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High‐altitude pulmonary edema (HAPE) is a life‐threatening disease, and autophagy deficiency is implicated in the pathogenesis of HAPE. Eleutheroside B (EB), which is the main bioactive component of Acanthopanax senticosus, exhibits various pharmacological activities. Our previous research demonstrated that autophagic structures were widely found in the ultrastructure of lung tissue in HAPE rats. However, whether EB regulates autophagy deficiency in HAPE remains unknown. This study aimed to investigate the protective effects of EB on hypobaric hypoxia‐induced HAPE and explore the underlying molecular mechanism of regulating autophagy. The rat model of high‐altitude pulmonary edema was replicated using a hypobaric hypoxic chamber. Rats were pretreated with EB or in combination with chloroquine or compound C. The pulmonary edema was assessed by the lung wet/dry ratio, total protein concentration in bronchoalveolar lavage fluid, and histological analysis. Inflammation and oxidative stress were measured using commercial biochemical kits. Autophagy and autophagic flux were evaluated by western blotting, transmission electron microscopy, and adeno‐associated virus‐mRFP‐GFP‐labeled tandem fluorescence LC3. The AMPK/mTOR signaling pathway was detected by western blotting. EB alleviated hypobaric hypoxia‐induced pulmonary edema, hypoxemia, acid–base imbalance in the blood, inflammation, and oxidative stress in a dose‐dependent manner. EB restored impaired autophagic flux by activating the AMPK/mTOR signaling pathway. However, chloroquine or compound C abolished eleutheroside B‐mediated autophagy flux restoration. EB has the potential to restore impaired autophagic flux in the lung of hypobaric hypoxia‐induced HAPE rats, which could be attributed to the activation of AMPK/mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

Small cell lung cancer (SCLC) is characterized by rapid development of chemoresistance and poor outcomes. Cyclin‐dependent kinase 4/6 inhibitors (CDK4/6is) are widely used in breast cancer and other cancer types. However, the molecular mechanisms of CDK4/6 in SCLC chemoresistance remain poorly understood. Here, Rb1flox/flox, Trp53flox/flox, Ptenflox/flox (RTP) and Rb1flox/flox, Trp53flox/flox, MycLSL/LSL (RPM) spontaneous SCLC mouse models, SCLC cell line‐derived xenograft (CDX) models, and SCLC patient‐derived xenograft (PDX) models are established to reveal the potential effects of CDK4/6is on SCLC chemoresistance. In this study, it is found that CDK4/6is palbociclib (PD) or ribociclib (LEE) combined with chemotherapeutic drugs significantly inhibit SCLC tumor growth. Mechanistically, CDK4/6is do not function through the classic Retionblastoma1 (RB) dependent axis in SCLC. CDK4/6is induce impair autophagy through the AMBRA1‐lysosome signaling pathway. The upregulated AMBRA1 protein expression leads to CDK6 degradation via autophagy, and the following TFEB and TFE3 nuclear translocation inhibition leading to the lysosome‐related genes levels downregulation. Moreover, it is found that the expression of CDK6 is higher in SCLC tumors than in normal tissue and it is associated with the survival and prognosis of SCLC patients. Finally, these findings demonstrate that combining CDK4/6is with chemotherapy treatment may serve as a potential therapeutic option for SCLC patients. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CVD) remains the major cause of morbidity and mortality around the world. Transcription factor EB (TFEB) is a master regulator of lysosome biogenesis and autophagy. Emerging studies revealed that TFEB also mediates cellular adaptation responses to various stimuli, such as mitochondrial dysfunction, pathogen infection and metabolic toxin. Based on its significant capability to modulate the autophagy-lysosome process (ALP), TFEB plays a critical role in the development of CVD. In this review, we briefly summarize that TFEB regulates cardiac dysfunction mainly through ameliorating lysosomal and mitochondrial dysfunction and reducing inflammation. [ABSTRACT FROM AUTHOR]

Urinary tract infections are primarily caused by uropathogenic Escherichia coli (UPEC). UPEC infects bladder epithelial cells (BECs) via fusiform vesicles and escapes into the cytosol by disrupting fusiform vesicle membrane using outer membrane phospholipase PldA, and establishes biofilm-like intracellular bacterial communities (IBCs) for protection from host immune clearance. Cytosolic UPEC is captured by autophagy to form autophagosomes, then transported to lysosomes, triggering the spontaneous exocytosis of lysosomes. The mechanism by which UPEC evades autophagy to recognize and form IBCs remains unclear. Here, we demonstrate that by inhibiting autophagic flux, UPEC PldA reduces the lysosome exocytosis of BECs. By reducing intracellular phosphatidylinositol 3-phosphate levels, UPEC PldA increases the accumulation of NDP52 granules and decreases the targeting of NDP52 to autophagy, hence stalling preautophagosome structures. Thus, our results uncover a critical role for PldA to inhibit autophagic flux, favoring UPEC escapes from lysosome exocytosis, thereby contributing to acute urinary tract infection. [ABSTRACT FROM AUTHOR]

Nine jatrophane diterpenoids were isolated from the whole plant Euphorbia helioscopia, including two new ones, helioscopnins A (1) and B (2). Comprehensive spectroscopic data analysis and ECD calculations elucidated their structures, including absolute configurations. All compounds were evaluated for bioactivity towards autophagic flux by flow cytometry using HM mCherry-GFP-LC3 cells. Compounds 1, 3, 4, 5, 8, and 9 significantly increased autophagic flux. [ABSTRACT FROM AUTHOR]

Ambient fine particulate matter (PM2.5), a vital environmental toxicant, not only adversely affects the cardiovascular and respiratory systems but also potentially exhibits an association with intestinal inflammation and colorectal cancer (CRC). The underlying molecular mechanisms of PM2.5 impacts on CRC are still unclear. In this study, we utilized collected ambient PM2.5 and standard reference material SRM2786 to investigate the toxic effects on the colon through in vivo chronic exposure mouse and in vitro cell culture models. We employed a chronic mouse exposure model to clarify the colonic injury and gut microbiome biomarkers. Prolonged exposure to PM2.5 via oropharyngeal aspiration led to a significant rise in colonic epithelial proliferation and reduced colon length in mice. It triggered characteristics indicative of gut microbiota dysbiosis linked to inflammatory bowel disease. The gut microbiome alternations may serve as a biomarker indicating the colonic health impacts of PM2.5 exposure. PM2.5 and SRM2786-induced cytotoxicity manifested as autophagy dysregulation-mediated abnormal proliferation, IL-8 production, p62/SQSTM1 accumulation, and lysosomal membrane damage in human colon cells WiDr and Caco-2. Both PM2.5 and SRM2786 exposures led to the accumulation of p62/SQSTM1 and compromised lysosomal membrane integrity, showing impaired autophagic flux in WiDr and Caco-2 cells. Finally, we examined the correlations between atmospheric PM2.5 data and biomarkers of colonic inflammation in human population. The serum level of IL-8 was significantly correlated with regional anthropogenic pollutants. In conclusion, our findings elucidate that ambient PM2.5 exhibits adverse effects on colon health manifested as inflammation, aberrant proliferation, and gut dysbiosis, potentially mediated through autophagy dysregulation, thereby highlighting the importance of further research on the impact of environmental pollutants on gastrointestinal health.

Abstract Small cell lung cancer (SCLC) is characterized by rapid development of chemoresistance and poor outcomes. Cyclin‐dependent kinase 4/6 inhibitors (CDK4/6is) are widely used in breast cancer and other cancer types. However, the molecular mechanisms of CDK4/6 in SCLC chemoresistance remain poorly understood. Here, Rb1flox/flox, Trp53flox/flox, Ptenflox/flox (RTP) and Rb1flox/flox, Trp53flox/flox, MycLSL/LSL (RPM) spontaneous SCLC mouse models, SCLC cell line‐derived xenograft (CDX) models, and SCLC patient‐derived xenograft (PDX) models are established to reveal the potential effects of CDK4/6is on SCLC chemoresistance. In this study, it is found that CDK4/6is palbociclib (PD) or ribociclib (LEE) combined with chemotherapeutic drugs significantly inhibit SCLC tumor growth. Mechanistically, CDK4/6is do not function through the classic Retionblastoma1 (RB) dependent axis in SCLC. CDK4/6is induce impair autophagy through the AMBRA1‐lysosome signaling pathway. The upregulated AMBRA1 protein expression leads to CDK6 degradation via autophagy, and the following TFEB and TFE3 nuclear translocation inhibition leading to the lysosome‐related genes levels downregulation. Moreover, it is found that the expression of CDK6 is higher in SCLC tumors than in normal tissue and it is associated with the survival and prognosis of SCLC patients. Finally, these findings demonstrate that combining CDK4/6is with chemotherapy treatment may serve as a potential therapeutic option for SCLC patients.

Cardiovascular disease (CVD) remains the major cause of morbidity and mortality around the world. Transcription factor EB (TFEB) is a master regulator of lysosome biogenesis and autophagy. Emerging studies revealed that TFEB also mediates cellular adaptation responses to various stimuli, such as mitochondrial dysfunction, pathogen infection and metabolic toxin. Based on its significant capability to modulate the autophagy-lysosome process (ALP), TFEB plays a critical role in the development of CVD. In this review, we briefly summarize that TFEB regulates cardiac dysfunction mainly through ameliorating lysosomal and mitochondrial dysfunction and reducing inflammation.

Background Information: Two pore channels (TPCs) are voltage‐gated ion channel superfamily members that release Ca2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy. Results: We showed TPC2 protein localizes in lysosome‐like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi‐tipped structures attached to a common base, while tpc2OE cells showed faster development with numerous small‐sized aggregates and wiry fruiting bodies. The tpc2OE cells showed higher intracellular cAMP levels as compared to the tpc2− cells while pinocytosis was found to be higher in the tpc2− cells. Also, TPC2 regulates cell‐substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2− cells showed a bias towards the prestalk/stalk region while tpc2OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell‐type patterning and loss of distinct boundary between the prestalk/prespore regions. Conclusion: TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell‐type patterning. Significance: Decreased calcium due to deletion of tpc2 inhibit autophagic flux. [ABSTRACT FROM AUTHOR]

Hernandezine (Her), a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum , is recognized for its range of biological activities inherent to this herbal medicine. Despite its notable properties, the anti-cancer effects of Her have remained largely unexplored. In this study, we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms. Furthermore, Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems, leading to LC3 lipidation. Our findings revealed that Her caused damage to the mitochondrial membrane, with the damaged mitochondria undergoing mitophagy, as evidenced by the elevated expression of mitophagy markers. Conversely, Her disrupted autophagic flux, demonstrated by the upregulation of p62 and accumulation of autolysosomes, as observed in the RFP-GFP-LC3 reporter assay. Initially, we determined that Her did not prevent the fusion of autophagosomes and lysosomes. However, it inhibited the maturation of cathepsin D and increased lysosomal pH, indicating an impairment of lysosomal function. The use of the early-stage autophagy inhibitor, 3-methyladenine (3-MA), did not suppress LC3II, suggesting that Her also induces noncanonical autophagy in autophagosome formation. The application of Bafilomycin A1, an inhibitor of noncanonical autophagy, diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her, thereby augmenting Her-induced cell death. These observations imply that while autophagy initially plays a protective role, the disruption of the autophagic process by Her promotes programmed cell death. This study provides the first evidence of Her's dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death. These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death, offering potential avenues for enhancing cancer prevention and therapeutic strategies. [ABSTRACT FROM AUTHOR]

Chickens are a major source of meat and eggs in human food and have significant economic value. Cadmium (Cd) is a common environmental pollutant that can contaminate feed and drinking water, leading to kidney injury in livestock and poultry, primarily by inducing the generation of free radicals. It is necessary to develop potential medicines to prevent and treat Cd-induced nephrotoxicity in poultry. Luteolin (Lut) is a natural flavonoid compound mainly extracted from peanut shells and has a variety of biological functions to defend against oxidative damage. In this study, we aimed to demonstrate whether Lut can alleviate kidney injury under Cd exposure and elucidate the underlying molecular mechanisms. Renal histopathology and cell morphology were observed. The indicators of renal function, oxidative stress, DNA damage and repair, NAD+ content, SIRT1 activity, and autophagy were analyzed. In vitro data showed that Cd exposure increased ROS levels and induced oxidative DNA damage and repair, as indicated by increased 8-OHdG content, increased γ-H2AX protein expression, and the over-activation of the DNA repair enzyme PARP-1. Cd exposure decreased NAD+ content and SIRT1 activity and increased LC3 II, ATG5, and particularly p62 protein expression. In addition, Cd-induced oxidative DNA damage resulted in PARP-1 over-activation, reduced SIRT1 activity, and autophagic flux blockade, as evidenced by reactive oxygen species scavenger NAC application. The inhibition of PARP-1 activation with the pharmacological inhibitor PJ34 restored NAD+ content and SIRT1 activity. The activation of SIRT1 with the pharmacological activator RSV reversed Cd-induced autophagic flux blockade and cell injury. In vivo data demonstrated that Cd treatment caused the microstructural disruption of renal tissues, reduced creatinine, and urea nitrogen clearance, raised MDA content, and decreased the activities or contents of antioxidants (GSH, T-SOD, CAT, and T-AOC). Cd treatment caused oxidative DNA damage and PARP-1 activation, decreased NAD+ content, decreased SIRT1 activity, and impaired autophagic flux. Notably, the dietary Lut supplement observably alleviated these alterations in chicken kidney tissues induced by Cd. In conclusion, the dietary Lut supplement alleviated Cd-induced chicken kidney injury through its potent antioxidant properties by relieving the oxidative DNA damage-activated PARP-1-mediated reduction in SIRT1 activity and repairing autophagic flux blockade. [ABSTRACT FROM AUTHOR]

The role of autophagy in pulmonary microvascular endothelial cells (PMVECs) is controversial in LPS-induced acute lung injury (ALI). Mixed lineage kinase domain–like pseudokinase (MLKL) has recently been reported to maintain cell survival by facilitating autophagic flux in response to starvation rather than its well-recognized role in necroptosis. Using a mouse PMVEC and LPS-induced ALI model, we showed that in PMVECs, MLKL was phosphorylated (p-MLKL) and autophagic flux was accelerated at the early stage of LPS stimulation (1–3 h), manifested by increases in concentrations of lipidated MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β; LC3-II), decreases in concentrations of SQSTM1/p62 (sequestosome 1), and fusion of the autophagosome and lysosome by pHluorin-mKate2-human LC3 assay, which were all reversed by either MLKL inhibitor or siRNA MLKL. In mice, the inhibition of MLKL increased vascular permeability and aggravated mouse ALI upon 3-hour LPS stimulation. The p-MLKL induced by short-term LPS formed multimers to facilitate the closure of the phagophore by HaloTag-LC3 autophagosome completion assay. The charged multivesicular body protein 2A (CHMP2A) is essential in the process of phagophore closure into the nascent autophagosome. In agreement with the p-MLKL change, CHMP2A concentrations markedly increased during 1–3-hour LPS stimulation. CHMP2A knockdown blocked autophagic flux upon LPS stimulation, whereas CHMP2A overexpression boosted autophagic flux and attenuated mouse ALI even in the presence of MLKL inhibitor. We propose that the activated MLKL induced by short-term LPS facilitates autophagic flux by accelerating the closure of the phagophore via CHMP2A, thus protecting PMVECs and alleviating LPS-induced ALI. [ABSTRACT FROM AUTHOR]

Cancer continues to be a prominent global threat, and current chemotherapy options often involve tubulin-targeting compounds disrupting microtubule function. Autophagy's role in cancer varies across cell types, tumor stages, and carcinogenic stimuli. Recent attention has focused on autophagy as a response to antimitotic drugs. Our study spotlights TubA, a myxobacteria-derived peptide with potent antimitotic properties, demonstrating notable antiproliferative effects on diverse cancer cell lines at low concentrations. Remarkably, TubA proved more effective than TubB, another myxobacterial compound. In MCF-7 cells, TubA induced autophagy, increasing LC3-I lipidation to LC3-II and enhancing lysosomal activity and acidity. Intriguingly, TubA triggered an intrinsic apoptotic pathway mediated by autophagy, evidenced by increased cathepsin B activity, cytosolic leakage, and subsequent apoptosis through cytochrome c release. In summary, our findings indicate that TubA promotes cell death via cytotoxic autophagic activity in the MCF-7 cancer cell line.

(Macro)autophagy is a cellular degradation system for unnecessary materials, such as aggregate-prone TDP-43, a central molecule in neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Abemaciclib (Abe) and vacuolin-1 (Vac) treatments are known to induce vacuoles characterized by an autophagosome and a lysosome component, suggesting that they facilitate autophagosome-lysosome fusion. However, it remains unknown whether Abe and Vac suppress the accumulation of aggregate-prone TDP-43 by accelerating autophagic flux. In the present study, the Abe and Vac treatment dose-dependently reduced the GFP/RFP ratio in SH-SY5Y neuroblastoma cells stably expressing the autophagic flux marker GFP-LC3-RFP-LC3ΔG. Abe and Vac also increased the omegasome marker GFP-ATG13 signal and the autophagosome marker mCherry-LC3 localized to the lysosome marker LAMP1-GFP. The Abe and Vac treatment decreased the intracellular level of the lysosome marker LAMP1-GFP in SH-SY5Y cells stably expressing LAMP1-GFP, but did not increase the levels of LAMP1-GFP, the autophagosome marker LC3-II, or the multivesicular body marker TSG101 in the extracellular vesicle-enriched fraction. Moreover, Abe and Vac treatment autophagy-dependently inhibited GFP-tagged aggregate-prone TDP-43 accumulation. The results of a PI(3)P reporter assay using the fluorescent protein tagged-2 × FYVE and LAMP1-GFP indicated that Abe and Vac increased the intensity of the PI(3)P signal on lysosomes. A treatment with the VPS34 inhibitor wortmannin (WM) suppressed Abe-/Vac-facilitated autophagic flux and the degradation of GFP-tagged aggregate-prone TDP-43. Collectively, these results suggest that Abe and Vac degrade aggregate-prone TDP-43 by accelerating autophagosome formation and autophagosome-lysosome fusion through the formation of PI(3)P.

Rotaviruses (RV) are a major cause of severe gastroenteritis, particularly in neonatal piglets. Despite the availability of effective vaccines, the development of antiviral therapies for RV remains an ongoing challenge. Retinoic acid (RA), a metabolite of vitamin A, has been shown to have anti-oxidative and antiviral properties. However, the mechanism by which RA exerts its intestinal-protective and antiviral effects on RV infection is not fully understood. The study investigates the effects of RA supplementation in Duroc × Landrace × Yorkshire (DLY) piglets challenged with RV. Thirty-six DLY piglets were assigned into six treatments, including a control group, RA treatment group with two concentration gradients (5 and 15 mg/d), RV treatment group, and RV treatment group with the addition of different concentration gradients of RA (5 and 15 mg/d). Our study revealed that RV infection led to extensive intestinal architecture damage, which was mitigated by RA treatment at lower concentrations by increasing the villus height and villus height/crypt depth ratio (P

The brain is, after the adipose tissue, the organ with the greatest amount of lipids and diversity in their composition in the human body. In neurons, lipids are involved in signaling pathways controlling autophagy, a lysosome-dependent catabolic process essential for the maintenance of neuronal homeostasis and the function of the primary cilium, a cellular antenna that acts as a communication hub that transfers extracellular signals into intracellular responses required for neurogenesis and brain development. A crosstalk between primary cilia and autophagy has been established; however, its role in the control of neuronal activity and homeostasis is barely known. In this review, we briefly discuss the current knowledge regarding the role of autophagy and the primary cilium in neurons. Then we review the recent literature about specific lipid subclasses in the regulation of autophagy, in the control of primary cilium structure and its dependent cellular signaling in physiological and pathological conditions, specifically focusing on neurons, an area of research that could have major implications in neurodevelopment, energy homeostasis, and neurodegeneration.

Background and Purpose: Autophagy is a protective factor for controlling neuronal damage, while necroptosis promotes neuroinflammation after spinal cord injury (SCI). DADLE (D‐Ala2, D‐Leu5]‐enkephalin) is a selective agonist for delta (δ) opioid receptor and has been identified as a promising drug for neuroprotection. The aim of this study was to investigate the mechanism/s by which DADLE causes locomotor recovery following SCI. Experimental approach: Spinal cord contusion model was used and DADLE was given by i.p. (16 mg·kg−1) in mice for following experiments. Motor function was assessed by footprint and Basso mouse scale (BMS) score analysis. Western blotting used to evaluate related protein expression. Immunofluorescence showed the protein expression in each cell and its distribution. Network pharmacology analysis was used to find the related signalling pathways. Key Results: DADLE promoted functional recovery after SCI. In SCI model of mice, DADLE significantly increased autophagic flux and inhibited necroptosis. Concurrently, DADLE restored autophagic flux by decreasing lysosomal membrane permeabilization (LMP). Additionally, chloroquine administration reversed the protective effect of DADLE to inhibit necroptosis. Further analysis showed that DADLE decreased phosphorylated cPLA2, overexpression of cPLA2 partially reversed DADLE inhibitory effect on LMP and necroptosis, as well as the promotion autophagy. Finally, AMPK/SIRT1/p38 pathway regulating cPLA2 is involved in the action DADLE on SCI and naltrindole inhibited DADLE action on δ receptor and on AMPK signalling pathway. Conclusion and Implication: DADLE causes its neuroprotective effects on SCI by promoting autophagic flux and inhibiting necroptosis by decreasing LMP via activating δ receptor/AMPK/SIRT1/p38/cPLA2 pathway. [ABSTRACT FROM AUTHOR]

Impaired autophagy is known to cause mitochondrial dysfunction and heart failure, in part due to altered mitophagy and protein quality control. However, whether additional mechanisms are involved in the development of mitochondrial dysfunction and heart failure in the setting of deficient autophagic flux remains poorly explored. Here, we show that impaired autophagic flux reduces nicotinamide adenine dinucleotide (NAD+) availability in cardiomyocytes. NAD+ deficiency upon autophagic impairment is attributable to the induction of nicotinamide N-methyltransferase (NNMT), which methylates the NAD+ precursor nicotinamide (NAM) to generate N-methyl-nicotinamide (MeNAM). The administration of nicotinamide mononucleotide (NMN) or inhibition of NNMT activity in autophagy-deficient hearts and cardiomyocytes restores NAD+ levels and ameliorates cardiac and mitochondrial dysfunction. Mechanistically, autophagic inhibition causes the accumulation of SQSTM1, which activates NF-κB signaling and promotes NNMT transcription. In summary, we describe a novel mechanism illustrating how autophagic flux maintains mitochondrial and cardiac function by mediating SQSTM1-NF-κB-NNMT signaling and controlling the cellular levels of NAD+. Synopsis: How autophagy safeguards cardiac structure and function to prevent heart failure remains unclear. This genetic work links a novel connection between deficient autophagic flux in cardiomyocytes to reduced availability of the cellular metabolite NAD+, suggesting novel tractable avenues for intervention. Depletion of autophagy-related protein 3 (ATG3) in cardiomyocytes causes cardiac dysfunction and early mortality in mice. ATG3 depletion reduces mitochondrial metabolism and biogenesis, preceding age-dependent cardiac dysfunction. Reduced autophagy leads to accumulation of SQSTM1, activation of NF-κB subunit RELA, and increased transcription of the NAD+ metabolic enzyme NNMT. Increased NNMT catalyzes the competing conversion of NAD+ precursor NAM to MeNAM, leading to NAD+ deficiency. Autophagy induction prevents NNMT induction and NNMT inhibition or NMN supplementation restore cardiac and mitochondrial dysfunction when autophagy is defective. Deficient autophagy reduces NAD+ availability in cardiomyocytes via a SQSTM1-NF-κB-NNMT axis, contributing to mitochondrial dysfunction and heart failure. [ABSTRACT FROM AUTHOR]

PARP (poly(ADP-ribose) polymerase) inhibitors have demonstrated promising clinical activity in multiple homologous recombination (HR) deficiency tumors. However, glioblastoma (GBM) patients have obtained little benefit from PARP inhibitors alone. PARP inhibition shows considerable promise when used together with other therapeutic agents. Thus, novel combination therapies may enhance PARP inhibitor efficacy and overcome resistance mechanisms in GBM. Herein, we report that concurrent treatment with the PARP inhibitor olaparib and XPO1 (exportin 1) inhibitor KPT330 showed synergetic anticancer effects on GBM cells. Mechanistically, in the nucleus, we show that KPT330 induced the nuclear retention of SQSTM1 (sequestosome 1) and further inhibited the ubiquitination of the DNA repair signal H2AX (H2A.X variant histone) mediated by olaparib, thus inhibiting DNA damage response and repair in GBM. Moreover, in the cytoplasm, KPT330 blocked the activation of autophagic flux caused by olaparib reagent, downregulated the expression of LAPTM4B (lysosomal protein transmembrane 4 beta) and induced the dysfunction of lysosomes, thereby preventing the degradation of autophagosome, and ultimately promoted cell death. Furthermore, in the LN229-luc mouse orthotopic xenograft model, combination treatment showed significantly increased antitumor efficacy compared to each monotherapy. These data illustrate the application prospects of combined oral administration of olaparib and KPT330 for the treatment of glioblastoma. AO: acridine orange; ATM: ATM serine/threonine kinase; CHEK1: checkpoint kinase 1; CHEK2: checkpoint kinase 2; CI: combination index; DMSO: dimethyl sulfoxide; DSBs: double-strand breaks; GBM: glioblastoma; HR: homologous recombination; H2AX: H2A.X variant histone; IHC: immunohistochemistry; LAPTM4B: lysosomal protein transmembrane 4 beta; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PARP: poly(ADP-ribose) polymerase; RAD51: RAD51 recombinase; SQSTM1: sequestosome 1; SSBs: single-strand breaks; RNF168: ring finger protein 168; XPO1: exportin 1. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder caused by the depletion of the ubiquitously expressed survival motor neuron (SMN) protein. While the genetic cause of SMA has been well documented, the exact mechanism(s) by which SMN depletion results in disease progression remain elusive. A wide body of evidence has highlighted the involvement and dysregulation of autophagy in SMA. Autophagy is a highly conserved lysosomal degradation process which is necessary for cellular homeostasis; defects in the autophagic machinery have been linked with a wide range of neurodegenerative disorders, including amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. The pathway is particularly known to prevent neurodegeneration and has been suggested to act as a neuroprotective factor, thus presenting an attractive target for novel therapies for SMA patients. In this review, (a) we provide for the first time a comprehensive summary of the perturbations in the autophagic networks that characterize SMA development, (b) highlight the autophagic regulators which may play a key role in SMA pathogenesis and (c) propose decreased autophagic flux as the causative agent underlying the autophagic dysregulation observed in these patients. [ABSTRACT FROM AUTHOR]

Nitrogen‐doped graphene (C2N), a novel graphene‐based materials, has been proposed as a potential alternative to graphene oxide (GO) in biomedical applications. However, due to the challenges in synthesizing C2N, reports in the biomedical field are currently rare. Here, we have modified the reported procedure and successfully synthesized C2N nanoparticles at 120°C, which we refer to as C2N‐120. The toxicity and biocompatibility of GO and C2N‐120 were evaluated using a mouse model injected with GO/C2N‐120 via the tail vein, as well as cell models treated with GO/C2N‐120. In vivo studies revealed that GO/C2N‐120 showed similar distribution patterns after tail vein injection. The liver, spleen, and lung are the major nanoparticle uptake organs of GO and C2N‐120. However, GO deposition in the major nanoparticle uptake organs was more significant than that of C2N‐120. In addition, GO deposition caused structural abnormalities, increased apoptotic cells, and enhanced macrophage infiltration whereas C2N‐120 exhibited fewer adverse effects. In vitro experiments were conducted using different cell lines treated with GO/C2N‐120. Unlike GO which induced mitochondrial damage, oxidative stress, inflammatory response, autophagic flux blockage and cell apoptosis, C2N‐120 showed lower cytotoxicity in cell models. Our data demonstrated that C2N‐120 exhibits higher biocompatibility than GO, both in vivo and in vitro, suggesting its potential for biomedical application in the future. [ABSTRACT FROM AUTHOR]

Progressive accumulation of amyloid-β (Aβ) aggregates in extracellular plaques is a characteristic hallmark of Alzheimer disease (AD). Aβ is also found in intraneuronal deposits and associated with alterations of the endo-lysosomal system and impairment of macroautophagy/autophagy. Here, we assessed the effect of Aβ phosphorylation on neuronal autophagy and the endo-lysosomal pathway. Analysis of APP-PSEN1dE9 transgenic mice revealed a phosphorylation-state dependent intraneuronal accumulation of Aβ species in endo-lysosomal and autophagy-related compartments. Cell biological studies further demonstrate a differential uptake and sorting of phosphorylated Aβ variants in cultured neurons, and phosphorylation-state specific effects of Aβ variants on neuronal autophagy and lysosomal function. While Aβ phosphorylated at serine residue 8 accumulated in autophagosomes, Aβ phosphorylated at serine residue 26 showed efficient transport to lysosomes. The selective sorting of phosphorylated Aβ species caused differential impairment of vesicular transport and lysosomal function associated with neurotoxicity. Thus, the relative occurrence of phosphorylated Aβ species and their intraneuronal accumulation could contribute to AD pathogenesis, and to the commonly observed aberrations of the vesicular transport system already at the early stages of the disease. AD: Alzheimer disease; APP: amyloid beta precursor protein; ATG: autophagy related; Aβ: amyloid-β; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; EEA1: early endosome antigen 1; FA: formic acid; GFP: green fluorescent protein; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP2: microtubule-associated protein 2; nmAβ: non-modified amyloid-β; npAβ: non-phosphorylated amyloid-β; pAβ: phosphorylated amyloid-β; p-Ser26Aβ: amyloid-β phosphorylated at serine residue 26; p-Ser8Aβ: amyloid-β phosphorylated at serine residue 8; RAB: RAB, member RAS oncogene family; RFP: red fluorescent protein; SQSTM1/p62: sequestome 1; YFP: yellow fluorescent protein. [ABSTRACT FROM AUTHOR]

Queen bee acid (QBA), which is exclusively found in royal jelly, has anti-inflammatory, antihypercholesterolemic, and antiangiogenic effects. A recent study demonstrated that QBA enhances autophagic flux in the heart. Considering the significant role of autophagy in the development of myocardial ischemia/reperfusion (I/R) injury, we investigated the effect of pretreatment with QBA on myocardial damage. In an in vivo model, left coronary artery blockage for 30 min and reperfusion for 2 h were used to induce myocardial I/R. In an in vitro model, neonatal rat cardiomyocytes (NRCs) were exposed to 3 h of hypoxia and 3 h of reoxygenation (H/R). Our results showed that pretreatment with QBA increased the cell viability of cardiomyocytes exposed to H/R in a dose-dependent manner, and the best protective concentration of QBA was 100 μM. Next, we noted that QBA pretreatment (24h before H/R) enhanced autophagic flux and attenuated mitochondrial damage, cardiac oxidative stress and apoptosis in NRCs exposed to H/R injury, and these effects were weakened by cotreatment with the autophagy inhibitor bafilomycin A1 (Baf). In addition, similar results were observed when QBA (10 mg/kg) was injected intraperitoneally into I/R mice 30 min before ischemia. Compared to mice subjected to I/R alone, those treated with QBA had decreased myocardial infarct area and increased cardiac function, whereas, these effects were partly reversed by Baf. Notably, in NRCs exposed to H/R, tandem fluorescent mRFP-GFP-LC3 assays indicated increased autophagosome degradation due to the increase in autophagic flux upon QBA treatment, but coinjection of Baf blocked autophagic flux. In this investigation, no notable adverse effects of QBA were detected in either cellular or animal models. Our findings suggest that QBA pretreatment mitigates myocardial I/R injury by eliminating dysfunctional mitochondria and reducing reactive oxygen species via promoting autophagic flux.

Although carbon monoxide (CO)-based treatments have demonstrated the high cancer efficacy by promoting mitochondrial damage and core-region penetrating ability, the efficiency was often compromised by protective autophagy (mitophagy). Herein, cannabidiol (CBD) is integrated into biomimetic carbon monoxide nanocomplexes (HMPOC@M) to address this issue by inducing excessive autophagy. The biomimetic membrane not only prevents premature drugs leakage, but also prolongs blood circulation for tumor enrichment. After entering the acidic tumor microenvironment, carbon monoxide (CO) donors are stimulated by hydrogen oxide (H2O2) to disintegrate into CO and Mn2+. The comprehensive effect of CO/Mn2+ and CBD can induce ROS-mediated cell apoptosis. In addition, HMPOC@M-mediated excessive autophagy can promote cancer cell death by increasing autophagic flux via class III PI3K/BECN1 complex activation and blocking autolysosome degradation via LAMP1 downregulation. Furthermore, in vivo experiments showed that HMPOC@M+ laser strongly inhibited tumor growth and attenuated liver and lung metastases by downregulating VEGF and MMP9 proteins. This strategy may highlight the pro-death role of excessive autophagy in TNBC treatment, providing a novel yet versatile avenue to enhance the efficacy of CO treatments. Importantly, this work also indicated the applicability of CBD for triple-negative breast cancer (TNBC) therapy through excessive autophagy.

Autophagic flux refers to a series of dynamic process of autophagic bilayer membrane formation, autophagosome formation, autophagolysosomes formation and degradation. The etiology of cataract is complex, including congenital abnormalities in lens development due to genetic mutations, oxidative damage due to aging, abnormalities in glucose metabolism due to diabetes, and proliferation of lens epithelial cells(LECs)stimulated by postoperative inflammatory factor, all of which are associated with the development of cataracts. A growing number of research in recent years have discovered that altering the status of LECs can contribute to the pathophysiological process of cataract by regulating autophagic flux. This review summarized the impacts of autophagic flux regulation on cataract.

The role of fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) demethylase, in non-small cell lung cancer (NSCLC) has recently received widespread attention. However the underlying mechanisms of FTO-mediated autophagy regulation in NSCLC progression remain elusive. In this study, we found that FTO was significantly upregulated in NSCLC, and downregulation of FTO suppressed the growth, invasion and migration of NSCLC cells by inducing autophagy. FTO knockdown resulted in elevated m6A levels in NSCLC cells. Methylated RNA immunoprecipitation sequencing showed that sestrin 2 (SESN2) was involved in m6A regulation during autophagy in NSCLC cells. Interestingly, m6A modifications in exon 9 of SESN2 regulated its stability. FTO deficiency promoted the binding of insulin-like growth factor 2 mRNA-binding protein 1 to SESN2 mRNA, enhancing its stability and elevating its protein expression. FTO inhibited autophagic flux by downregulating SESN2, thereby promoting the growth, invasion and migration of NSCLC cells. Besides, the mechanism by which FTO blocked SESN2-mediated autophagy activation was associated with the AMPK-mTOR signaling pathway. Taken together, these findings uncover an essential role of the FTO–autophagy–SESN2 axis in NSCLC progression.

The male reproductive dysfunction accounts for 50% of infertile couples in the world. Cadmium (Cd) is one of the most harmful heavy metals to both the environment and inhabitants. Accumulating data suggest that Cd could cause male infertility. Sertoli cell (SC) is a somatic cell of testis and a key regulator of spermatogenesis by providing physical and nutritional support for developing sperm. Many studies showed that Cd induced dysfunction of SCs was directly related to male reproductive damage. However, the mechanism of SCs injury caused by Cd remains to be clarified. We found that Cd treatment caused a significant increase of apoptosis in SCs cells, accompanied by a marked increase in the production of ROS. These results were associated with the formation of mitochondria-containing autophagosomes and increased expression of LC3-II in vitro. Interestingly, our results showed that Cd did not promote but inhibited the fusion of mitochondria-containing autophagosomes with lysosomes by reducing the function of lysosomes. Together, this study provides insight into the negative effects of Cd, which interferes with autophagic flux and induces the apoptosis of SCs.

Background/aim: Doxorubicin (DOX) is a highly effective chemotherapy medicine commonly used to treat breast cancer; yet, despite its clinical efficacy, it usually causes chemobrain. As a result, we investigated the neuroprotective effects of Diosmin (DIOS) on DOX-induced chemobrain in male rat brains. Animals and methods: In the experimental protocol, Rats were divided into 4 groups: group I received solvent and saline for 56 days, group II received Dios and a concomitant dose of saline for 56 days, group III received DOX intraperitoneally on 7th, 14th, 21st, 28th, 35th, 42nd, 49th and 56th days, and Group IV received DIOS (40 mg/kg P.O.) for 56 days and DOX was administered one hour after DIOS oral administration. The novel-object recognition memory test (NORT) was used to assess non-spatial memory function. Following that, brain neurochemical status were assessed. Results: DIOS increased cognitive performance in DOX-treated rats by enhancing exploration of unfamiliar objects. DIOS protects the brain from DOX-mediated alterations in oxidative status, as well as brain metabolic enzyme indicators. It also decreased MMP-6, IL-6, IL-β1, TNF-α and COX-2 expression, reduced apoptotic markers levels, boosted mTOR protein levels, lowered beclin-1, restored brain metabolic enzyme activities, increased neurotransmitter as well as cathepsin levels, and avoided the rise in α-synuclein and PON1 enzyme activity. Conclusion: In conclusion, DIOS protects rats' brains against DOX-induced chemobrain via oxidative stress inhibition, autophagy modulation, and down-regulation of inflammatory and apoptotic markers.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder caused by the depletion of the ubiquitously expressed survival motor neuron (SMN) protein. While the genetic cause of SMA has been well documented, the exact mechanism(s) by which SMN depletion results in disease progression remain elusive. A wide body of evidence has highlighted the involvement and dysregulation of autophagy in SMA. Autophagy is a highly conserved lysosomal degradation process which is necessary for cellular homeostasis; defects in the autophagic machinery have been linked with a wide range of neurodegenerative disorders, including amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. The pathway is particularly known to prevent neurodegeneration and has been suggested to act as a neuroprotective factor, thus presenting an attractive target for novel therapies for SMA patients. In this review, (a) we provide for the first time a comprehensive summary of the perturbations in the autophagic networks that characterize SMA development, (b) highlight the autophagic regulators which may play a key role in SMA pathogenesis and (c) propose decreased autophagic flux as the causative agent underlying the autophagic dysregulation observed in these patients.

Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma. Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most common malignancies worldwide and remains a major clinical challenge. Periplocin, a major bioactive component of the traditional Chinese herb Cortex periplocae, has recently been reported to be a potential anticancer drug. However, the mechanism of action is poorly understood. Here, we show that periplocin exhibits promising anticancer activity against CRC both in vitro and in vivo. Mechanistically, periplocin promotes lysosomal damage and induces apoptosis in CRC cells. Notably, periplocin upregulates LGALS3 (galectin 3) by binding and preventing LGALS3 from Lys210 ubiquitination-mediated proteasomal degradation, leading to the induction of excessive lysophagy and resultant exacerbation of lysosomal damage. Inhibition of LGALS3-mediated lysophagy attenuates periplocin-induced lysosomal damage and growth inhibition in CRC cells, suggesting a critical role of lysophagy in the anticancer effects of periplocin. Taken together, our results reveal a novel link between periplocin and the lysophagy machinery, and indicate periplocin as a potential therapeutic option for the treatment of CRC. Abbreviations: 3-MA: 3-methyladenine; ACACA/ACC1: acetyl-CoA carboxylase alpha; AMPK: adenosine monophosphate-activated protein kinase; AO: Acridine orange; ATG5: autophagy related 5; ATG7: autophagy related 7; CALM: calmodulin; CHX: cycloheximide; CRC: colorectal cancer; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; ESCRT: endosomal sorting complex required for transport; LAMP1: lysosomal associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MKI67/Ki-67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; P2RX4/P2X4: purinergic receptor P2X 4; PARP1/PARP: poly(ADP-ribose) polymerase 1; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TRIM16: tripartite motif containing 16. [ABSTRACT FROM AUTHOR]

Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this context, it was demonstrated that autophagy plays an important role in megakaryopoiesis, the development and differentiation of hematopoietic progenitor cells into megakaryocytes. Furthermore, in recent years, autophagic proteins were detected in platelets, anucleate cells generated by megakaryocytes, responsible for hemostasis, thrombosis, and a key cell in inflammation and host immune responses. In the last decade studies have indicated the occurrence of autophagy in platelets. Moreover, autophagy in platelets was subsequently demonstrated to be involved in platelet aggregation, adhesion, and thrombus formation. Here, we review the current knowledge about autophagy in platelets, its function, and clinical implications. However, at the advent of platelet autophagy research, additional discoveries derived from evolving work will be required to precisely define the contributions of autophagy in platelets, and to expand the ever increasing physiologic and pathologic roles these remarkable and versatile blood cells play. • Autophagy plays an important role in megakaryopoiesis. • Autophagosome formation and autophagy are essential in platelet biology. • Autophagy in platelets is involved in platelet aggregation, adhesion, and thrombus formation. • The importance of platelet autophagic functions is demonstrated in clinically relevant conditions and disease states, such as diabetes, hypoxia/ischemia, ITP, and sepsis. • Elaborate methods are available to study autophagy in platelets reliably and in great detail. [ABSTRACT FROM AUTHOR]

SQSTM1/p62 (sequestosome 1) is a multifunctional protein that serves as a receptor for selective autophagy and scaffold. In selective autophagy, p62 functions as a bridge between polyubiquitinated proteins and autophagosomes. Further, p62 acts as a signaling hub for many cellular pathways including mTORC1, NF-κB, and Keap1-Nrf2. Post-translational modifications of p62, such as ubiquitination and phosphorylation, are known to determine its binding partners and regulate their intracellular functions. However, the mechanism of p62 deubiquitination remains unclear. In this study, we found that ubiquitin-specific protease 13 (USP13), a member of the USP family, directly binds p62 and removes ubiquitin at Lys7 (K7) of the PB1 domain. USP13-mediated p62 deubiquitination enhances p62 protein stability and facilitates p62 oligomerization, resulting in increased autophagy and degradation of Keap1, which is a negative regulator of the antioxidant response that promotes Nrf2 activation. Thus, USP13 can be considered a therapeutic target as a deubiquitination enzyme of p62 in autophagy-related diseases. [Display omitted] • USP13 is a deubiquitination enzyme of p62/SQSTM1. • USP13 enhances p62 stability by removing K48 and K63 ubiquitin at residue p62 K7. • p62-mediated autophagy is promoted by USP13. • USP13 induces p62-Keap1 binding, leading to free Nrf2 enter the nucleus for antioxidant gene expression. [ABSTRACT FROM AUTHOR]

Although carbon monoxide (CO)-based treatments have demonstrated the high cancer efficacy by promoting mitochondrial damage and core-region penetrating ability, the efficiency was often compromised by protective autophagy (mitophagy). Herein, cannabidiol (CBD) is integrated into biomimetic carbon monoxide nanocomplexes (HMPOC@M) to address this issue by inducing excessive autophagy. The biomimetic membrane not only prevents premature drugs leakage, but also prolongs blood circulation for tumor enrichment. After entering the acidic tumor microenvironment, carbon monoxide (CO) donors are stimulated by hydrogen oxide (H 2 O 2) to disintegrate into CO and Mn2+. The comprehensive effect of CO/Mn2+ and CBD can induce ROS-mediated cell apoptosis. In addition, HMPOC@M-mediated excessive autophagy can promote cancer cell death by increasing autophagic flux via class III PI3K/BECN1 complex activation and blocking autolysosome degradation via LAMP1 downregulation. Furthermore, in vivo experiments showed that HMPOC@M+ laser strongly inhibited tumor growth and attenuated liver and lung metastases by downregulating VEGF and MMP9 proteins. This strategy may highlight the pro-death role of excessive autophagy in TNBC treatment, providing a novel yet versatile avenue to enhance the efficacy of CO treatments. Importantly, this work also indicated the applicability of CBD for triple-negative breast cancer (TNBC) therapy through excessive autophagy. Cannabidiol (CBD) is integrated into biomimetic carbon monoxide nanocomplexes (HMPOC@M) to fight against triple-negative breast cancer (TNBC) by inducing excessive autophagy. [Display omitted] [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy has both negative and positive aspects in the development of many diseases. Yet, its exact role and specific mechanism in the onset of medication-related osteonecrosis of the jaw (MRONJ) is still not fully understood. Retarded gingiva healing is the primary clinical manifestation in patients with MRONJ. In this study, we aimed to explore the relationship between autophagy and apoptosis in MRONJ gingival epithelium and search for a method to prevent this disease. First, we examined clinical samples from patients diagnosed with MRONJ and healthy controls, finding that autophagy-related markers MAP1LC3/LC3 and SQSTM1/p62 synchronously increased, thus suggesting that autophagic flux was suppressed in MRONJ. Moreover, mRNA sequencing analysis and TUNEL assay showed that the process of apoptosis was upregulated in patients and animals with MRONJ, indicating autophagy and apoptosis participate in the development of MRONJ. Furthermore, the level of autophagy and apoptosis in zoledronic acid (ZA)-treated human keratinocytes cell lines (HaCaT cells) was concentration dependent in vitro. In addition, we also found that RAB7 (RAB7, member RAS oncogene family) activator ML098 could rescue MRONJ gingival lesions in mice by activating the autophagic flux and downregulating apoptosis. To sum up, this study demonstrated that autophagic flux is impaired in the gingival epithelium during MRONJ, and the rescued autophagic flux could prevent the occurrence of MRONJ. Abbreviations: ACTB: actin beta; Baf-A1: bafilomycin A1; CASP3: caspase 3; CASP8: caspase 8; CT: computed tomography; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; HaCaT cells: human keratinocytes cell lines; H&E: hematoxylin and eosin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MRONJ: medication-related osteonecrosis of the jaw; PARP: poly(ADP-ribose) polymerase; RAB7: RAB7, member RAS oncogene family; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; ZA: zoledronic acid. [ABSTRACT FROM AUTHOR]

Realgar is a traditional Chinese medicine that contains arsenic. It has been reported that the abuse of medicine-containing realgar has potential central nervous system (CNS) toxicity, but the toxicity mechanism has not been elucidated. In this study, we established an in vivo realgar exposure model and selected the end product of realgar metabolism, DMA, to treat SH-SY5Y cells in vitro. Many assays, including behavioral, analytical chemistry, and molecular biology, were used to elucidate the roles of the autophagic flux and the p62-NRF2 feedback loop in realgar-induced neurotoxicity. The results showed that arsenic could accumulate in the brain, causing cognitive impairment and anxiety-like behavior. Realgar impairs the ultrastructure of neurons, promotes apoptosis, perturbs autophagic flux homeostasis, amplifies the p62-NRF2 feedback loop, and leads to p62 accumulation. Further analysis showed that realgar promotes the formation of the Beclin1-Vps34 complex by activating JNK/c-Jun to induce autophagy and recruit p62. Meanwhile, realgar inhibits the activities of CTSB and CTSD and changes the acidity of lysosomes, leading to the inhibition of p62 degradation and p62 accumulation. Moreover, the amplified p62-NRF2 feedback loop is involved in the accumulation of p62. Its accumulation promotes neuronal apoptosis by upregulating the expression levels of Bax and cleaved caspase-9, resulting in neurotoxicity. Taken together, these data suggest that realgar can perturb the crosstalk between the autophagic flux and the p62-NRF2 feedback loop to mediate p62 accumulation, promote apoptosis, and induce neurotoxicity. Realgar promotes p62 accumulation to produce neurotoxicity by perturbing the autophagic flux and p62-NRF2 feedback loop crosstalk [ABSTRACT FROM AUTHOR]

Abstract Background Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation. This event causes muscle mass loss and decreased strength and muscle function, a syndrome known as sarcopenia. Recently, our laboratory described secondary sarcopenia in a chronic cholestatic liver disease (CCLD) mouse model. Interestingly, the administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is an effective therapy for cholestatic hepatic alterations. However, the effect of UDCA on skeletal muscle mass and functionality has never been evaluated, nor the possible involved mechanisms. Methods We assessed the ability of UDCA to generate sarcopenia in C57BL6 mice and develop a sarcopenic-like phenotype in C2C12 myotubes and isolated muscle fibers. In mice, we measured muscle strength by a grip strength test, muscle mass by bioimpedance and mass for specific muscles, and physical function by a treadmill test. We also detected the fiber’s diameter and content of sarcomeric proteins. In C2C12 myotubes and/or isolated muscle fibers, we determined the diameter and troponin I level to validate the cellular effect. Moreover, to evaluate possible mechanisms, we detected puromycin incorporation, p70S6K, and 4EBP1 to evaluate protein synthesis and ULK1, LC3 I, and II protein levels to determine autophagic flux. The mitophagosome-like structures were detected by transmission electron microscopy. Results UDCA induced sarcopenia in healthy mice, evidenced by decreased strength, muscle mass, and physical function, with a decline in the fiber’s diameter and the troponin I protein levels. In the C2C12 myotubes, we observed that UDCA caused a reduction in the diameter and content of MHC, troponin I, puromycin incorporation, and phosphorylated forms of p70S6K and 4EBP1. Further, we detected increased levels of phosphorylated ULK1, the LC3II/LC3I ratio, and the number of mitophagosome-like structures. These data suggest that UDCA induces a sarcopenic-like phenotype with decreased protein synthesis and autophagic flux. Conclusions Our results indicate that UDCA induces sarcopenia in mice and sarcopenic-like features in C2C12 myotubes and/or isolated muscle fibers concomitantly with decreased protein synthesis and alterations in autophagic flux.

Chickens are a major source of meat and eggs in human food and have significant economic value. Cadmium (Cd) is a common environmental pollutant that can contaminate feed and drinking water, leading to kidney injury in livestock and poultry, primarily by inducing the generation of free radicals. It is necessary to develop potential medicines to prevent and treat Cd-induced nephrotoxicity in poultry. Luteolin (Lut) is a natural flavonoid compound mainly extracted from peanut shells and has a variety of biological functions to defend against oxidative damage. In this study, we aimed to demonstrate whether Lut can alleviate kidney injury under Cd exposure and elucidate the underlying molecular mechanisms. Renal histopathology and cell morphology were observed. The indicators of renal function, oxidative stress, DNA damage and repair, NAD+ content, SIRT1 activity, and autophagy were analyzed. In vitro data showed that Cd exposure increased ROS levels and induced oxidative DNA damage and repair, as indicated by increased 8-OHdG content, increased γ-H2AX protein expression, and the over-activation of the DNA repair enzyme PARP-1. Cd exposure decreased NAD+ content and SIRT1 activity and increased LC3 II, ATG5, and particularly p62 protein expression. In addition, Cd-induced oxidative DNA damage resulted in PARP-1 over-activation, reduced SIRT1 activity, and autophagic flux blockade, as evidenced by reactive oxygen species scavenger NAC application. The inhibition of PARP-1 activation with the pharmacological inhibitor PJ34 restored NAD+ content and SIRT1 activity. The activation of SIRT1 with the pharmacological activator RSV reversed Cd-induced autophagic flux blockade and cell injury. In vivo data demonstrated that Cd treatment caused the microstructural disruption of renal tissues, reduced creatinine, and urea nitrogen clearance, raised MDA content, and decreased the activities or contents of antioxidants (GSH, T-SOD, CAT, and T-AOC). Cd treatment caused oxidative DNA damage and PARP-1 activation, decreased NAD+ content, decreased SIRT1 activity, and impaired autophagic flux. Notably, the dietary Lut supplement observably alleviated these alterations in chicken kidney tissues induced by Cd. In conclusion, the dietary Lut supplement alleviated Cd-induced chicken kidney injury through its potent antioxidant properties by relieving the oxidative DNA damage-activated PARP-1-mediated reduction in SIRT1 activity and repairing autophagic flux blockade.

Venetoclax (VCX) is a BCL2 inhibitor approved for treating B cell-derived leukemia, including chronic lymphocytic leukemia (CLL). VCX’s role in apoptosis induction is well-defined, whereas its other mechanistic roles need to be clarified. Autophagy is an intracellular degradation process involving the lysosome, playing a fundamental role in cancer cell survival and death. In this study, we aim to investigate VCX’s function in autophagy. Following the measurement of autophagic flux by western blot and fluorescent microscopy, we demonstrate that VCX is a novel autophagy inhibitor and reduces the level of the essential autophagy pathway protein ATG12 (autophagy-related 12). VCX sensitizes B cell lines and primary CLL cells to cell death induced by amino acid starvation or ibrutinib. These findings provide a rationale for VCX treatment in combination therapies that inhibit the pro-cell survival function of autophagy in B cell-derived malignancies. Abbreviations: ATG12, autophagy-related 12; BCL2, BCL2 apoptosis regulator; CLL, chronic lymphocytic leukemia; CQ, chloroquine; IBR, ibrutinib; BTK, Bruton’s tyrosine kinase; LC3B, MAP1LC3B (microtubule-associated protein 1 light chain 3 beta); VCX, venetoclax; BECN1, beclin 1

Cadmium has been reported to accumulate primarily in spermatogonia and spermatocytes. Exposure to cadmium results in male reproductive toxicity via germ-cell apoptosis and impaired autophagy. Apoptosis and autophagy are two physiologically conserved events that maintain cellular homeostasis. However, the precise role of autophagy in cadmium-induced apoptosis of male germ cells has yet to be addressed. The present study aimed to investigate the impact of cadmium exposure on the cytotoxicity of GC-2 spd cells, a mouse spermatocyte cell line. The results showed that cadmium exposure caused apoptotic cell death and the accumulation of autophagosomes, along with the up-regulation of ATG proteins in GC-2 spd cells. It was demonstrated that the cadmium-induced accumulation of autophagosomes contributes to the apoptosis of GC-2 spd cells. This notion is supported by the findings that the autophagy inhibitor 3-MA reduced accumulation of autophagosomes and apoptotic cell death. Conversely, the apoptosis inhibitor Z-VAD-FMK inhibited apoptosis but had little effect on the accumulation of autophagosomes. Cadmium may impede the fusion of autophagosomes with lysosomes, leading to the autophagosome buildup. Additionally, we found that the JNK pathway mediates transcriptional induction of several autophagy-related (ATG) genes involved in autophagosome formation. The cadmium-activated JNK pathway regulates apoptosis by mediating the autophagosome formation. Treatment of cells with the JNK inhibitor SP600125 attenuated the accumulation of autophagosomes, the upregulated expression of autophagosome-associated proteins and apoptotic cell death induced by cadmium. Overall, these findings suggest that cadmium enhances apoptosis of GC-2 spd cells by activating the JNK pathway and inhibiting autophagic flux.

Autophagy is a highly conserved self-degradation process that involves the degradation and recycling of cellular components and organelles. Although the involvement of autophagy in metabolic changes during fruit ripening has been preliminarily demonstrated, the variations in autophagic flux and specific functional roles in tomato fruit ripening remain to be elucidated. In this study, we analyzed the variations in autophagic flux during tomato fruit ripening. The results revealed differential expression of the SlATG8 family members during tomato fruit ripening. Transmission electron microscopy observations and dansylcadaverine (MDC) staining confirmed the presence of autophagy at the cellular level in tomato fruits. Furthermore, the overexpression of SlATG8f induced the formation of autophagosomes, increased autophagic flux within tomato fruits, and effectively enhanced the expression of ATG8 proteins during the color-transition phase of fruit ripening, thus promoting tomato fruit maturation. SlATG8f overexpression also led to the accumulation of vitamin C (VC) and soluble solids while reducing acidity in the fruit. Collectively, our findings highlight the pivotal role of SlATG8f in enhancing tomato fruit ripening, providing insights into the mechanistic involvement of autophagy in this process. This research contributes to a better understanding of the key factors that regulate tomato fruit quality and offers a theoretical basis for tomato variety improvement. [ABSTRACT FROM AUTHOR]

Ewing sarcoma family tumors (ESFTs) are a group of aggressive tumors mainly affecting children and young people. A compound derived from Curcuma wenyujin plant or lemon grass, β-elemene, has exhibited antitumor effects to ESFT cells, the mechanism of which remains to be clarified further. Autophagy is involved in the antitumor effects of various drugs, whereas the role of autophagy in the antitumor effects of β-elemene persists controversial. Herein we found that β-elemene treatment inhibited the viability of ESFT cells in a dose-dependent manner. The increase of LC3-II level and the decrease of p62 level were observed in β-elemene-treated cells, as well as the increase of autolysosomes, which indicated the promotion of autophagic flux. Sequentially the autophagy inhibition using 3-MA treatment or ATG5 depletion significantly reversed the viability repression and apoptosis induction by β-elemene treatment. In addition, autophagy was found to be important in the toxic effects induced by the combination treatment of β-elemene and IGF1R inhibition in ESFT cells. Our data suggested an essential role of autophagy in β-elemene-induced apoptosis in ESFT cells, which is anticipated to provide novel insights to the development of ESFT treatments. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) chemotherapy in cancer patients increases the risk of the occurrence of cardiac dysfunction and even results in congestive heart failure. Despite the great progress of pathology in DOX-induced cardiomyopathy, the underlying molecular mechanisms remain elusive. Here, we investigate the protective effects and the underlying mechanisms of melatonin in DOX-induced cardiomyopathy. Our results clearly show that oral administration of melatonin prevented the deterioration of cardiac function caused by DOX treatment, which was evaluated by left ventricular ejection fraction and fractional shortening as well as cardiac fibrosis. The ejection fraction and fractional shortening in the DOX group were 49.48% and 25.5%, respectively, while melatonin treatment increased the ejection fraction and fractional shortening to 60.33 and 31.39 in wild-type mice. Cardiac fibrosis in the DOX group was 3.97%, while melatonin reduced cardiac fibrosis to 1.95% in wild-type mice. Sirt3 is a mitochondrial deacetylase and shows protective effects in diverse cardiovascular diseases. Therefore, to test whether Sirt3 is a key factor in protection, Sirt3 knockout mice were used, and it was found that the protective effects of melatonin in DOX-induced cardiomyopathy were partly abolished. Further analysis revealed that Sirt3 and its downstream molecule TFEB were downregulated in response to DOX treatment, while melatonin administration was able to significantly enhance the expressions of Sirt3 and TFEB. Our in vitro study demonstrated that melatonin enhanced lysosomal function by increasing the Sirt3-mediated increase at the TFEB level, and the accumulation of autolysosomes induced by DOX treatment was attenuated. Thus, autophagic flux disrupted by DOX treatment was restored by melatonin supplementation. In summary, our results demonstrate that melatonin protects the heart against DOX injury by the restoration of autophagic flux via the activation of the Sirt3/TFEB signaling pathway. [ABSTRACT FROM AUTHOR]

Diallyl sulfide (DAS), as a major component of garlic extracts, has been shown to inhibit growth of hepatocellular carcinoma cells (HCC), but the underlying mechanism is still elusive. In this study, we aimed to explore the involvement of autophagy in DAS‐induced growth inhibition of HepG2 and Huh7 hepatocellular carcinoma cells. We studied growth of DAS‐treated HepG2 and Huh7 cells using the MTS and clonogenic assays. Autophagic flux was examined by immunofluorescence and confocal microscopy. The expression levels of autophagy‐related proteins AMPK, mTOR, p62, LC3‐II, LAMP1, and cathepsin D in the HepG2 and Huh7 cells treated with DAS as well as the tumors formed by HepG2 cells in the nude mice in the presence or absence of DAS were examined using western blotting and immunohistochemistry analysis. We found that DAS treatment induced activation of AMPK/mTOR, and accumulation of LC3‐II and p62 both in vivo and in vitro. DAS inhibited autophagic flux through blocking the fusion of autophagosomes with lysosomes. Furthermore, DAS induced an increase in lysosomal pH and inhibition of Cathepsin D maturation. Co‐treatment with an autophagy inhibitor (Chloroquine, CQ) further enhanced the growth inhibitory activity of DAS in HCC cells. Thus, our findings indicate that autophagy is involved in DAS‐mediated growth inhibition of HCC cells both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases. [ABSTRACT FROM AUTHOR]

IntroductionAcquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC.MethodsWe aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC.ResultsWe found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018).ConclusionOur results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

Lipotoxicity has been shown to induce the loss of functional β-cell mass and lead to type 2 diabetes, but the mechanism remains unknown. In this study, we aim to explore the role of secretagogin (SCGN) in lipotoxicity-induced β-cell injury. Our results indicate that ox-LDL treatment leads to autophagic cell death, as evidenced by decreased cell viability, aggravated cell apoptosis, and the accumulation of the p62 protein in MIN6 cells. LysoTracker Red staining, TEM and mRFP-GFP-LC3 assays demonstrate that autophagic flux is blocked in ox-LDL-treated MIN6 cells. Intriguingly, SCGN is significantly decreased in MIN6 cells under lipotoxic conditions. Additionally, siRNA-guided SCGN knockdown blocks autophagic flux triggered by rapamycin, while SCGN restoration alleviates autophagic flux retardation and mitigates cell apoptosis. The physical interaction between SCGN and SNAP29 is validated by bioinformatics analysis, coimmunoprecipitation assay and SCGN knockdown test. Downregulation of SCGN expression reduces the interaction of these two proteins. Taken together, our results indicate that ox-LDL treatment induces apoptotic β-cell death by blocking autophagic flux dependent on SCGN downregulation. SCGN administration prevents lipotoxic β-cell injury and may be a potential therapeutic strategy to promote β-cell expansion in type 2 diabetes.

Alzheimer disease (AD) is a devastating condition associated with loss of proteostasis control and autophagy failure, leading to progressive neuronal cell death. However, the relationship between the activity in macroautophagy (MA) and chaperone-mediated autophagy (CMA) in the context of cargo clearance remains poorly dissected. We have found that cell death is observed as early as 24 h into amyloid-induced toxicity, despite enhanced and fully functional autophagy activity even at late stages of injury progression, whereas inhibition of autophagosome degradation indeed shifts apoptosis onset to an earlier point in time. A prolonged intermittent fasting (IF) regimen during severe paraquat-induced neuronal injury enhances both MA- and CMA-associated protein clearance in the brain differentially and region-specifically. These results have a number of practical implications that point towards a major focus to decrease proteinaceous cargo burden, while enhancing autophagy flux early and late in the pathogenesis, so as to rapidly offset the disequilibrium between cargo and machinery flux. In this punctum, we summarize these findings.