Visualizing lysosomes hypochlorous acid in Parkinson's disease models by a novel fluorescent probe.

Heightened oxidative stress is the principal driver behind the altered metabolism of neurotransmitters within the brains of Parkinson's disease (PD). Hypochlorous acid (HClO), a variant of reactive oxygen species (ROS), plays a crucial role in several lysosomal activities. An irregular concentration of HClO may result in significant molecular damage and contribute to the onset of neurodegenerative disorders. Despite this, the precise role of lysosomal HClO in PD remains unclear, due to its fast reactivity and low levels. This is further complicated by the lack of effective in situ imaging techniques for accurately tracking its dynamics. Therefore, it is of great significance to use effective tools to map the lysosomal HClO during the pathological process of PD. In this study, we propose a fluorogenic probe named Lys-PTZ-HClO for the specific and sensitive detection of HClO. Lys-PTZ-HClO exhibits features like a fast response time (10 s) and a low detection limit (0.72 µM). Benefiting from its superior properties, the probe was used to visualize the basal HClO levels, and the variation of HClO levels in lysosomal of living cells. More importantly, this probe was successfully applied for the first time to reveal increased lysosomal HClO in a cellular model of PD.

Cytosolic serpins act in a cytoprotective feedback loop that limits ESX-1-dependent death of Mycobacterium marinum-infected macrophages.

Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo. Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g, two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture. IMPORTANCE: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo. The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection.

Methuosis Inducer SGI-1027 Cooperates with Everolimus to Promote Apoptosis and Pyroptosis by Triggering Lysosomal Membrane Permeability in Renal Cancer.

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.

Identification of lysosome-related hub genes as potential biomarkers and immune infiltrations of moyamoya disease by multiple bioinformatics methods and machine-learning strategies.

Background: Moyamoya disease (MMD), characterized by chronic cerebrovascular pathology, poses a rare yet significant clinical challenge, associated with elevated rates of mortality and disability. Despite intensive research endeavors, the exact biomarkers driving its pathogenesis remain enigmatic. Methods: The expression patterns of GSE189993 and GSE141022 were retrieved from the Gene Expression Omnibus (GEO) repository to procure differentially expressed genes (DEGs) between samples afflicted with MMD and those under control conditions. The Least Absolute Shrinkage and Selection Operator (LASSO), Support Vector Machine with Recursive Feature Elimination (SVM-RFE), and Random Forest (RF) algorithms were employed for identifying candidate diagnostic genes associated with MMD. Subsequently, these candidate genes underwent validation in an independent cohort (GSE157628). The CMAP database was ultimately employed to forecast drugs pertinent to MMD for clinical translation. Results: A collective of 240 DEGs were discerned. Functional enrichment scrutiny unveiled the enrichment of the cholesterol metabolism pathway, salmonella infection pathway, and allograft rejection pathway within the MMD cohort. EPDR1, DENND3, and NCSTN emerged as discerned diagnostic biomarkers for MMD. The CMAP database was ultimately employed to scrutinize the ten most auspicious pharmaceutical compounds for managing MMD. Finally, after validation through in vitro experiments, EPDR1, DENND3, and NCSTN were identified as the key genes. Conclusion: EPDR1, DENND3, and NCSTN have emerged as potential novel biomarkers for MMD. The involvement of T lymphocytes, neutrophilic granulocytes, dendritic cells, natural killer cells, and plasma cells could be pivotal in the pathogenesis and advancement of MMD.

Localization of silica nanoparticles to lysosome causes lysosomal dysfunction in JEG-3 cells.

Nanoparticles have useful functions due to the characteristics conferred on them by an increase in their specific surface area, and they have already been put into practical use in products in various industrial fields. Although exposure to nanoparticles in daily life is unavoidable for pregnant women, studies that evaluate the toxicity of nanoparticles in pregnant women are lacking. To redress this, we have focused on the placenta and have previously revealed that nanoparticles can show placental toxicity. However, there is still little knowledge regarding the behavior of nanoparticles within placental cells, which would enable us to understand their mode of action. Here, we tried to clarify the intracellular localization of silica nanoparticles in placental cells and how this affects placental toxicity. We analyzed the uptake of silica nanoparticles with a diameter of 10 nm (nSP10) into JEG-3 cells, a human choriocarcinoma cell line. Flow cytometry analysis showed that nSP10 labelled with red fluorescence were taken up into JEG-3 cells, and that pre-treatment with the endocytosis inhibitor cytochalasin D inhibited their uptake, suggesting that nSP10 are taken up into JEG-3 cells by the endocytic pathway. Moreover, confocal microscopy revealed that nSP10 are prominently localized in lysosomes. Staining with LysoTracker showed that nSP10 treatment increased the acidic compartment of JEG-3 cells, suggesting lysosome accumulation and swelling. These results indicate that nSP10 taken into placental cells are transferred to lysosomes and may cause lysosomal dysfunction.

Danon Disease Presenting with Slowly Progressive Cardiomyopathy and Harboring a Novel Missense Variant in the Lysosome-associated Membrane Protein Type 2 (LAMP-2) gene: A Case Report.

Danon disease (DD) is a rare lysosomal storage disorder resulting from pathogenic variants of the lysosome-associated membrane protein type 2 (LAMP-2) gene. The disease is characterized by severe cardiomyopathy, which rapidly progresses to end-stage heart failure. This case, with DD caused by a missense variant, exhibited slow progressive cardiomyopathy and survived for an extended period despite being a male. A pathological analysis revealed that only a minority of the samples exhibited autophagic vacuoles with unique sarcolemmal features (AVSFs), which are typical of DD. Importantly, LAMP-2 expression was absent and the myocardial tissue contained a substantial amount of p62-positive aggregates.

UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

Wilms' tumor is a malignant neoplasm where current medical advancements have significantly improved survival rates; however, challenges persist such as the resistance of the tumor to chemotherapy drugs like doxorubicin. This necessitates higher dosages, leading to decreased sensitivity. However, using high doses of doxorubicin can have late effects on the heart. Unc-13 homolog B (UNC13B) may be involved in the drug resistance in several tumors, yet its role in modulating drug sensitivity in Wilms' tumor remains unexplored. UNC13B levels were quantified using reverse transcription-qPCR and Western blotting. The half-maximal inhibitory concentration for doxorubicin, vincristine, and actinomycin-D was determined using CCK-8 assays. Cell cycle and apoptosis were analyzed using flow cytometry, and lysosomal changes were observed using Lyso-Tracker staining. The present study initially evaluated UNC13B expression levels in the Wilms' tumor 17.94 cell line. Additionally, through short hairpin RNA-mediated knockdown, changes in doxorubicin sensitivity in 17.94 Wilms' tumor cells were assessed. Concurrently, preliminary investigations into the role of UNC13B in regulating lysosomes was performed, revealing a significant positive association between UNC13B levels and lysosome formation in the 17.94 cell line. Lysosomes likely serve a role in the sensitivity of Wilms' tumor cell lines to drugs. Elevated UNC13B expression was observed in the 17.94 Wilms' tumor cell line compared to normal kidney cells. UNC13B knockdown also resulted in increased apoptosis levels upon doxorubicin treatment. Immunofluorescence revealed UNC13B localization within cellular vesicles, and its knockdown significantly decreased lysosome levels. Overall, the findings of the present study demonstrate that UNC13B regulates the sensitivity of the Wilms' tumor 17.94 cell line to doxorubicin by modulating lysosome formation within cells. The results suggest that UNC13B is likely an enriched target involved in lysosomal regulation in certain tumors, offering a new approach for optimizing chemotherapy in Wilms' tumor and other cancers with high UNC13B expression.

Super-resolution imaging lysosome vesicles and establishing a gallbladder-visualizable zebrafish model via a fluorescence probe.

Advanced probes for imaging viscous lipids microenvironment in vitro and in vivo are desirable for the study of membranous organelles and lipids traffic. Herein, a reaction-based dihydroquinoline probe (DCQ) was prepared via linking a diethylamino coumarin fluorophore with a N-methylquinoline moiety. DCQ is stable in low viscous aqueous mediums and exhibits green fluorescence, which undergoes fast autoxidation in high viscous mediums to form a fluorescent product with deep-red to near-infrared (NIR) emission, rendering the ability for dual-color imaging. Living cell imaging indicated that DCQ can effectively stain lysosomal membranes with deep-red fluorescence. Super-resolution imaging of lysosome vesicles has been achieved by DCQ and stimulated emission depletion (STED) microscopy. In addition, DCQ realizes multiple organs imaging in zebrafish, whose dual-color emission can perfectly discriminate zebrafish's yolk sac, digestive tract and gallbladder. Most importantly, DCQ has been successfully used to establish a gallbladder-visualizable zebrafish model for the evaluation of drug stress.

Long-term effects of luteolin in a mouse model of nephropathic cystinosis.

In infantile nephropathic cystinosis, variants of the CTNS gene cause accumulation of cystine in lysosomes, causing progressive damage to most organs. Patients usually present before 1 year of age with signs of renal Fanconi syndrome. Cysteamine therapy allows cystine clearance from lysosomes and delays kidney damage but does not prevent progression to end-stage kidney disease, suggesting that pathways unrelated to cystine accumulation are also involved. Among these, impaired autophagy, altered endolysosomal trafficking, and increased apoptosis have emerged in recent years as potential targets for new therapies. We previously showed that luteolin, a flavonoid compound, improves these abnormal pathways in cystinotic cells and in zebrafish models of the disease. Herein, we have investigated if prolonged luteolin treatment ameliorates kidney damage in a murine model of cystinosis. To this end, we have treated Ctns-/- mice from 2 to 8 months with 150 mg/kg/day of luteolin. No significant side effects were observed. Compared to untreated animals, analyses of kidney cortex samples obtained after sacrifice showed that luteolin decreased p62/SQSTM1 levels (p <0.001), improved the number, size, and distribution of LAMP1-positive structures (p <0.02), and decreased tissue expression of cleaved caspase 3 (p <0.001). However, we did not observe improvements in renal Fanconi syndrome and kidney inflammation. Kidney function remained normal during the time of the study. These results indicate that luteolin has positive effects on the apoptosis and endo-lysosomal defects of cystinotic proximal tubular cells. However, these beneficial effects did not translate into improvement of renal Fanconi syndrome.

Targeted degradation of membrane and extracellular proteins with LYTACs.

Targeted protein degradation technology has gained substantial momentum over the past two decades as a revolutionary strategy for eliminating pathogenic proteins that are otherwise refractory to treatment. Among the various approaches developed to harness the body's innate protein homeostasis mechanisms for this purpose, lysosome targeting chimeras (LYTACs) that exploit the lysosomal degradation pathway by coupling the target proteins with lysosome-trafficking receptors represent the latest innovation. These chimeras are uniquely tailored to degrade proteins that are membrane-bound and extracellular, encompassing approximately 40% of all proteome. Several novel LYTAC formulas have been developed recently, providing valuable insights for the design and development of therapeutic degraders. This review delineates the recent progresses of LYTAC technology, its practical applications, and the factors that dictate target degradation efficiency. The potential and emerging trends of this technology are discussed as well. LYTAC technology offers a promising avenue for targeted protein degradation, potentially revolutionizing the therapeutic landscape for numerous diseases.

Heat-killed probiotic Levilactobacillus brevis MKAK9 and its exopolysaccharide promote longevity by modulating aging hallmarks and enhancing immune responses in Caenorhabditis elegans.

BACKGROUND: Proteostasis is a critical aging hallmark responsible for removing damaged or misfolded proteins and their aggregates by improving proteasomal degradation through the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS). Research on the impact of heat-killed probiotic bacteria and their structural components on aging hallmarks and innate immune responses is scarce, yet enhancing these effects could potentially delay age-related diseases. RESULTS: This study introduces a novel heat-killed Levilactobacillus brevis strain MKAK9 (HK MKAK9), along with its exopolysaccharide (EPS), demonstrating their ability to extend longevity by improving proteostasis and immune responses in wild-type Caenorhabditis elegans. We elucidate the underlying mechanisms through a comprehensive approach involving mRNA- and small RNA sequencing, proteomic analysis, lifespan assays on loss-of-function mutants, and quantitative RT-PCR. Mechanistically, HK MKAK9 and its EPS resulted in downregulation of the insulin-like signaling pathway in a DAF-16-dependent manner, enhancing protein ubiquitination and subsequent proteasomal degradation through activation of the ALP pathway, which is partially mediated by microRNA mir-243. Importantly, autophagosomes engulf ubiquitinylated proteins, as evidenced by increased expression of the autophagy receptor sqst-3, and subsequently fuse with lysosomes, facilitated by increased levels of the lysosome-associated membrane protein (LAMP) lmp-1, suggesting the formation of autolysosomes for degradation of the selected cargo. Moreover, HK MKAK9 and its EPS activated the p38 MAPK pathway and its downstream SKN-1 transcription factor, which are known to regulate genes involved in innate immune response (thn-1, ilys-1, cnc-2, spp-9, spp-21, clec-47, and clec-266) and antioxidation (sod-3 and gst-44), thereby reducing the accumulation of reactive oxygen species (ROS) at both cellular and mitochondrial levels. Notably, SOD-3 emerged as a transcriptional target of both DAF-16 and SKN-1 transcription factors. CONCLUSION: Our research sets a benchmark for future investigations by demonstrating that heat-killed probiotic and its specific cellular component, EPS, can downregulate the insulin-signaling pathway, potentially improving the autophagy-lysosome pathway (ALP) for degrading ubiquitinylated proteins and promoting organismal longevity. Additionally, we discovered that increased expression of microRNA mir-243 regulates insulin-like signaling and its downstream ALP pathway. Our findings also indicate that postbiotic treatment may bolster antioxidative and innate immune responses, offering a promising avenue for interventions in aging-related diseases.

Endolysosomal channel TMEM175 mediates antitoxin activity of DABMA.

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.

TFEB activator tanshinone IIA and derivatives derived from Salvia miltiorrhiza Bge. Attenuate hepatic steatosis and insulin resistance.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bge. (SMB) is an herbal medicine extensively used for improving metabolic disorders, including Nonalcoholic fatty liver disease (NAFLD). However, the potential material basis and working mechanism still remained to be elucidated. AIM OF THE STUDY: To find potential ingredients for therapy of NAFLD by high content screening and further verify the efficacy on restoring hepatic steatosis and insulin resistance, and clarify the potential working mechanism. MATERIALS AND METHODS: The mouse transcription factor EB (Tfeb) in preadipocytes was knocked out by CRISPR-Cas9 gene editing. High content screening of TFEB nuclear translocation was performed to identify TFEB activators. The effect of candidate compounds on reducing lipid accumulation was evaluated using Caenorhabditis elegans (C. elegans). Then the role of Salvia miltiorrhiza extract (SMB) containing Tanshinone IIA and the derivatives were further investigated on high-fat diet (HFD) fed mice. RNA-seq was performed to explore potential molecular mechanism of SMB. Finally, the gut microbiota diversity was evaluated using 16S rRNA sequencing to investigate the protective role of SMB on regulating gut microbiota homeostasis. RESULTS: Knockout of Tfeb led to excessive lipid accumulation in adipocytes while expression of TFEB homolog HLH-30 in C. elegans (MAH240) attenuated lipid deposition. Screening of TFEB activators identified multiple candidates from Salvia miltiorrhiza, all of them markedly induced lysosome biogenesis in HepG2 cells. One of the candidate compounds Tanshinone IIA significantly decreased lipid droplet deposition in HFD fed C. elegans. Administration of SMB on C57BL/6J mice via gastric irrigation at the dose of 15 g/kg/d markedly alleviated hepatic steatosis, restored serum lipid profile, and glucose tolerance. RNA-seq showed that gene expression profile was altered and the genes related to lipid metabolism were restored. The disordered microbiome was remodeled by SMB, Firmicutes and Actinobacteriotawere notably reduced, Bacteroidota and Verrucomicrobiota were significantly increased. CONCLUSION: Taken together, the observations presented here help address the question concerning what were the main active ingredients in SMB for alleviating NAFLD, and established that targeting TFEB was key molecular basis for the efficacy of SMB.

Palbociclib-derived multifunctional molecules for lysosomal targeting and diagnostic-therapeutic integration.

Aim: Lysosomal pH changes are associated with drug resistance, cell growth and invasion of tumors, but effective and specific real-time monitoring of lysosomal pH compounds for cancer therapy is lacking. Materials & methods: Here, based on the covalent linkage of the anticancer drug palbociclib and fluorescent dye fluorescein isothiocyanate (FITC), we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Results & discussion: Pal-FITC fluoresces is 20-fold stronger than that of FITC and shows a linear response in the pH range of 4.0-8.2 (R2 = 0.9901). Pal-FITC blocks cells in G1 phase via Cyclin D-CDK4/6-Rb. Conclusion: Our study provides new strategies for tumor-targeted imaging and personalized therapy.

Based on the covalent linkage of the anticancer drug and the fluorescent dye, we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Pal-FITC responded linearly in the pH range of 4.0­8.2. In addition, Pal-FITC was able to effectively treat lung cancer without toxic side effects on normal cells. It has a significant cell cycle blocking phenomenon and blocks G1 phase cells via Cyclin D-CDK4/6-Rb. Our study provides a new strategy for tumor-targeted imaging and personalized therapy.

Optogenetic manipulation of lysosomal physiology and autophagic activity.

Lysosomes are essential degradative organelles and signaling hubs within cells, playing a crucial role in the regulation of macroautophagy/autophagy. Dysfunction of lysosomes and impaired autophagy are closely associated with the development of various neurodegenerative diseases. Enhancing lysosomal activity and boosting autophagy levels holds great promise as effective strategies for treating these diseases. However, there remains a lack of methods to dynamically regulate lysosomal activity and autophagy levels in living cells or animals. In our recent work, we applied optogenetics to manipulate lysosomal physiology and function, developing three lysosome-targeted optogenetic tools: lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2. These new actuators enable light-dependent regulation of key aspects such as lysosomal membrane potential, lumenal pH, hydrolase activity, degradation processes, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy via the MTOR pathway while it promotes Aß clearance through autophagy induction in cellular models of Alzheimer disease. Furthermore, lyso-ChR2 activation reduces Aß deposition and alleviates Aß-induced paralysis in Caenorhabditis elegans models of Alzheimer disease. Our lysosomal optogenetic actuators offer a novel method for dynamically regulating lysosomal physiology and autophagic activity in living cells and animals.

Recycling the recyclers: lysophagy emerges as a new pharmacological target for retinal degeneration.

Dysregulated macroautophagy/autophagy is one of the hallmarks of aging and has also been linked to higher incidence of several age-associated diseases such as age-related macular degeneration (AMD). The main cell type affected in AMD is the retinal pigment epithelium (RPE), and this disease can lead to central vision loss. Despite affecting around 8.7% of the population between 45-85 years, its etiopathogenesis remains unknown. In our recent manuscript using the pharmacological sodium iodate (SI) model of AMD we identified severe lysosomal membrane permeabilization (LMP) in the RPE, that leads to autophagy flux blockage and proteostasis defects. Treatment with the natural compound urolithin A (UA) reduces RPE cell death and alleviates vision loss, concurrent with full autophagy restoration. While UA was initially described as a specific mitophagy inducer, we now show that it is also able to promote SQSTM1/p62-dependent lysophagy in the context of lysosomal damage and LMP. Genetic downregulation of SQSTM1/p62 fully abolishes the effect of UA on lysophagy while mitophagy stimulation remains unaffected. In summary, these findings highlight the wide range of pathways modulated by UA and its potential implementation in the management of AMD and other diseases involving lysosomal damage.

Integrative analysis reveals key lysosomal genes as potential therapeutic targets in Alzheimer's disease.

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, TGFß, 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.

A lysosome-located and rhodamine-based fluorescence probe for recognizing hydrogen polysulfide.

Hydrogen polysulfide (H2Sn, n≥2), as a kind of active sulfur species (RSS), has become a hot topic in RSS. It can regulate the biological activity of many proteins through S-sulfhydrylation of cysteine residues (protein Cys-SSH), and has a protective effect on cells. Although there have been some studies on hydrogen polysulfide, its production, degradation pathway and regulation mechanism still need further be researched. In presented study, an original lysosome-localized fluorescent probe for determining H2Sn was developed utilizing rhodamine as the fluorogen. The probe used morpholine as the locating unit of lysosomes and chose 2-fluoro-5-nitrobenzoate as the recognizing group. Before adding H2Sn, the proposed probe displayed a spironolactone structure and emitted very weak fluorescence. After adding H2Sn, a conjugated xanthene was formed and the probe demonstrated green fluorescence. When the H2Sn concentration was varied from 6.0×10-7 mol·L-1 to 10.0×10-5 mol·L-1, the fluorescence intensity of the probe was linearly dependent on the H2Sn concentration. And the detection limit was 1.5×10-7 mol·L-1. The presented probe owned a fast response speed, good selectivity, excellent sensitivity and broad pH work scope. In addition, the probe had been well utilized to sense endogenic and exogenic H2Sn in lysosomes.

Host-specific SRSF7 regulates polymerase activity and replication of influenza A virus.

Avian influenza viruses crossing the host barrier to infect humans have caused great panic in human society and seriously threatened public health. Herein, we revealed that knockdown of SRSF7 significantly down-regulated influenza virus titers and viral protein expression. We further observed for the first time that human SRSF7, but not avian SRSF7, significantly inhibited polymerase activity (PB2627E). Molecular mapping demonstrated that amino acids 206 to 228 of human SRSF7 play a decisive role in regulating the polymerase activity, which contains the amino acid motif absent in avian SRSF7. Importantly, our results illustrated that the PB2627K-encoding influenza virus induces SRSF7 protein degradation more strongly via the lysosome pathway and not via the proteasome pathway. Functional enrichment analysis of SRSF7-related KEGG pathways indicated that SRSF7 is closely related to cell growth and death. Lastly, our results showed that knocking down SRSF7 interferes with normal polymerase activity. Taken together, our results advance our understanding of interspecies transmission and our findings point out new targets for the development of drugs preventing or treating influenza virus infection.

Lysosomal TBK1 responds to amino acid availability to relieve Rab7-dependent mTORC1 inhibition.

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.