. Nicotinamide riboside supplementation decreased mitochondrial ROS, while capacity for lysosomal degradation remained unchanged in sAD fibroblasts relative to healthy control fibroblasts. These findings provide insight into molecular mechanisms involving the dysregulation of lysosome and autophagy/mitophagy pathways that may contribute significantly to clinical signs and pathological features of sAD. [Display omitted] • Mitochondrial reactive oxygen species (ROS) level is elevated in the AD fibroblasts. • The systematic changes of mitochondrial morphology and bioenergetics changes exist in AD fibroblasts. • Lysosome signaling is increased but lysosome degradation capacity is decreased in AD fibroblasts. • Supplementation with NAD + precursor nicotinamide riboside (NR) can reduce mitochondrial ROS level in AD fibroblasts. [ABSTRACT FROM AUTHOR]

Summary: In this work, deferoxamine (DFO) was injected into Tibetan sheep meat (distilled water as control) to reveal the effects of lysosomal Fe2+ on mitochondrial apoptosis and muscle tenderness. The reactive oxygen species (ROS) content, lysosomal membrane stability, cathepsins activity, mitochondrial membrane permeability transition pore (MPTP), mitochondrial membrane potential (MMP), cytochrome c (Cyt‐c) reduction level, caspase activity, myofibril fragmentation index (MFI) and shear force of Oula Tibetan sheep meat were measured at different ageing times. The results indicated that the ROS content, cathepsins activity, MPTP opening degree, lysosomal membrane stability, caspase‐3/9 activity and MFI of Tibetan sheep meat were significantly decreased after DFO treatment, whilst MMP, shear force and Cyt‐c reduction levels were noticeably increased. Furthermore, correlation analysis and principal component analysis showed that mitochondrial damage was positively correlated with meat tenderness. In sum, the study suggests that lysosomal Fe2+ may promote mitochondrial apoptosis and improve the tenderness of Tibetan sheep meat. [ABSTRACT FROM AUTHOR]

Abstract Acquired drug resistance is a major challenge in the management of cancer, which underscores the need for discovery and development of novel therapeutic strategies. We report here the mechanism of the anti-cancer activity of a small coordinate complex composed of the rare earth metal praseodymium (Pr) and mercaptopyridine oxide (MPO; pyrithione). Exposure of cancer cells to relatively low concentrations of the conjugate Pr-MPO (5 µM) significantly impairs cell survival in a p53-independent manner and irrespective of the drug resistant phenotype. Mechanistically, Pr-MPO-induced cell death is caspase-independent, not inhibitable by necrostatin, but associated with the appearance of autophagy markers. However, further analysis revealed incomplete autophagic flux, thus suggesting altered integrity of lysosomal machinery. Supporting the lysosomal targeting activity are data demonstrating increased lysosomal Ca2+ accumulation and alkalinization, which coincides with cytosolic acidification (drop in pHc from 7.75 to 7.00). In parallel, an increase in lysosomal activity of glycosidase alpha acid (GAA), involved in passive glycogen breakdown, correlates with rapid depletion of glucose stores upon Pr-MPO treatment. This is associated with swift cataplerosis of TCA cycle intermediates, loss of NAD+/NADH and increase in pyruvate dehydrogenase (PDH) activity to compensate for pyruvate loss. Addition of exogenous pyruvate rescued cell survival. Notably, lysosomal impairment and metabolic catastrophe triggered by Pr-MPO are suggestive of Zn2+-mediated cytotoxicity, which is confirmed by the ability of Zn2+ chelator TPEN to block Pr-MPO-mediated anti-tumor activity. Together, these results highlight the ability of the small molecule lanthanide conjugate to target the cells’ waste clearing machinery as well as mitochondrial metabolism for Zn2+-mediated execution of cancer cells, which could have therapeutic potential against cancers with high metabolic activity.

Metabolic-associated steatotic liver disease (MASLD) and its pathological version, metabolic dysfunction-associated steatohepatitis (MASH), are becoming the main leading causes of chronic liver disease almost worldwide and are the fastest growing aetiology of hepatocellular carcinoma (HCC), especially in the Western countries. The combination of high incidence and morbidity with limited treatment options for both MASH and HCC highlights an urgent need for the discovery of novel therapeutic candidates to inform drug development. The importance of lysosomes and cathepsins, their most abundant hydrolases, has been overlooked for decades. They were considered organelles only involved in the recycling of macromolecules, with cathepsins simply being their effectors. Contrary to this traditional view, recent findings have shed new light on the lysosome and its enzymes as drivers of essential cellular processes, such as apoptosis and autophagy. Bringing lysosomal activity and the regulation of cathepsins into the spotlight of MASH and HCC research can open new avenues for the development of novel drugs based on targeting cathepsin-driven lysosomal activity and its associated pathological processes. This review comprehensively summarises the current knowledge on the role and contribution of lysosomal cathepsins to MASLD/MASH and HCC progression.

Abstract Acute pancreatitis (AP) is a multifaceted inflammatory disorder stemming from the aberrant activation of trypsin within the pancreas. Despite the contribution of various factors to the pathogenesis of AP, such as trypsin activation, dysregulated increases in cytosolic Ca2+ levels, inflammatory cascade activation, and mitochondrial dysfunction, the precise molecular mechanisms underlying the disease are still not fully understood. Mitophagy, a cellular process that preserves mitochondrial homeostasis under stress, has emerged as a pivotal player in the context of AP. Research suggests that augmenting mitophagy can mitigate pancreatic injury by clearing away malfunctioning mitochondria. Elucidating the role of mitophagy in AP may pave the way for novel therapeutic strategies. This review article aims to synthesize the current research findings on mitophagy in AP and underscore its significance in the clinical management of the disorder.

Abstract Lysosomes are acidic organelles involved in crucial intracellular functions, including the degradation of organelles and protein, membrane repair, phagocytosis, endocytosis, and nutrient sensing. Given these key roles of lysosomes, maintaining their homeostasis is essential for cell viability. Thus, to preserve lysosome integrity and functionality, cells have developed a complex intracellular system, called lysosome quality control (LQC). Several stressors may affect the integrity of lysosomes, causing Lysosomal membrane permeabilization (LMP), in which membrane rupture results in the leakage of luminal hydrolase enzymes into the cytosol. After sensing the damage, LQC either activates lysosome repair, or induces the degradation of the ruptured lysosomes through autophagy. In addition, LQC stimulates the de novo biogenesis of functional lysosomes and lysosome exocytosis. Alterations in LQC give rise to deleterious consequences for cellular homeostasis. Specifically, the persistence of impaired lysosomes or the malfunctioning of lysosomal processes leads to cellular toxicity and death, thereby contributing to the pathogenesis of different disorders, including neurodegenerative diseases (NDs). Recently, several pieces of evidence have underlined the importance of the role of lysosomes in NDs. In this review, we describe the elements of the LQC system, how they cooperate to maintain lysosome homeostasis, and their implication in the pathogenesis of different NDs. Graphical Abstract

Abstract Background Cystinosis is a lysosomal storage disorder characterized by an autosomal recessive phenotype. Intermediate cystinosis, which progresses slowly and causes renal failure, accounts for approximately 5% of all cystinosis cases. Patients with intermediate cystinosis may not exhibit the typical symptoms of cystinosis, such as Fanconi syndrome and ocular symptoms. Because of its diverse clinical presentation and rarity, intermediate cystinosis can be difficult to diagnose. Additionally, few patients can tolerate cystine-depleting drugs, such as cysteamine, because of their complicated administration schedules and side effects. We report a case of intermediate cystinosis that was treated with cysteamine for 10 years. Case presentation Urinary abnormalities were first diagnosed when the patient was 3 years of age during a health examination specifically for 3-year-old children, which is unique to Japan. Cystinosis was diagnosed when the patient was 12 years of age. Cysteamine therapy was initiated and regular cystine concentration measurements were performed. Although proteinuria persisted, the patient’s renal function progressed slowly. Two renal biopsies were performed, and multinucleated podocytes and cystine crystals without focal segmental glomerulosclerosis lesions were observed in the biopsy specimens. The patient’s renal function remained stable. Conclusions This case of intermediate cystinosis was treated with cysteamine over the course of 10 years. Intermediate cystinosis requires an appropriate diagnosis and long-term treatment.

Introduction: β-Elemene (β-ELE), derived from Curcuma wenyujin, has anticancer effect on non-small cell lung cancer (NSCLC). However, the potential target and detail mechanism were still not clear. TFEB is the master regulator of lysosome biogenesis. Ferroptosis, a promising strategy for cancer therapy could be triggered via suppression on glutathione peroxidase 4 (GPX4). Weather TFEB-mediated lysosome degradation contributes to GPX4 decline and how β-ELE modulates on this process are not clear. Objectives: To observe the action of β-ELE on TFEB, and the role of TFEB-mediated GPX4 degradation in β-ELE induced ferroptosis. Methods: Surface plasmon resonance (SPR) and molecular docking were applied to observe the binding affinity of β-ELE on TFEB. Activation of TFEB and lysosome were observed by immunofluorescence, western blot, flow cytometry and qPCR. Ferroptosis induced by β-ELE was observed via lipid ROS, a labile iron pool (LIP) assay and western blot. A549TFEB KO cells were established via CRISPR/Cas9. The regulation of TFEB on GPX4 and ferroptosis was observed in β-ELE treated A549WT and A549TFEB KO cells, which was further studied in orthotopic NOD/SCID mouse model. Results: β-ELE can bind to TFEB, notably activate TFEB, lysosome and transcriptional increase on downstream gene GLA, MCOLN1, SLC26A11 involved in lysosome activity in EGFR wild-type NSCLC cells. β-ELE increased GPX4 ubiquitination and lysosomal localization, with the increase on lysosome degradation of GPX4. Furthermore, β-ELE induced ferroptosis, which could be promoted by TFEB overexpression or compromised by TFEB knockout. Genetic knockout or inactivation of TFEB compromised β-ELE induced lysosome degradation of GPX4, which was further demonstrated in orthotopic NSCLC NOD/SCID mice model. Conclusion: This study firstly demonstrated that TFEB promoted GPX4 lysosome degradation contributes to β-ELE induced ferroptosis in EGFR wild-type NSCLC, which gives a clue that TFEB mediated GPX4 degradation would be a novel strategy for ferroptosis induction and NSCLC therapy.

Endocytosis is a quintessential feature of eukaryotes, and the emergence of endocytosis played a major role in the origin and evolution of eukaryotes. During the early evolution of eukaryotes, phagocytosis and the digestion of prey (i.e., bacteria) combined with the endocytosis of macromolecules opened a new source of nutrients beyond osmotrophy. Pathogenic and commensal protozoa have retained endocytosis as a major mechanism of nutrient acquisition even though, in theory, nutrients could be obtained from the host through osmotrophy. Nearly all pathogenic protozoa exhibit endocytosis and have lysosomal-like compartments that function as digestive vacuoles, and endocytosis appears to play a major role in the acquisition of nutrients. Cryptosporidium is a possible exception that may not exhibit endocytosis. Phagotrophy, however, is only observed in parasites of the intestinal lumen and appears to have been lost in blood and tissue parasites. Overall, the basic features of endocytosis and lysosomes are similar to other eukaryotes. Nonetheless, adaptation to the host has generated some novel features that are specific to certain protozoan lineages.

Abstract Elucidating the molecular mechanism of autophagy was a landmark in understanding not only the physiology of cells and tissues, but also the pathogenesis of diverse diseases, including diabetes and metabolic disorders. Autophagy of pancreatic β‐cells plays a pivotal role in the maintenance of the mass, structure and function of β‐cells, whose dysregulation can lead to abnormal metabolic profiles or diabetes. Modulators of autophagy are being developed to improve metabolic profile and β‐cell function through the removal of harmful materials and rejuvenation of organelles, such as mitochondria and endoplasmic reticulum. Among the known antidiabetic drugs, glucagon‐like peptide‐1 receptor agonists enhance the autophagic activity of β‐cells, which might contribute to the profound effects of glucagon‐like peptide‐1 receptor agonists on systemic metabolism. In this review, the results from studies on the role of autophagy in β‐cells and their implication in the development of diabetes are discussed. In addition to non‐selective (macro)autophagy, the role and mechanisms of selective autophagy and other minor forms of autophagy that might occur in β‐cells are discussed. As β‐cell failure is the ultimate cause of diabetes and unresponsiveness to conventional therapy, modulation of β‐cell autophagy might represent a future antidiabetic treatment approach, particularly in patients who are not well managed with current antidiabetic therapy.

Background: Cystinosis is a lysosomal storage disorder characterized by an autosomal recessive phenotype. Intermediate cystinosis, which progresses slowly and causes renal failure, accounts for approximately 5% of all cystinosis cases. Patients with intermediate cystinosis may not exhibit the typical symptoms of cystinosis, such as Fanconi syndrome and ocular symptoms. Because of its diverse clinical presentation and rarity, intermediate cystinosis can be difficult to diagnose. Additionally, few patients can tolerate cystine-depleting drugs, such as cysteamine, because of their complicated administration schedules and side effects. We report a case of intermediate cystinosis that was treated with cysteamine for 10 years. Case presentation: Urinary abnormalities were first diagnosed when the patient was 3 years of age during a health examination specifically for 3-year-old children, which is unique to Japan. Cystinosis was diagnosed when the patient was 12 years of age. Cysteamine therapy was initiated and regular cystine concentration measurements were performed. Although proteinuria persisted, the patient's renal function progressed slowly. Two renal biopsies were performed, and multinucleated podocytes and cystine crystals without focal segmental glomerulosclerosis lesions were observed in the biopsy specimens. The patient's renal function remained stable. Conclusions: This case of intermediate cystinosis was treated with cysteamine over the course of 10 years. Intermediate cystinosis requires an appropriate diagnosis and long-term treatment. [ABSTRACT FROM AUTHOR]

Defective mitochondria and autophagy, as well as accumulation of lipid and iron in WDR45 mutant fibroblasts, is related to beta-propeller protein-associated neurodegeneration (BPAN). In this study, we found that enlarged lysosomes in cells derived from patients with BPAN had low enzyme activity, and most of the enlarged lysosomes had an accumulation of iron and oxidized lipid. Cryo-electron tomography revealed elongated lipid accumulation, and spectrometry-based elemental analysis showed that lysosomal iron and oxygen accumulation superimposed with lipid aggregates. Lysosomal lipid aggregates superimposed with autofluorescence as free radical generator, lipofuscin. To eliminate free radical stress by iron accumulation in cells derived from patients with BPAN, we investigated the effects of the iron chelator, 2,2′-bipyridine (bipyridyl, BIP). To study whether the defects in patient-derived cells can be rescued by an iron chelator BIP, we tested whether the level of iron and reactive oxygen species (ROS) in the cells and genes related to oxidative stress were rescued BIP treatment. Although BIP treatment decreased some iron accumulation in the cytoplasm and mitochondria, the accumulation of iron in the lysosomes and levels of cellular ROS were unaffected. In addition, the change of specific RNA levels related to free radical stress in patient fibroblasts was not rescued by BIP. To alleviate free radical stress, we investigated whether l -serine can regulate abnormal structures in cells derived from patients with BPAN through the regulation of free radical stress. l -serine treatment alleviated increase of enlarged lysosomes and iron accumulation and rescued impaired lysosomal activity by reducing oxidized lipid accumulation in the lysosomes of the cells. Lamellated lipids in the lysosomes of the cells were identified as lipofuscin through correlative light and electron microscopy, and l -serine treatment reduced the increase of lipofuscin. These data suggest that l -serine reduces oxidative stress-mediated lysosomal lipid oxidation and iron accumulation by rescuing lysosomal activity. [Display omitted] • WDR45 mutant fibroblasts show abnormal iron and lipid accumulation in lysosomes. • WDR45 mutant fibroblasts show dysfunctional lysosome activity. • The iron chelator was unable to regulate the accumulation of iron and lipids in the lysosome. • l -serine can regulate iron and lipid accumulation through lysosomal activity. [ABSTRACT FROM AUTHOR]

Lysosomes serve as catabolic centers and signaling hubs in cells, regulating a multitude of cellular processes such as intracellular environment homeostasis, macromolecule degradation, intracellular vesicle trafficking and autophagy. Alterations in lysosomal level and function are crucial for cellular adaptation to external stimuli, with lysosome dysfunction being implicated in the pathogenesis of numerous diseases. Osteoclasts (OCs), as multinucleated cells responsible for bone resorption and maintaining bone homeostasis, have a complex relationship with lysosomes that is not fully understood. Dysregulated function of OCs can disrupt bone homeostasis leading to the development of various bone disorders. The regulation of OC differentiation and bone resorption for the treatment of bone disease have received considerable attention in recent years, yet the role and regulation of lysosomes in OCs, as well as the potential therapeutic implications of intervening in lysosomal biologic behavior for the treatment of bone diseases, remain relatively understudied. This review aims to elucidate the mechanisms involved in lysosomal biogenesis and to discuss the functions of lysosomes in OCs, specifically in relation to differentiation, bone resorption, and autophagy. Finally, we explore the potential therapeutic implication of targeting lysosomes in the treatment of bone metabolic disorders. [ABSTRACT FROM AUTHOR]

Herein, we report a novel flavin analogue as singular chemical component for lysosome bioimaging, and inherited photosensitizer capability of the flavin core was demonstrated as a promising candidate for photodynamic therapy (PDT) application. Fine‐tuning the flavin core with the incorporation of methoxy naphthyl appendage provides an appropriate chemical design, thereby offering photostability, selectivity, and lysosomal colocalization, along with the aggregation‐induced emissive nature, making it suitable for lysosomal bioimaging applications. Additionally, photosensitization capability of the flavin core with photostable nature of the synthesized analogue has shown remarkable capacity for generating reactive oxygen species (ROS) within cells, making it a promising candidate for photodynamic therapy (PDT) application. [ABSTRACT FROM AUTHOR]

Metformin is a widely employed drug in type 2 diabetes. In addition to warranting good short- and long-term glycemic control, metformin displays many intriguing properties as protection against cardiovascular and neurodegenerative diseases, anti-tumorigenic and longevity promotion. In addition to being a low-cost drug, metformin is generally well tolerated. However, despite the enthusiastic drive to aliment these novel studies, many contradictory results suggest the importance of better elucidating the complexity of metformin action in different tissues/cells to establish its possible employment in neurodegenerative diseases. This review summarises recent data identifying lysosomal-dependent processes and lysosomal targets, such as endosomal Na+/H+ exchangers, presenilin enhancer 2 (PEN2), the lysosomal pathway leading to AMP-activated protein kinase (AMPK) activation, and the transcription factor EB (TFEB), modulated by metformin. Lysosomal dysfunctions resulting in autophagic and lysosomal acidification and biogenesis impairment appear to be hallmarks of many inherited and acquired neurodegenerative diseases. Lysosomes are not yet seen as a sort of cellular dump but are crucial in determining key signalling paths and processes involved in the clearance of aggregated proteins. Thus, the possibility of pharmacologically modulating them deserves great interest. Despite the potentiality of metformin in this context, many additional important issues, such as dosing, should be addressed in the future. [ABSTRACT FROM AUTHOR]

Two-pore channels are pathophysiologically important Na+-and Ca2+-permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P2, they are more Na+-selective than when gated by the Ca2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P2 in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na+ permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na+ to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na+ over Ca2+. Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions. [ABSTRACT FROM AUTHOR]

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation. [ABSTRACT FROM AUTHOR]

The cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway has a crucial role in combating pathogen infection. However, its aberrant activation is involved in several human disorders. Lysosomes are emerging as key negative regulators of cGAS-STING signaling. Here, we discuss the lysosomal control of cGAS-STING signaling and its implication in human disorders. [ABSTRACT FROM AUTHOR]

Aim: Understanding the physiological role of ATP6V1A, a component of the cytosolic V1 domain of the proton pump vacuolar ATPase, in regulating neuronal development and function. Methods: Modeling loss of function of Atp6v1a in primary murine hippocampal neurons and studying neuronal morphology and function by immunoimaging, electrophysiological recordings and electron microscopy. Results: Atp6v1a depletion affects neurite elongation, stabilization, and function of excitatory synapses and prevents synaptic rearrangement upon induction of plasticity. These phenotypes are due to an overall decreased expression of the V1 subunits, that leads to impairment of lysosomal pH‐regulation and autophagy progression with accumulation of aberrant lysosomes at neuronal soma and of enlarged vacuoles at synaptic boutons. Conclusions: These data suggest a physiological role of ATP6V1A in the surveillance of synaptic integrity and plasticity and highlight the pathophysiological significance of ATP6V1A loss in the alteration of synaptic function that is associated with neurodevelopmental and neurodegenerative diseases. The data further support the pivotal involvement of lysosomal function and autophagy flux in maintaining proper synaptic connectivity and adaptive neuronal properties. [ABSTRACT FROM AUTHOR]

Senescence in bone marrow mesenchymal stem cells (BMSCs), triggered by excessive oxidative stress, plays a crucial role in the onset of postmenopausal osteoporosis. Recent studies underscore the importance of mitochondrial rehabilitation and quality control as key determinants in the modulation of oxidative stress and cellular senescence. MitoTEMPO, a mitochondria‐targeted antioxidant, has been shown to mitigate the heightened levels of reactive oxygen species (ROS). In our research, we observed that BMSCs from ovariectomized (OVX) rats displayed premature senescence, which was attributed to combined mitochondrial and lysosomal dysfunction, a condition that worsens with extended estrogen deprivation. Treatment with MitoTEMPO effectively reversed these effects, reinstating lysosomal functionality and suppressing the mitochondrial unfolded protein response (UPRmt). Subsequent in vivo experiments corroborated these observations, revealing that MitoTEMPO administration in OVX rats curtailed trabecular bone loss and reduced the expression of p53, HSP60, and CLPP in the trabecular bone region of the proximal tibia. Overall, our findings suggest that MitoTEMPO holds promise as a therapeutic agent to counteract senescence in OVX‐BMSCs, offering a potential strategy for treating postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin (Grn), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome‐related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes. [ABSTRACT FROM AUTHOR]