Search Results (26)

Lutein is a dietary xanthophyll carotenoid valued for its antioxidant properties that has been shown to benefit liver health and reduce hepatic lipid accumulation; however, this lipid-lowering effect cannot be fully attributed to its antioxidant activity, and the underlying mechanism remains poorly understood. Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by excessive hepatic lipid accumulation, oxidative stress, and limited therapeutic options. Transcription factor EB (TFEB) coordinates the autophagy–lysosomal pathways involved in cellular lipid clearance, including lipophagy and lysosomal exocytosis, and is sensitive to the cellular redox state; however, whether the antioxidant lutein modulates TFEB-regulated lipid homeostasis remains unclear. HepG2 cells were exposed to free fatty acids (FFAs) to induce intracellular lipid accumulation and co-treated with lutein. TFEB localization and the expression of TFEB-related genes were assessed by immunofluorescence and qPCR, respectively. Immunofluorescence was also used to evaluate lipid droplet accumulation, LC3 content, and LAMP1 localization. Lipid droplet ultrastructure was analyzed by transmission electron microscopy, and extracellular triglyceride levels were measured as a functional readout of lipid extrusion. Lutein attenuated lipid droplet accumulation in FFA-treated cells, increased nuclear TFEB immunoreactivity, upregulated LC3 mRNA expression, and enhanced LC3 colocalization with lipid droplets. Chloroquine abolished the lipid-lowering effect of lutein, supporting an autophagy-dependent mechanism. In addition, lutein increased the abundance of LAMP1-positive compartments, while ultrastructural analysis and elevated extracellular triglyceride levels suggested enhanced lipid extrusion. These findings position the antioxidant lutein as a candidate natural compound whose lipid-lowering action is associated with TFEB nuclear translocation/activation and with the autophagy–lysosomal pathway, warranting further investigation in MASLD. Full article

Background: Atherosclerosis preferentially develops at arterial regions exposed to low shear stress (LSS), highlighting the critical role of local hemodynamic forces in disease initiation and progression. Emerging evidence indicates that endothelial lipid metabolism is a key determinant of vascular homeostasis; however, whether LSS directly regulates endothelial lipid droplets’ (LDs) dynamics remains unclear. In particular, the mechano-transduction pathways linking shear stress to lysosome-mediated lipid processing within the endothelium have yet to be defined. Methods: Complementary in vitro flow systems and in vivo atheroprone models were employed to examine the effects of LSS on endothelial lipid metabolism. Endothelial LDs accumulation, lysosome-dependent lipophagy, and atherosclerotic lesion development were systematically assessed under LSS conditions. Mechanistically, molecular profiling and rapamycin-mediated functional rescue were conducted to delineate the role of the KLF4/TFEB/ATP1A1 signaling axis in LSS-induced impairment of lysosome-dependent lipophagy. Results: We found that LSS induced pathological accumulation of LDs in vascular endothelial cells, accompanied by a marked suppression of lysosome-dependent lipophagy. Elucidation of the mechanism showed that LSS downregulated the shear-responsive transcription factor KLF4, resulting in aberrant phosphorylation of transcription factor EB (TFEB) and impaired TFEB nuclear translocation. Consequently, the TFEB transcriptional program governing lysosomal function was disrupted, including reduced expression of the TFEB target ATP1A1, leading to defective lysosomal acidification and blockade of lipid autophagic flux. Restoration of the KLF4/TFEB/ATP1A1 axis reactivated lipophagy, alleviated endothelial lipid burden, and significantly attenuated atherosclerotic lesion development. Conclusions: Our findings demonstrate that disruption of the KLF4/TFEB/ATP1A1 signaling pathway mediates LSS-induced impairment of endothelial lipophagy, thereby driving pathological LDs accumulation. This highlights the potential of restoring this axis as a therapeutic strategy to attenuate atherosclerotic progression. Full article

Lysosomes are crucial for the function of fetal vacuolated enterocytes in neonatal piglets, yet how they are regulated by miRNAs remains poorly defined. Therefore, this study aimed to elucidate how miRNAs govern lysosomal homeostasis in the developing intestine. Using a neonatal piglet model of lysosomal dysfunction induced by imipramine (IMI), we identified ssc-miR-214-3p as a key down-regulated miRNA implicated in lysosomal pathways. In IPEC-J2 enterocytes, the miR-214-3p mimic ameliorated IMI cytotoxicity by restoring cell viability and migration while suppressing apoptosis. Further analysis revealed that miR-214-3p directly reversed the lysosomal defects triggered by IMI treatment. Specifically, it alleviated lysosomal alkalinization and markedly restored acid phosphatase (ACP) activity, indicating a recovery of the acidic hydrolytic environment. This restoration was also accompanied by the preservation of lysosomal membrane integrity and a consequent reduction in the nuclear translocation of transcription factor EB (TFEB). Furthermore, cathepsin D (CTSD) was validated as a direct target of miR-214-3p by luciferase assay, and its overexpression reversed the protective effects of the mimic on lysosomal acidification and lysosome-associated membrane protein 1 (LAMP1) levels. Collectively, our findings reveal a novel miR-214-3p/CTSD axis that regulates lysosomal homeostasis during neonatal intestinal maturation, providing a potential therapeutic target for porcine intestinal disorders. Full article

Neurodegenerative disorders are increasingly linked to a progressive decline in lysosomal function. Activating Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, has therefore emerged as a promising therapeutic strategy to enhance cellular clearance in these conditions. In this study, we identified KHS-101 as a novel TFEB activator through a high-throughput screen of blood–brain-barrier-permeable small molecules. We demonstrated that KHS-101 promotes TFEB nuclear translocation, enhances lysosomal biogenesis and proteolytic activity, and increases autophagic flux. Furthermore, KHS-101 significantly accelerates the degradation of pathogenic A53T mutant α-synuclein in a cellular model of Parkinson’s disease, suggesting its potential to mitigate α-synuclein-mediated proteotoxicity and hold neuroprotective potential. Our findings identify KHS-101 as a potent TFEB activator and highlight the therapeutic potential of modulating the autophagy-lysosomal pathway for treating Parkinson’s disease and related disorders. Full article

Hepatitis E virus (HEV) is a zoonotic pathogen that can infect pregnant women and cause adverse pregnancy outcomes, including miscarriage and preterm delivery. The previous study demonstrated that HEV genotype 3 (HEV-3) inhibits complete autophagic flux in both mouse placental tissue and human trophoblast cells (JEG-3), evidenced by reduced expression of ATG proteins (including LC3, Beclin1, ATG4B, ATG5, and ATG9A) and accumulation of p62. However, the specific regulatory pathway involved remains unclear. Thus, eukaryotic expression vectors for HEV open reading frames (ORFs) were constructed, and ORF2 and ORF3 proteins were transiently overexpressed in JEG-3 cells via liposome transfection. While both ORF2 and ORF3 significantly reduced LC3B protein levels (p < 0.01), only ORF2 induced p62 accumulation (p < 0.01), indicative of autophagic inhibition, which indicates that ORF2 was the key viral protein mediating autophagy suppression in JEG-3. The results of WB and RT-qPCR showed that ORF2 suppressed the PI3K/Akt/mTOR pathway while enhancing nuclear translocation of TFEB (p < 0.01) and AMPK phosphorylation (p < 0.01), suggesting paradoxical activation of upstream autophagy regulators. Through co-transfection of mCherry-LC3 with ORF2, co-localization studies, and AlphaFold 3-based intermolecular interaction predictions, we propose that ORF2 directly binds LC3B to block autophagosome formation. Finally, co-immunoprecipitation confirmed physical interaction between HEV ORF2 and LC3B, elucidating the molecular mechanism of HEV-induced autophagy suppression in trophoblasts. These findings reveal the molecular mechanism by which HEV inhibits autophagy leading to miscarriage in mice, providing new insights into HEV-induced reproductive damage. Full article

Hepatocellular carcinoma (HCC) remains a therapeutic challenge due to metabolic plasticity and drug resistance. Oncolytic viruses (OVs), such as thymidine kinase-deleted vaccinia virus (oncoVV), selectively lyse tumors while stimulating antitumor immunity, however, their metabolic interplay with cancer cells is poorly understood. Here, we engineered an oncoVV-expressing Aphrocallistes vastus lectin (oncoVV-AVL) and uncovered its unique ability to exploit the ACSS2/TFEB axis, driving metabolic competition in HCC. In vitro, oncoVV-AVL triggered cell autophagy and lipid accumulation (3.4–5.7-fold upregulation of FASN and ACC1) while suppressing glucose uptake (41–63% higher extracellular glucose and 33–34% reduced lactate). Mechanistically, oncoVV-AVL upregulated acetyl-CoA synthetase 2 (ACSS2), promoting its nuclear translocation and interaction with transcription factor EB (TFEB) to concurrently activate lipogenesis and autophagic flux. The pharmacological inhibition of ACSS2 abolished these effects, confirming its central role. In vivo, oncoVV-AVL suppressed tumor growth while inducing lipid deposition (2-fold triglyceride increase), systemic hypoglycemia (42% glucose reduction), and autophagy activation (elevated LC3B-II/I ratios). This study establishes ACSS2 as a metabolic checkpoint in OV therapy, providing a rationale for combining oncolytic virotherapy with metabolic modulators in HCC. Full article

Lysosomal storage diseases (LSDs) are characterized by the accumulation of undegraded substrates within lysosomes, often associated with oxidative stress and impaired lysosomal function. In this study, we investigate the role of the c-Abl/TFEB pathway in different LSDs: Gaucher, Niemann-Pick type A (NPA), and Niemann-Pick type C (NPC). Our findings identify c-Abl activation (p-c-Abl) as a common pathogenic mechanism in these disorders. We demonstrate that c-Abl phosphorylates TFEB at Tyr173, leading to its cytoplasmic retention. Using pharmacological models of Gaucher, NPA and NPC in SH-SY5Y neuronal cells and HeLa cells, we assess the effects of the c-Abl inhibitors Imatinib and Neurotinib, as well as the antioxidant α-Tocopherol (α-TOH), on TFEB nuclear translocation and p-c-Abl protein levels. Additionally, we explore the effects of c-Abl inhibitors in cholesterol accumulation in LSDs neuronal models. Our results show that treatment with c-Abl inhibitors or α-TOH promotes TFEB nuclear translocation, enhances lysosomal clearance, and reduces cholesterol accumulation in all three LSD models. These findings highlight the c-Abl/TFEB pathway as a potential therapeutic target for LSDs and potentially other neurodegenerative disorders associated with lysosomal dysfunction. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Consequent to the loss of these cells, neuromuscular functions decline, causing progressive weakness, muscle wasting, and paralysis, leading to death in 2 to 5 years. More than 90% of ALS cases are sporadic, while the remaining 10% of cases are familial, due to mutations in 40 different genes. One of the most common genes to be mutated in ALS is TARDBP (transactive response DNA binding protein 43), which encodes TDP-43 (TAR DNA-binding protein 43). A mutation in exon 6 of TARDBP causes the aminoacidic substitution G376D in the C-terminal region of TDP-43, leading to its cytoplasmic mislocalization and aggregation. In fibroblasts derived from patients carrying this mutation, we found a strong increase in lysosome number, with overexpression and higher nuclear translocation of the transcription factor TFEB. In contrast, lysosomal functionality was deeply compromised. Interestingly, lysosomal activity was unaffected at an early stage of the disease, worsening in more advanced stages. Moreover, we observed the same pathological phenotype in iPSC (induced pluripotent stem cells)-derived patient motor neurons carrying the G376D mutation. Therefore, this mutation compromises the functionality of lysosomes, possibly contributing to neurodegeneration. Full article

AdipoRon is a selective adiponectin receptor agonist that inhibits vascular remodeling by promoting the differentiation of arterial smooth muscle cells (SMCs). Our recent studies have demonstrated that activation of TFEB and its downstream autophagy–lysosomal signaling contribute to adipoRon-induced differentiation of SMCs. The present study was designed to examine whether acid sphingomyelinase (ASM; gene symbol Smpd1) is involved in mediating adipoRon-induced activation of TFEB–autophagy signaling and inhibition of proliferation/migration in arterial SMCs. Our results showed that adipoRon induced ASM expression and ceramide production in Smpd1^+/+ SMCs, which were abolished in Smpd1^−/− SMCs. Compared to Smpd1^+/+ SMCs, Smpd1^−/− SMCs exhibited less TFEB nuclear translocation and activation of autophagy signaling induced by adipoRon stimulation. SMC differentiation was further characterized by retarded wound healing, reduced proliferation, F-actin reorganization, and MMP downregulation. The results showed that Smpd1^−/− SMCs were less responsive to adipoRon-induced differentiation than Smpd1^+/+ SMCs. Mechanistically, adipoRon increased the expression of protein phosphatases such as calcineurin and PP2A in Smpd1^+/+ SMCs. The calcineurin inhibitor FK506/cyclosporin A or PP2A inhibitor okadaic acid significantly attenuated adipoRon-induced activation of TFEB–autophagy signaling. In addition, adipoRon-induced expressions of calcineurin and PP2A were not observed in Smpd1^−/− SMCs. However, activation of calcineurin by lysosomal TRPML1-Ca²⁺ channel agonist ML-SA1 rescued the activation of TFEB–autophagy signaling and the effects of adipoRon on cell differentiation in Smpd1^−/− SMCs. Taken together, these data suggested that ASM regulates adipoRon-induced SMC differentiation through TFEB activation. This study provided novel mechanistic insights into the therapeutic effects of adipoRon on TFEB signaling and pathological vascular remodeling. Full article

Acidosis, a common feature of cerebral ischemia and hypoxia, results in neuronal damage and death. This study aimed to investigate the protective effects and mechanisms of action of melatonin against acidosis-induced neuronal damage. SH-SY5Y cells were exposed to an acidic environment to simulate acidosis, and a photothrombotic (PT) infarction model was used to establish an animal model of cerebral ischemia of male C57/BL6J mice. Both in vivo and in vitro studies demonstrated that acidosis increased cytoplasmic transcription factor EB (TFEB) levels, reduced nuclear TFEB levels, and suppressed autophagy, as evidenced by elevated p62 levels, a higher LC3-II/LC3-I ratio, decreased synapse-associated proteins (PSD-95 and synaptophysin), and increased neuronal apoptosis. In contrast, melatonin promoted the nuclear translocation of TFEB, enhanced autophagy, and reversed neuronal apoptosis. Moreover, the role of TFEB in melatonin’s neuroprotective effects was validated by modulating TFEB nuclear translocation. In conclusion, melatonin mitigates acidosis-induced neuronal damage by promoting the nuclear translocation of TFEB, thereby enhancing autophagy. These findings offer new insights into potential treatments for acidosis. Full article

Metabolic syndrome (MetS) is a cluster of metabolic abnormalities, including visceral obesity, dyslipidemia, and insulin resistance. In this regard, visceral white adipose tissue (vWAT) plays a critical role, influencing energy metabolism, immunomodulation, and oxidative stress. Adipose-derived stem cells (ADSCs) are key players in these processes within vWAT. While second-generation antipsychotics (SGAs) have significantly improved treatments for mental health disorders, their chronic use is associated with an increased risk of MetS. In this study, we explored the impact of SGAs on ADSCs to better understand their role in MetS and identify potential therapeutic targets. Our findings reveal that olanzapine disrupts lipid droplet formation during adipogenic differentiation, impairing insulin receptor endocytosis, turnover, and signaling. SGAs also alter the endolysosomal compartment, leading to acidic vesicle accumulation and increased lysosomal biogenesis through TFEB activation. PKCζ is crucial for the SGA-induced nuclear translocation of TFEB and acidic vesicle formation. Notably, inhibiting PKCζ restored insulin receptor tyrosine phosphorylation, normalized receptor turnover, and improved downstream signaling following olanzapine treatment. This activation of PKCζ by olanzapine is driven by increased phosphatidic acid synthesis via phospholipase D (PLD), following G protein-coupled receptor (GPCR) signaling activation. Overall, olanzapine and clozapine disrupt endolysosomal homeostasis and insulin signaling in a PKCζ-dependent manner. These findings highlight SGAs as valuable tools for uncovering cellular dysfunction in vWAT during MetS and may guide the development of new therapeutic strategies to mitigate the metabolic side effects of these drugs. Full article

Among the several mechanisms accounting for endocrine resistance in breast cancer, autophagy has emerged as an important player. Previous reports have evidenced that tamoxifen (Tam) induces autophagy and activates transcription factor EB (TFEB), which regulates the expression of genes controlling autophagy and lysosomal biogenesis. However, the mechanisms by which this occurs have not been elucidated as yet. This investigation aims at dissecting how TFEB is activated and contributes to Tam resistance in luminal A breast cancer cells. TFEB was overexpressed and prominently nuclear in Tam-resistant MCF7 cells (MCF7-TamR) compared with their parental counterpart, and this was not dependent on alterations of its nucleo-cytoplasmic shuttling. Tam promoted the release of lysosomal Ca²⁺ through the major transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) and two-pore channels (TPCs), which caused the nuclear translocation and activation of TFEB. Consistently, inhibiting lysosomal calcium release restored the susceptibility of MCF7-TamR cells to Tam. Our findings demonstrate that Tam drives the nuclear relocation and transcriptional activation of TFEB by triggering the release of Ca²⁺ from the acidic compartment, and they suggest that lysosomal Ca²⁺ channels may represent new druggable targets to counteract the onset of autophagy-mediated endocrine resistance in luminal A breast cancer cells. Full article

The accumulation of exogenous silver nanoparticles (AgNPs) will terminally bring about liver injury, including cell death, where DNA methylation tends to be a crucial epigenetic modulator. The change in the cell autophagy level verified to be closely associated with hepatocyte death has been followed with wide interest. But the molecular toxicological mechanisms of AgNPs in relation to DNA methylation, autophagy, and cell death remain inconclusive. To address the issue above, in LO2 cells treated with increasing concentrations of AgNPs (0, 5, 10, and 20 μg/mL), a cell cytotoxicity assay was performed to analyze the level of cell death, which also helped to choose an optimal concentration for next experiments. An immunofluorescence assay was used to determine the autophagic flux as well as TFEB translocation, with qRT-PCR and western blot being used to analyze the expression level of autophagy-related genes and proteins. According to our findings, in the determination of cell viability, 20 μg/mL (AgNPs) was adopted as the best working concentration. LO2 cell death, autophagy, and TFEB nuclear translocation were induced by AgNPs, which could be inhibited by lysosome inhibitor chloroquine (CQ) or siRNA specific for TFEB. Moreover, AgNP exposure led to DNA hypermethylation, with DNMT1 taking part mainly, which could be obviously prevented by 5-Aza-2′-deoxycytidine (5-AzaC) or trichostatin A (TSA) treatment or DNMT1 knockout in LO2 cells. Our studies suggest that through TFEB-dependent cell autophagy, increased DNMT1 may facilitate cell death induced by AgNPs. Full article

Dysregulated biological behaviors of trophoblast cells can result in recurrent spontaneous abortion (RSA)—whose underlying etiology still remains insufficient. Autophagy, a conserved intracellular physiological process, is precisely monitored throughout whole pregnancy. Although the exact mechanism or role remains elusive, epigenetic modification has emerged as an important process. Herein, we found that a proportion of RSA patients exhibited higher levels of autophagy in villus tissues compared to controls, accompanied with impaired histone deacetylase (HDAC) expression. The purpose of this study is to explore the connection between HDACs and autophagy in the pathological course of RSA. Mechanistically, using human trophoblast cell models, treatment with HDAC inhibitor (HDACI)-trichostatin A (TSA) can induce autophagy by promoting nuclear translocation and transcriptional activity of the central autophagic regulator transcription factor EB (TFEB). Specifically, overactivated autophagy is involved in the TSA-driven growth inhibition of trophoblast, which can be partially reversed by the autophagy inhibitor chloroquine (CQ) or RNA interference of TFEB. In summary, our results reveal that abnormal acetylation and autophagy levels during early gestation may be associated with RSA and suggest the potential novel molecular target TFEB for RSA treatment. Full article

Human colon carcinomas, including HCT116 cells, often exhibit high autophagic flux under nutrient deprivation or hypoxic conditions. Mitochondrial ROS (mROS) is known as a ‘molecular switch’ for regulating the autophagic pathway, which is critical for directing cancer cell survival or death. In early tumorigenesis, autophagy plays important roles in maintaining cellular homeostasis and contributes to tumor growth. However, the relationships between mROS and the autophagic capacities of HCT116 cells are poorly understood. Ubiquinol cytochrome c reductase binding protein (UQCRB) has been reported as a biomarker of colorectal cancer, but its role in tumor growth has not been clarified. Here, we showed that UQCRB is overexpressed in HCT116 cells compared to CCD18co cells, a normal colon fibroblast cell line. Pharmacological inhibition of UQCRB reduced mROS levels, autophagic flux, and the growth of HCT116 tumors in a xenograft mouse model. We further investigated mutant UQCRB-overexpressing cell lines to identify functional links in UQCRB-mROS-autophagy. Notably, an increasing level of mROS caused by UQCRB overexpression released Ca²⁺ by the activation of lysosomal transient receptor potential mucolipin 1 (TRPML1) channels. This activation induced transcription factor EB (TFEB) nuclear translocation and lysosome biogenesis, leading to autophagy flux. Collectively, our study showed that increasing levels of mROS caused by the overexpression of UQCRB in human colon carcinoma HCT116 cells could be linked to autophagy for cell survival. Full article