Increased Autophagosome Formation Research Articles

Role of heat shock transcription factor 1(HSF1)-upregulated macrophage in ameliorating pressure overload-induced heart failure in mice

In order to explore the role of macrophages in HSF1-mediated alleviation of heart failure, mice model of pressure overload-induced heart failure was established using transverse aortic constriction (TAC). Changes in cardiac function and morphology were studied in TAC and SHAM groups using ultrasonic device, tissue staining, electron microscopy, real-time quantitative polymerase chain reaction (RT-QPCR), and Western blotting. We found that mice in the TAC group showed evidence of impaired cardiac function and aggravation of fibrosis on ultrasonic and histopathological examination when compared to those in the SHAM group. The expressions of HSF1, LC3II/LC3I, Becline-1 and HIF-1, as well as autophagosome formation in TAC group were greater than that in SHAM group. On sub-group analyses in the TAC group, improved cardiac function and alleviation of fibrosis was observed in the HSF1 TG subgroup as compared to that in the wild type subgroup. Expressions of LC3II/LC3I, Becline-1 and HIF-1, too showed an obvious increase; and increased autophagosome formation was observed on electron microscopy. Opposite results were observed in the HSF1 KO subgroup. These results collectively suggest that in the pressure overload heart failure model, HSF1 promoted formation of macrophages by inducing upregulation of HIF-1 expression, through which heart failure was ameliorated.

Mitofusin 2 Exerts a Protective Role in Ischemia Reperfusion Injury Through Increasing Autophagy

Background/Aims: Autophagy is essential for maintaining cellular homeostasis and the survival of terminally differentiated cells as neurons. In this study, we aim to investigate whether mitofusin 2, a mitochondrial fusion protein, mediates autophagy in cerebral ischemia/reperfusion (I/R) injury. Methods: Primary cultured neurons were treated with oxygen-glucose deprivation/reperfusion to mimic cerebral I/R injury in vitro. Autophagosomes were visualized upon TEM. Autophagy-markers were then detected to monitor autophagy by western-blot and real-time PCR, and the autophagic flux was tracked with a mRFP-GFP-LC3 construct by fluorescence as well as autophagy inhibitors and agonists. The up- and downregulation of Mfn2 were through transfecting a lentivirusexpression vector respectively. And neuronal injury was detected by cell counting kit and TUNEL assay. Results: Results showed I/R increased autophagosome formation and inhibited autolysosome degradation. Furthermore, use of autophagy related agents demonstrated that I/R injury was caused by insufficient autophagy and aggravated by impaired autophagic degradation. The results also indicated that mitofusin 2 could ameliorate I/R injury through increasing autophagosome formation and promoting the fusion of autophagosomes and lysosomes. In contrast, downregulation of mitofusin 2 aggravated the I/R injury by inhibiting autophagosome formation and the fusion of autophagosomes and lysosomes. Additionly, mitofusin 2 overexpression did not lead to autolysosome accumulation induced by I/R. Conclusions: In summary, this study explicitly demonstrated that mitofusin 2 could ameliorate I/R injury mainly through promoting autophagy, which represented a potential novel strategy for neuroprotection against cerebral I/R damage.

Aliskiren Improves Ischemia- and Oxygen Glucose Deprivation-Induced Cardiac Injury through Activation of Autophagy and AMP-Activated Protein Kinase.

Aliskiren is a direct renin inhibitor that has been effective in anti-hypertension. We investigated whether aliskiren could improve the ischemia-induced cardiac injury and whether the autophagy was involved in this effect. A myocardial infarction (MI) model was created by the ligation of the left anterior coronary artery in C57J/BL6 mice. They were treated for 1, 3, 7, and 14 days with vehicle or aliskiren (25 mg/kg/day via subcutaneous injection). In vivo, the MI mice exhibited worse cardiac function by echocardiographic assessment and showed larger myocardial scarring by light microscopy, whereas aliskiren treatment reversed these effects, which were also associated with the changes in caspase-3 and Bcl-2 expression as well as in the number of apoptotic cells. Aliskiren increased autophagy, as demonstrated by LC3B-II expression and transmission electron microscopy. Furthermore, H9c2 cardiomyocytes were employed as an in vitro model to examine the effects of aliskiren on apoptosis and autophagy under oxygen glucose deprivation (OGD)-induced injury. Aliskiren significantly increased cell viability in a dose-dependent manner. The beneficial effects of aliskiren were associated with decreased apoptosis and mitochondrial membrane potential as well as increased autophagy via increased autophagosome formation. We also found that aliskiren-induced cardiomyocyte survival occurred via AMP-activated protein kinase (AMPK)-dependent autophagy. Taken together, these results indicated that aliskiren increased cardiomyocyte survival through increased autophagosomal formation and decreased apoptosis and necrosis via modulating AMPK expression. AMPK-dependent autophagy may represent a novel mechanism for aliskiren in ischemic cardiac disease therapy.

Impaired placental autophagy in placental malaria.

BackgroundPlacental malaria is a major cause of low birthweight, principally due to impaired fetal growth. Intervillositis, a local inflammatory response to placental malaria, is central to the pathogenesis of poor fetal growth as it impairs transplacental amino acid transport. Given the link between inflammation and autophagy, we investigated whether placental malaria-associated intervillositis increased placental autophagy as a potential mechanism in impaired fetal growth.MethodsWe examined placental biopsies collected after delivery from uninfected women (n = 17) and from women with Plasmodium falciparum infection with (n = 14) and without (n = 7) intervillositis. Western blotting and immunofluorescence staining coupled with advanced image analysis were used to quantify the expression of autophagic markers (LC3-II, LC3-I, Rab7, ATG4B and p62) and the density of autophagosomes (LC3-positive puncta) and lysosomes (LAMP1-positive puncta).ResultsPlacental malaria with intervillositis was associated with higher LC3-II:LC3-I ratio, suggesting increased autophagosome formation. We found higher density of autophagosomes and lysosomes in the syncytiotrophoblast of malaria-infected placentas with intervillositis. However, there appear to be no biologically relevant increase in LC3B/LAMP1 colocalization and expression of Rab7, a molecule involved in autophagosome/lysosome fusion, was lower in placental malaria with intervillositis, indicating a block in the later stage of autophagy. ATG4B and p62 expression showed no significant difference across histological groups suggesting normal autophagosome maturation and loading of cargo proteins into autophagosomes. The density of autophagosomes and lysosomes in the syncytiotrophoblast was negatively correlated with placental amino acid uptake.ConclusionsPlacental malaria-associated intervillositis is associated with dysregulated autophagy that may impair transplacental amino acid transport, possibly contributing to poor fetal growth.

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

ABSTRACTBacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl4 injection alone, CCl4 plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.

Autophagy regulates DUOX1 localization and superoxide production in airway epithelial cells during chronic IL-13 stimulation

The airway epithelium is a broad interface with the environment, mandating well-orchestrated responses to properly modulate inflammation. Classically, autophagy is a homeostatic pathway triggered in response to external cellular stresses, and is elevated in chronic airway diseases. Recent findings highlight the additional role of autophagy in vesicle trafficking and protein secretion, implicating autophagy pathways in complex cellular responses in disease. Th2 cytokines, IL-13 and IL-4, are increased in asthma and other airway diseases contributing to chronic inflammation. Previously, we observed that IL-13 increases reactive oxygen species (ROS) in airway epithelial cells in an autophagy-dependent fashion. Here, we tested our hypothesis that autophagy is required for IL-13-mediated superoxide production via the NADPH oxidase DUOX1. Using a mouse model of Th2-mediated inflammation induced by OVA-allergen, we observed elevated lung amounts of IL-13 and IL-4 accompanied by increased autophagosome levels, determined by LC3BII protein levels and immunostaining. ROS levels were elevated and DUOX1 expression was increased 70-fold in OVA-challenged lungs. To address the role of autophagy and ROS in the airway epithelium, we treated primary human tracheobronchial epithelial cells with IL-13 or IL-4. Prolonged, 7-day treatment increased autophagosome formation and degradation, while brief activation had no effect. Under parallel culture conditions, IL-13 and IL-4 increased intracellular superoxide levels as determined by electron paramagnetic resonance (EPR) spectroscopy. Prolonged IL-13 activation increased DUOX1, localized at the apical membrane. Silencing DUOX1 by siRNA attenuated IL-13-mediated increases in superoxide, but did not reduce autophagy activities. Notably, depletion of autophagy regulatory protein ATG5 significantly reduced superoxide without diminishing total DUOX1 levels. Depletion of ATG5, however, diminished DUOX1 localization at the apical membrane. The findings suggest non-canonical autophagy activity regulates DUOX1-dependent localization required for intracellular superoxide production during Th2 inflammation. Thus, in chronic Th2 inflammatory airway disease, autophagy proteins may be responsible for persistent intracellular superoxide production.

MiR-96 attenuates status epilepticus-induced brain injury by directly targeting Atg7 and Atg16L1

Status epilepticus (SE) can cause brain damage and lead to neural dysfunction. Developing novel targets for SE therapy and diagnosis is important and necessary. Previously, we found several differentially expressed microRNAs (miRNAs) in the developing hippocampus following SE, including the autophagy-related miR-96. In the present study, we employed immunofluorescence staining and Western blot analysis to assess the expression of autophagy-related 7 (Atg7) and Atg16L1 and the status of autophagosome formation in the hippocampus of immature rats with SE. Additional in vivo intervention was also performed to investigate the potential therapeutic function of miR-96 in developing rats with SE. We found that Atg7 and Atg16L1 were up-regulated in the neurons after SE, together with an increase in autophagosome formation. Meanwhile, overexpression of miR-96 significantly prevented brain damage in SE rats by inhibiting Atg7 and Atg16L1 expression and autophagosome formation in the hippocampus. Furthermore, Rapamycin negated miR-96 mediated brain injury attenuation through inducing autophagosome formation. Our study indicates that miR-96 might be a potential target for therapy of pediatric SE.

High mobility group A1 protein modulates autophagy in cancer cells

High Mobility Group A1 (HMGA1) is an architectural chromatin protein whose overexpression is a feature of malignant neoplasias with a causal role in cancer initiation and progression. HMGA1 promotes tumor growth by several mechanisms, including increase of cell proliferation and survival, impairment of DNA repair and induction of chromosome instability. Autophagy is a self-degradative process that, by providing energy sources and removing damaged organelles and misfolded proteins, allows cell survival under stress conditions. On the other hand, hyper-activated autophagy can lead to non-apoptotic programmed cell death. Autophagy deregulation is a common feature of cancer cells in which has a complex role, showing either an oncogenic or tumor suppressor activity, depending on cellular context and tumor stage. Here, we report that depletion of HMGA1 perturbs autophagy by different mechanisms. HMGA1-knockdown increases autophagosome formation by constraining the activity of the mTOR pathway, a major regulator of autophagy, and transcriptionally upregulating the autophagy-initiating kinase Unc-51-like kinase 1 (ULK1). Consistently, functional experiments demonstrate that HMGA1 binds ULK1 promoter region and negatively regulates its transcription. On the other hand, the increase in autophagosomes is not associated to a proportionate increase in their maturation. Overall, the effects of HMGA1 depletion on autophagy are associated to a decrease in cell proliferation and ultimately impact on cancer cells viability. Importantly, silencing of ULK1 prevents the effects of HMGA1-knockdown on cellular proliferation, viability and autophagic activity, highlighting how these effects are, at least in part, mediated by ULK1. Interestingly, this phenomenon is not restricted to skin cancer cells, as similar results have been observed also in HeLa cells silenced for HMGA1. Taken together, these results clearly indicate HMGA1 as a key regulator of the autophagic pathway in cancer cells, thus suggesting a novel mechanism through which HMGA1 can contribute to cancer progression.

Oxidative stress plays a key role in butyrate-mediated autophagy via Akt/mTOR pathway in hepatoma cells

Hepatocellular Carcinoma (HCC) is one of the most aggressive forms of cancer, responsible for a number of deaths in humans. Butyrate, one of the short chain fatty acids produced by the gut microbiota during anaerobic fermentation, was shown to be beneficial for inhibiting cancer growth. In this study, we showed that sodium butyrate induced autophagy via reactive oxygen species (ROS) in hepatoma cells. Butyrate (0–6 mM) incubation significantly increased intracellular ROS levels (45.2% compared to control), which in turn inhibited phosphorylation of akt and mTOR, leading to the upregulation of autophagic proteins, such as beclin 1, ATG 5, LC3-II, followed by the increased autophagosome formation (34.4% compared to control cells). Addition of a known antioxidant, N-acetyl cysteine (NAC), reversed these butyrate-induced ROS and autophagy. It was also found that butyrate-induced ROS enhanced MAPK activation, which was inhibited by NAC. In conclusion, our data showed that butyrate induced ROS, which in turn induced autophagy via inhibition of akt/mTOR pathway. Hence, butyrate could be considered as a potential candidate for HCC treatment.

Peretinoin, an acyclic retinoid, suppresses steatohepatitis and tumorigenesis by activating autophagy in mice fed an atherogenic high-fat diet.

The pathogenesis of non-alcoholic steatohepatitis (NASH) is still unclear and the prevention of the development of hepatocellular carcinoma (HCC) has not been established. We established an atherogenic and high-fat diet mouse model that develops hepatic steatosis, inflammation, fibrosis, and liver tumors at a high frequency. Using two NASH-HCC mouse models, we showed that peretinoin, an acyclic retinoid, significantly improved liver histology and reduced the incidence of liver tumors. Interestingly, we found that peretinoin induced autophagy in the liver of mice, which was characterized by the increased co-localized expression of microtubule-associated protein light chain 3B-II and lysosome-associated membrane protein 2, and increased autophagosome formation and autophagy flux in the liver. These findings were confirmed using primary mouse hepatocytes. Among representative autophagy pathways, the autophagy related (Atg) 5-Atg12-Atg16L1 pathway was impaired; especially, Atg16L1 was repressed at both the mRNA and protein level. Decreased Atg16L1 mRNA expression was also found in the liver of patients with NASH according to disease progression. Promoter analysis revealed that peretinoin activated the promoter of Atg16L1 by increasing the expression of CCAAT/enhancer-binding-protein-alpha. Interestingly, Atg16L1 overexpression in HepG2 cells inhibited palmitate-induced NF-kB activation and interleukin-6-induced STAT3 activation. We showed that Atg16L1 induced the de-phosphorylation of Gp130, a receptor subunit of interleukin-6 family cytokines, which subsequently repressed phosphorylated-STAT3 (Tyr705) levels, and this process might be independent of autophagy function. Thus, peretinoin prevents the progression of NASH and the development of HCC through activating the autophagy pathway by increased Atg16L1 expression, which is an essential regulator of autophagy and anti-inflammatory proteins.

Phospholipase C δ1 negatively regulates autophagy in colorectal cancer cells

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Kirsten rat sarcoma viral oncogene homolog (KRAS) is frequently mutated in CRC, and KRAS mutations promote cell motility, growth, and survival. We previously revealed that the expression of phospholipase C (PLC) δ1, one of the most basal PLCs, is down-regulated in colon adenocarcinoma, and that the KRAS signaling pathway suppresses PLCδ1 expression. Although recent studies revealed that KRAS mutations activate autophagy in cancer cells, a relation between PLCδ1 and autophagy remains unclear. Here, we found that PLCδ1 overexpression suppresses the formation of autophagosomes, which are key structures of autophagy, whereas endogenous PLCδ1 knockdown increases autophagosome formation in CRC cells. We also showed that PLCδ1 overexpression promotes cell death under nutrient deprivation. Furthermore, PLCδ1 overexpression suppresses the autophagy induced by the anti-cancer drug oxaliplatin and promotes cell death under oxaliplatin treatment. These data suggest that PLCδ1 negatively regulates autophagy, and PLCδ1 suppression contributes to the tolerance of CRC cells harboring KRAS mutations to nutrient deprivation and anti-cancer drug treatment.

Imiquimod-induced autophagy is regulated by ER stress-mediated PKR activation in cancer cells

BackgroundAutophagy is a highly conserved cellular catabolic pathway for degradation and recycling of intracellular components in response to nutrient starvation or environmental stress. Endoplasmic reticulum (ER) homeostasis can be disturbed by physiological and pathological influences, resulting in accumulation of misfolded and unfolded proteins in the ER lumen, a condition referred to as ER stress. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, possesses anti-tumor and anti-viral activities in vitro and in vivo. ObjectiveIMQ has been reported to promote the apoptosis of THP-1-derived macrophages through an ER stress-dependent pathway. However, the role of ER stress in IMQ-induced autophagy is unknown. In this study, we investigated the relationship between ER stress and IMQ-induced autophagy. MethodsThe expression of LC3, P62, p-PERK, Grp78, p-elF2α and IRE1α proteins were determined by immunoblotting. The relationship between ER stress and IMQ-induced autophagy were analyzed by ER stress inhibitors, a PERK inhibitor and the genetic silencing of PERK. The role of double-strand RNA-dependent protein kinase (PKR) activation in IMQ-induced autophagy was assessed by inhibiting PKR and genetically silencing PKR. The IMQ-induced autophagy was evaluated by immunoblotting and EGFP-LC3 puncta formation. ResultsIMQ induced reactive oxygen species (ROS) production in cancer cells. Additionally, IMQ markedly induced ER stress via ROS production and increased autophagosome formation in a dose- and time-dependent manner in both TLR7/8-expressing and TLR7/8-deficient cancer cells. Pharmacological or genetic inhibition of ER stress dramatically reduced LC3-II expression and EGFP-LC3 puncta formation in IMQ-treated cancer cells. IMQ-induced autophagy was markedly reduced by depletion and/or inhibition of PKR, a downstream effector of ER stress. ConclusionIMQ-induced autophagy is dependent on PKR activation, which is mediated by ROS-triggered ER stress. These findings might provide useful information for basic research and for the clinical application of IMQ.

Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy

Non-thermal plasma (NTP) is a potential new therapeutic modality for cancer. However, its mechanism of action remains unclear. Herein, we studied the effect of NTP on mesothelioma cells and fibroblasts to understand its anti-proliferative efficacy. Interestingly, NTP demonstrated greater selective anti-proliferative activity against mesothelioma cells relative to fibroblasts than cisplatin, which is used for mesothelioma treatment. The anti-proliferative effect of NTP was enhanced by pre-incubation with the cellular iron donor, ferric ammonium citrate (FAC), and inhibited by iron chelation using desferrioxamine (DFO). Three oxidative stress probes (CM-H2DCFDA, MitoSOX and C11-BODIPY) demonstrated reactive oxygen species (ROS) generation by NTP, which was inhibited by DFO. Moreover, NTP decreased transferrin receptor-1 and increased ferritin-H and -L chain expression that was correlated with decreased iron-regulatory protein expression and RNA-binding activity. This regulation was potentially due to increased intracellular iron in lysosomes, which was demonstrated via the Fe(II)-selective probe, HMRhoNox-M, and was consistent with autophagic-induction. Immunofluorescence using LysoTracker and Pepstatin A probes demonstrated increased cellular lysosome content, which was confirmed by elevated LAMP1 expression. The enhanced lysosomal biogenesis after NTP could be due to the observed increase in fluid-phase endocytosis and early endosome formation. These results suggest NTP acts as a stressor, which results in increased endocytosis, lysosome content and autophagy. In fact, NTP rapidly increased autophagosome formation, as judged by increased LC3B-II expression, which co-localized with LAMP1, indicating autophagolysosome formation. Autophagic-induction by NTP was confirmed using electron microscopy. In summary, NTP acts as a cellular stressor to rapidly induce fluid-phase endocytosis, lysosome biogenesis and autophagy.

Complement protein C1q modulates lipoprotein metabolism in macrophage foam cells

Abstract In the atherosclerotic lesion, macrophages ingest high levels of damaged, modified, low density lipoproteins (LDL), which accumulate in lipid droplets or defective lysosomes, generating macrophage foam cells which contribute to plaque instability and rupture. C1q, the recognition component of the classical complement cascade, opsonizes modified forms of LDL such as oxidized LDL, and promotes ingestion by macrophages. C1q has been shown to be protective in an atherosclerosis model in vivo and shown to improve macrophage foam cell survival in vitro. Autophagy is a catabolic pathway that plays a role in cellular survival and is an important process in many phagocytic cells, such as macrophages. We tested the hypothesis that C1q modulates macrophage lysosomal maturation during ingestion of oxLDL and increases autophagy in an atheroprotective manner to improve macrophage survival. Levels of autophagy initiation and elongation proteins, Beclin 1 and Atg5, are not modulated by C1q, but levels of autophagosome-associated proteins, LC3B-II and p62, were increased in immunoblot. Quantitative PCR revealed that the increase in p62 protein levels was not due to an increase in transcription. Immunofluorescence microscopy verified that the increase in LC3B and p62 by C1q resulted in an increase in autophagosome formation during oxLDL clearance. TEM verified the formation of double membrane vesicles. C1q also increased lysosomal acidification in macrophages during ingestion of oxLDL. These atheroprotective alterations to lipoprotein metabolism may be important in slowing disease progression, and provide insight into the protective role of C1q in early atherosclerosis.

Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress.

Dihydroartemisinin is an effective antimalarial agent with multiple biological activities. In the present investigation, we elucidated its therapeutic potential and working mechanism on human tongue squamous cell carcinoma (TSCC). It was demonstrated that dihydroartemisinin could significantly inhibit cell growth in a dose- and time-dependent manner by the Cell Counting Kit-8 and colony formation assay in vitro. Meanwhile, autophagy was promoted in the Cal-27 cells treated by dihydroartemisinin, evidenced by increased LC3B-II level, increased autophagosome formation, and increased Beclin-1 level compared to dihydroartemisinin-untreated cells. Importantly, dihydroartemisinin caused DNA double-strand break with simultaneously increased γH2AX foci and oxidative stress; this inhibited the nuclear localization of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), finally leading to autophagic cell death. Furthermore, the antitumor effect of dihydroartemisinin-monotherapy was confirmed with a mouse xenograft model, and no kidney injury associated with toxic effect was observed after intraperitoneal injection with dihydroartemisinin for 3 weeks in vivo. In the present study, it was revealed that dihydroartemisinin-induced DNA double-strand break promoted oxidative stress, which decreased p-STAT3 (Tyr705) nuclear localization, and successively increased autophagic cell death in the Cal-27 cells. Thus, dihydroartemisinin alone may represent an effective and safe therapeutic agent for human TSCC.

The function and magnetic resonance imaging of immature dendritic cells under ultrasmall superparamagnetic iron oxide (USPIO)-labeling.

To investigate the effects of ultrasmall superparamagnetic iron oxide (USPIO) labeling on the maturity or immune tolerance of immature dendritic cells (imDCs) as the success of immunotherapy with immature dendritic cells is highly dependent on immune tolerance. The feasibility of tracking implanted USPIO-labeled imDCs in vivo by magnetic resonance imaging (MRI) was explored. The effects of USPIO labeling on the immune tolerance of imDCs was examined. USPIO when higher than 200μg/ml caused considerable damage to imDCs, induced imDC maturation, and impacted the immune tolerance of imDCs. USPIO labeling caused a dose-dependent increase in autophagosome formation in imDCs, and autophagy inhibitors prevented the maturation of imDCs while stimulating their immune tolerance. We speculate that high concentrations of USPIO can be used to induce imDC maturation, and that this process is likely mediated through an autophagy-related pathway.

PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy

ABSTRACTPrion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-type cells. Furthermore, prnp-null astrocytes exhibited reduced MVB formation and increased autophagosome formation. The reconstitution of PRNP expression at the cell membrane restored exosome secretion in PRNP-deficient astrocytes, whereas macroautophagy/autophagy inhibition via BECN1 depletion reestablished exosome release in these cells. Moreover, the PRNP octapeptide repeat domain was necessary to promote exosome secretion and to impair the formation of the CAV1-dependent ATG12–ATG5 cytoplasmic complex that drives autophagosome formation. Accordingly, higher levels of CAV1 were found in lipid raft domains instead of in the cytoplasm in prnp-null cells. Collectively, these findings demonstrate that PRNP supports CAV1-suppressed autophagy to protect MVBs from sequestration into phagophores, thus facilitating exosome secretion.

Glucose deprivation induced upregulation of phosphoenolpyruvate carboxykinase modulates virulence in Leishmania donovani.

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP+ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.

Knockdown of GSK3β increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells.

High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3β (GSK3β). To determine whether GSK3β affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308: , C249-C263]. Presently, we found that incubation of HAECs with excess nutrients (25mM glucose and 0.4mM palmitate) increased GSK3β activity and impaired lysosome acidification. Suppression of GSK3β in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3β attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3β increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3β inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3β may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis.

Effect of autophagy induced by dexamethasone on senescence in chondrocytes.

The aim of the current study was to explore the effects of dexamethasone (DXM) on autophagy and senescence in chondrocytes. Collagen II and aggrecan were examined in normal chondrocytes isolated from Sprague-Dawley rats. Following stimulation with DXM, LysoTracker Red staining, monodansylcadaverine (MDC) staining, green fluorescent protein-red fluorescent protein-light chain 3 (LC3) and western blotting were used to detect autophagy levels in the chondrocytes. Mechanistic target of rapamycin (mTOR) pathway-associated molecules were investigated by western blotting. Cell senescence was analyzed by senescence-associated (SA)-β-galactosidase (β-gal) staining. A dose-dependent increase in the number of autophagic vacuoles was observed in the DXM-treated chondrocytes, as demonstrated by LysoTracker Red and MDC staining. A dose-dependent increase in autophagosome formation was observed in the DXM-treated chondrocytes. Expression of LC3-II and beclin-1 was increased by DXM, in particular in the cells treated with DXM for 4 days. However, P62 expression was reduced as a result of treatment. SA-β-gal staining indicated that DXM increased cell senescence. Notably, DXM-induced cell senescence was exacerbated by the autophagic inhibitor 3-MA. Autophagy induced by DXM protected chondrocytes from senescence, and it is suggested that the mTOR pathway may be involved in the activation of DXM-induced autophagy.