Search Results (34)

Cellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive- and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus, and SARS-CoV-2, not only in mammalian and insect cells but also in precision-cut lung slices and neuronal organoids. ULK1 activation inhibited enveloped viruses but not EV71. However, receptor presentation at the cytoplasmic membrane remained unaffected, indicating that receptor binding was unchanged, while later stages of endocytosis were targeted via two distinct pathways. Drug-induced activation of the AMPK pathway reduced early endocytic factor TXNIP by suppressing translation. In contrast, the amounts of Rab5 and the late endosomal marker Rab7 decreased due to translation inactivation and ULK1-dependent proteasome activation within minutes. Furthermore, activation of AMPK hindered the late replication steps of SARS-CoV-2 by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction in early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acid and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses. Full article

Geniposidic 4-isoamyl ester (GENI) with anti-aging effects is a new iridoid glycoside derivative from Gardenia jasminoides Ellis found in our previous study. In this study, to indicate whether this compound has anti-Alzheimer’s disease (AD) effect, the galactose-induced AD mice and naturally aging mice with AD were used to do drug efficacy evaluation. Furthermore, the Western blot, small interfering RNA (siRNA), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CESTA), liquid chromatography-tandem mass spectrometry (LC/MS-MS), adenosine 5′-monophosphate-activated protein kinase (AMPK) mutants and surface plasmon resonance (SPR) analysis were utilized to clarify the mechanism of action and identify target protein of this molecule. GENI exerts anti-AD efficacy in galactose-induced AD mice and naturally aging mice with AD through neuroprotection and modification of autophagy and neuron inflammation. Moreover, AMPK as the target protein of GENI to produce an anti-AD effect is identified and the ASP148, ASP157, and ASP166 of the AMPK α subunit and lysine (LYS)148, aspartic acid (ASP)156, LYS309, and ASP316 in the AMPK γ subunit as binding sites are confirmed. Meanwhile, the AMPK/unc-51-like autophagy-activating kinase 1 (ULK1)/microtubule-associated protein 1 light chain 3 beta (LC3B) and AMPK/mammalian target of rapamycin (mTOR) signaling pathways involved in anti-AD effects of GENI. The findings provide a new perspective on treating neurodegenerative diseases by activating AMPK for the energy metabolism disorder. Full article

Endocannabinoids have attracted great interest for their ability to counteract the neuroinflammation underlying Alzheimer’s disease (AD). Our study aimed at evaluating whether this activity was also due to a rebalance of autophagic mechanisms in cellular and animal models of AD. We supplied URB597, an inhibitor of Fatty-Acid Amide Hydrolase (FAAH), the degradation enzyme of anandamide, to microglial cultures treated with Aβ_25-35, and to Tg2576 transgenic mice, thus increasing the endocannabinoid tone. The addition of URB597 did not alter cell viability and induced microglia polarization toward an anti-inflammatory phenotype, as shown by the modulation of pro- and anti-inflammatory cytokines, as well as M1 and M2 markers; moreover microglia, after URB597 treatment released higher levels of Bdnf and Nrf2, confirming the protective role underlying endocannabinoids increase, as shown by RT-PCR and immunofluorescence experiments. We assessed the number and area of amyloid plaques in animals administered with URB597 compared to untreated animals and the expression of autophagy key markers in the hippocampus and prefrontal cortex from both groups of mice, via immunohistochemistry and ELISA. After URB597 supply, we detected a reduction in the number and areas of amyloid plaques, as detected by Congo Red staining and a reshaping of microglia activation as shown by M1 and M2 markers’ modulation. URB597 administration restored autophagy in Tg2576 mice via an increase in BECN1 (Beclin1), ATG7 (Autophagy Related 7), LC3 (light chain 3) and SQSTM1/p62 (sequestrome 1) as well as via the activation of the ULK1 (Unc-51 Like Autophagy Activating Kinase 1) signaling pathway, suggesting that it targets mTOR/ULK1-dependent autophagy pathway. The potential of endocannabinoids to rebalance autophagy machinery may be considered as a new perspective for therapeutic intervention in AD. Full article

Hexavalent chromium (Cr(VI)), a pervasive industrial contaminant, is highly toxic to both humans and animals. However, its effects on turtles are largely unexplored. Our study aimed to investigate the toxic effects of Cr(VI) on the Reeves’ turtles (Mauremys reevesii) primary hepatocytes. We exposed hepatocytes to two concentrations (25 μM and 50 μM) of Cr(VI) for 24 h. The results showed that compared to controls, Cr(VI)-treated cells showed elevated antioxidant enzyme activity (catalase (CAT) and superoxide dismutase (SOD)) and increased reactive oxygen species (ROS) levels. Adenosine triphosphatae (ATP) levels decreased, indicating mitochondrial dysfunction. Additionally, we found significant changes in mitochondrial dynamics related genes, with downregulation of mitofusin 2 (Mfn2) and silent information regulator 1 (SIRT1) and a decrease in sirtuin 3 (SIRT3) and tumor protein 53 (p53) mRNA levels. Annexin V-FITC fluorescence staining-positive cells increased with higher Cr(VI) concentrations, marked by elevated bcl-2-associated X protein (Bax) and cysteinyl aspartate specific proteinase (Caspase3) mRNA levels and reduced B-cell lymphoma-2 (Bcl2) expression. Autophagy-related genes were also affected, with increased microtubule-associated protein 1 light chain 3 (LC3-I), microtubule-associated protein light chain 3II (LC3-II), unc-51-like autophagy-activating kinase 1 (ULK1), and sequestosome 1 (p62/SQSTM1) mRNA levels and decreased mammalian target of rapamycin (mTOR) and Beclin1 expression. Taken together, Cr(VI) promotes cell apoptosis and autophagy in turtle hepatocytes by inducing oxidative stress and disrupting mitochondrial function. These findings highlight the serious health risks posed by Cr(VI) pollution and emphasize the need for protecting wild turtle populations. Full article

Melanosomes are specialized membrane-bound organelles where melanin is synthesized and stored. The levels of melanin can be effectively reduced by inhibiting melanin synthesis or promoting melanosome degradation via autophagy. Ceramide, a key component in the metabolism of sphingolipids, is crucial for preserving the skin barrier, keeping it hydrated, and warding off the signs of aging. Our preliminary study indicated that a long-chain C22-ceramide compound (Ehux-C22) isolated from the marine microalga Emiliania huxleyi, reduced melanin levels via melanosomal autophagy in B16 cells. Recently, microRNAs (miRNAs) were shown to act as melanogenesis-regulating molecules in melanocytes. However, whether the ceramide Ehux-C22 can induce melanosome autophagy at the post-transcriptional level, and which potential autophagy-dependent mechanisms are involved, remains unknown. Here, miR-199a-3p was screened and identified as a novel upregulated miRNA in Ehux-C22-treated B16 cells. An in vitro high melanin expression model in cultured mouse melanoma cells (B16 cells) was established by using 0.2 μM alpha-melanocyte-stimulating hormone(α-MSH) and used for subsequent analyses. miR-199a-3p overexpression significantly enhanced melanin degradation, as indicated by a reduction in the melanin level and an increase in melanosome autophagy. Further investigation demonstrated that in B16 cells, Ehux-C22 activated miR-199a-3p and inhibited mammalian target of rapamycin(mTOR) level, thus activating the mTOR-ULK1 signaling pathway by promoting the expression of unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma-2 (Bcl-2), Beclin-1, autophagy-related gene 5 (ATG5), and microtubule-associated protein light chain 3 (LC3-II) and degrading p62. Therefore, the roles of Ehux-C22-regulated miR-199a-3p and the mTOR pathway in melanosomal autophagy were elucidated. This research may provide novel perspectives on the post-translational regulation of melanin metabolism, which involves the coordinated control of melanosomes. Full article

The β-thalassemias are inherited genetic disorders affecting the hematopoietic system. In β-thalassemias, more than 350 mutations of the adult β-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in β-thalassemia are CRISPR-Cas9-based genome editing of the β-globin gene, forcing “de novo” HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in β-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for β-thalassemia. Full article

A specialized membrane-bound organelle, named the melanosome, is central to the storage and transport of melanin as well as melanin synthesis in melanocytes. Although previous studies have linked melanosomal degradation to autophagy, the precise mechanisms remain elusive. Autophagy, a complex catabolic process involving autophagosomes and lysosomes, plays a vital role in cellular constituent degradation. In this study, the role of autophagy in melanosomal degradation was explored, employing a cell-based screening system designed to unveil key pathway regulators. We identified specific dipeptidyl peptidase-4 inhibitors, such as teneligliptin hydrobromide and retagliptin phosphate, as novel agents inducing melanophagy through a comprehensive screening of a ubiquitination-related chemical library. We found that treatment with teneligliptin hydrobromide or retagliptin phosphate not only diminishes melanin content elevated by alpha-melanocyte-stimulating hormone (α-MSH) but also triggers autophagy activation within B16F1 cells. In addition, the targeted inhibition of unc-51-like kinase (ULK1) significantly attenuated both the anti-pigmentation effects and autophagy induced by teneligliptin hydrobromide and retagliptin phosphate in α-MSH-treated cells. Collectively, our data demonstrate a new frontier in understanding melanosomal degradation, identifying teneligliptin hydrobromide and retagliptin phosphate as promising inducers of melanophagy via autophagy activation. This study contributes essential insights into cellular degradation mechanisms and offers potential therapeutic avenues in the regulation of pigmentation. Full article

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and ³H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo. Full article

p53, a crucial tumor suppressor and transcription factor, plays a central role in the maintenance of genomic stability and the orchestration of cellular responses such as apoptosis, cell cycle arrest, and DNA repair in the face of various stresses. Sestrins, a group of evolutionarily conserved proteins, serve as pivotal mediators connecting p53 to kinase-regulated anti-stress responses, with Sestrin 2 being the most extensively studied member of this protein family. These responses involve the downregulation of cell proliferation, adaptation to shifts in nutrient availability, enhancement of antioxidant defenses, promotion of autophagy/mitophagy, and the clearing of misfolded proteins. Inhibition of the mTORC1 complex by Sestrins reduces cellular proliferation, while Sestrin-dependent activation of AMP-activated kinase (AMPK) and mTORC2 supports metabolic adaptation. Furthermore, Sestrin-induced AMPK and Unc-51-like protein kinase 1 (ULK1) activation regulates autophagy/mitophagy, facilitating the removal of damaged organelles. Moreover, AMPK and ULK1 are involved in adaptation to changing metabolic conditions. ULK1 stabilizes nuclear factor erythroid 2-related factor 2 (Nrf2), thereby activating antioxidative defenses. An understanding of the intricate network involving p53, Sestrins, and kinases holds significant potential for targeted therapeutic interventions, particularly in pathologies like cancer, where the regulatory pathways governed by p53 are often disrupted. Full article

Age-related macular degeneration (AMD) is a largely incurable disease and an emerging problem in aging societies. It occurs in two forms, dry and wet (exudative, neovascular), which may cause legal blindness and sight loss. Currently, there is not any effective treatment for dry AMD. Meanwhile, repeated intravitreal injections with antibodies effective against vascular endothelial growth factor A (VEGFA) slow down wet AMD progression but are not free from complications. (-)-Epigallocatechin-3-gallate (EGCG) is an active compound of green tea, which exerts many beneficial effects in the retinal pigment epithelium and the neural retina. It has been reported to downregulate the VEGFA gene by suppressing its activators. The inhibition of mitogen-activated protein kinases 1 and 3 (MAPK1 and MAPK3) may lie behind the antiangiogenic action of EGCG mediated by VEGFA. EGCG exerts protective effects against UV-induced damage to retinal cells and improves dysfunctional autophagy. EGCG may also interact with the mechanistic target rapamycin (MTOR) and unc-51-like autophagy activating kinase (ULK1) to modulate the interplay between autophagy and apoptosis. Several other studies report beneficial effects of EGCG on the retina that may be related to wet AMD. Therefore, controlled clinical trials are needed to verify whether diet supplementation with EGCG or green tea consumption may improve the results of anti-VEGFA therapy in wet AMD. Full article

The Hedgehog receptor, Patched1 (PTCH1), is a well-known tumour suppressor. While the tumour suppressor’s activity is mostly ascribed to its function as a repressor of the canonical Smoothened/Gli pathway, its C-terminal domain (CTD) was reported to have additional non-canonical functions. One of them is the reduction of autophagic flux through direct interaction with the Unc-51, like the autophagy activating kinase (ULK) complex subunit autophagy-related protein-101 (ATG101). With the aim of investigating whether this function of PTCH1 is important in cancer cell fitness, we first identified frameshift mutations in the CTD of PTCH1 in cancer databases. We demonstrated that those mutations disrupt PTCH1 interaction with ATG101 and increase autophagic flux. Using deletion mutants of the PTCH1 CTD in co-immunoprecipitation studies, we established that the 1309–1447 region is necessary and sufficient for interaction with ATG101. We next showed that the three most common PTCH1 CTD mutations in endometrial, stomach and colon adenocarcinomas that cause frameshifts at S1203, R1308 and Y1316 lack the ability to interact with ATG101 and limit autophagic flux, determined by bafilomycin A1-sensitive accumulation of the autophagy markers LC3BII and p62. We next engineered PTCH1 indel mutations at S1223 by CRISPR/Cas9 in SW620 colon cancer cells. Comparison of two independent clones harbouring PTCH1 S1223fs mutations to their isogenic parental cell lines expressing wild-type PTCH1 showed a significant increase in basal and rapamycin-stimulated autophagic flux, as predicted by loss of ATG101 interaction. Furthermore, the PTCH1 CTD mutant cells displayed increased proliferation in the presence of rapamycin and reduced sensitivity to glycolysis inhibitors. Our findings suggest that loss of the PTCH1-ATG101 interaction by mutations in the CTD of PTCH1 in cancer might confer a selective advantage by stimulating autophagy and facilitating adaptation to nutrient deprivation conditions. Full article

Autophagy is a cellular process that removes damaged components of cells and recycles them as biochemical building blocks. Autophagy can also be induced to protect cells in response to intra- and extracellular stresses, including damage to cellular components, nutrient deprivation, hypoxia, and pathogenic invasion. Dysregulation of autophagy has been attributed to various diseases. In particular, autophagy protects cancer cells by supporting tumor cell survival and the development of drug resistance. Understanding the pathophysiological mechanisms of autophagy in cancer has stimulated the research on discovery and development of specific inhibitors targeting various stages of autophagy. In recent years, Unc-51-like autophagy-activating kinase (ULK) inhibitors have become an attractive strategy to treat cancer. This review summarizes recent discoveries and developments in small-molecule ULK inhibitors and their potential as anticancer agents. We focused on structural features, interactions with binding sites, and biological effects of these inhibitors. Overall, this review will provide guidance for using ULK inhibitors as chemical probes for autophagy in various cancers and developing improved ULK inhibitors that would enhance therapeutic benefits in the clinic. Full article

Abnormal vasorin (Vasn) expression occurs in multiple diseases, particularly liver cancers. Vasn knockout (KO) in mice causes malnutrition, a shortened life span, and decreased physiological functions. However, the causes and underlying mechanisms remain unknown. Here, we established Vasn KO C57BL/6J mice by using the CRISPR/Cas9 system. The animals were weighed, and histology, immunohistochemistry, electronic microscopy, and liver function tests were used to examine any change in the livers. Autophagy markers were detected by Western blotting. MicroRNA (miRNA) sequencing was performed on liver samples and analyses to study the signaling pathway altered by Vasn KO. Significant reductions in mice body and liver weight, accompanied by abnormal liver function, liver injury, and reduced glycogen accumulation in hepatocytes, were observed in the Vasn KO mice. The deficiency of Vasn also significantly increased the number of autophagosomes and the expression of LC3A/B-II/I but decreased SQSTM1/p62 levels in hepatocytes, suggesting aberrant activation of autophagy. Vasn deficiency inhibited glycogen-mediated mammalian target of rapamycin (mTOR) phosphorylation and activated Unc-51-like kinase 1 (ULK1) signaling, suggesting that Vasn deletion upregulates hepatocyte autophagy through the mTOR-ULK1 signaling pathway as a possible cause of diminished life span and health. Our results indicate that Vasn is required for the homeostasis of liver glycogen metabolism upstream of hepatocyte autophagy, suggesting research values for regulating Vasn in pathways related to liver physiology and functions. Overall, this study provides new insight into the role of Vasn in liver functionality. Full article

Two common γ-chain family cytokines IL-2 and IL-15 stimulate the same mammalian target of rapamycin complex-1 (mTORC1) signaling yet induce effector T (T_E) and memory T (T_M) cell differentiation via a poorly understood mechanism(s). Here, we prepared in vitro IL-2-stimulated T_E (IL-2/T_E) and IL-15-stimulated T_M (IL-15/T_M) cells for characterization by flow cytometry, Western blotting, confocal microscopy and Seahorse-assay analyses. We demonstrate that IL-2 and IL-15 stimulate strong and weak mTORC1 signals, respectively, which lead to the formation of CD62 ligand (CD62L)⁻ killer cell lectin-like receptor subfamily G member-1 (KLRG)⁺ IL-2/T_E and CD62L⁺KLRG⁻ IL-15/T_M cells with short- and long-term survival following their adoptive transfer into mice. The IL-15/mTORC1^Weak signal activates the forkhead box-O-1 (FOXO1), T cell factor-1 (TCF1) and Eomes transcriptional network and the metabolic adenosine monophosphate-activated protein kinase-α-1 (AMPKα1), Unc-51-like autophagy-activating kinase-1 (ULK1) and autophagy-related gene-7 (ATG7) axis, increasing the expression of mitochondrial regulators aquaporin-9 (AQP9), mitochondrial transcription factor-A (TFAM), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), carnitine palmitoyl transferase-1 (CPT1α), microtubule-associated protein light chain-3 II (LC3II), Complex I and ortic atrophy-1 (OPA1), leading to promoting mitochondrial biogenesis and fatty-acid oxidation (FAO). Interestingly, AMPKα1 deficiency abrogates these downstream responses to IL-15/mTORC1^Weak signaling, leading to the upregulation of mTORC1 and hypoxia-inducible factor-1α (HIF-1α), a metabolic switch from FAO to glycolysis and reduced cell survival. Taken together, our data demonstrate that IL-15/mTORC1^Weak signaling controls T-cell memory via activation of the transcriptional FOXO1-TCF1-Eomes and metabolic AMPKα1-ULK1-ATG7 pathways, a finding that may greatly impact the development of efficient vaccines and immunotherapies for the treatment of cancer and infectious diseases. Full article

Background: As a central organ of energy metabolism, the liver is closely related to selenium for its normal function and disease development. However, the underlying roles of mitochondrial energy metabolism and mitophagy in liver fibrosis associated with selenium remain unclear. Methods: 28 rats were randomly divided into normal, low-selenium, nano-selenium supplement-1, and supplement-2 groups for a 12-week intervention. We observed pathological and ultrastructural changes in the liver and analyzed the effects of selenium deficiency and nano-selenium supplementation on liver metabolic activities and crucial proteins expression of mammalian target of the rapamycin (mTOR) signaling pathway. Results: Selenium deficiency caused liver pathological damage and fibrosis with the occurrence of mitophagy by disrupting normal metabolic activities; meanwhile, the mTOR signaling pathway was up-regulated to enhance mitophagy to clear damaged mitochondria. Furthermore, nano-selenium supplements could reduce the severity of pathological damage and fibrosis in livers and maintain normal energy metabolic activity. With the increased concentrations of nano-selenium supplement, swelling mitochondria and mitophagy gradually decreased, accompanied by the higher expression of mTOR and phosphorylation-modified mTOR proteins and lower expression of unc-51 like autophagy activating kinase 1 (ULK1) and phosphorylation-modified ULK1 proteins. Conclusions: Mitophagy regulated by the mTOR signaling pathway plays a dual protective role on low-selenium inducing liver fibrosis and nano-selenium supplements preventing liver fibrosis. Mitochondrial energy metabolism plays an important role in these processes as well. Full article