Accumulation Of Autophagosomes Research Articles

The SsAtg1 Activating Autophagy Is Required for Sclerotia Formation and Pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that produces sclerotia. Sclerotia are essential components of the survival and disease cycle of this devastating pathogen. In this study, we analyzed comparative transcriptomics of hyphae and sclerotia. A total of 1959 differentially expressed genes, 919 down-regulated and 1040 up-regulated, were identified. Transcriptomes data provide the possibility to precisely comprehend the sclerotia development. We further analyzed the differentially expressed genes (DEGs) in sclerotia to explore the molecular mechanism of sclerotia development, which include ribosome biogenesis and translation, melanin biosynthesis, autophagy and reactivate oxygen metabolism. Among these, the autophagy-related gene SsAtg1 was up-regulated in sclerotia. Atg1 homologs play critical roles in autophagy, a ubiquitous and evolutionarily highly conserved cellular mechanism for turnover of intracellular materials in eukaryotes. Therefore, we investigated the function of SsAtg1 to explore the function of the autophagy pathway in S. sclerotiorum. Deficiency of SsAtg1 inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. Notably, ΔSsAtg1 was unable to form sclerotia and displayed defects in vegetative growth under conditions of nutrient restriction. Furthermore, the development and penetration of the compound appressoria in ΔSsAtg1 was abnormal. Pathogenicity analysis showed that SsAtg1 was required for full virulence of S. sclerotiorum. Taken together, these results indicate that SsAtg1 is a core autophagy-related gene that has vital functions in nutrient utilization, sclerotia development and pathogenicity in S. sclerotiorum.

Cryptosporidium parvum regulates HCT-8 cell autophagy to facilitate survival via inhibiting miR-26a and promoting miR-30a expression

BackgroundCryptosporidium parvum is an important zoonotic parasite, which not only causes economic losses in animal husbandry but also harms human health. Due to the lack of effective measures for prevention and treatment, it is important to understand the pathogenesis and survival mechanism of C. parvum. Autophagy is an important mechanism of host cells against parasite infection through key regulatory factors such as microRNAs and MAPK pathways. However, the regulatory effect of C. parvum on autophagy has not been reported. Here, we demonstrated that C. parvum manipulated autophagy through host cellular miR-26a, miR-30a, ERK signaling and P38 signaling for parasite survival.MethodsThe expression of Beclin1, p62, LC3, ERK and P38 was detected using western blotting in HCT-8 cells infected with C. parvum as well as treated with miR-26a-mimic, miR-30a-mimic, miR-26a-mimic or miR-30a-inhibitor post C. parvum infection. The qPCR was used to detect the expression of miR-26a and miR-30a and the number of C. parvum in HCT-8 cells. Besides, the accumulation of autophagosomes was examined using immunofluorescence.ResultsThe expression of Beclin1 and p62 was increased, whereas LC3 expression was increased initially at 0–8 h but decreased at 12 h and then increased again in C. parvum-infected cells. C. parvum inhibited miR-26a-mimic-induced miR-26a but promoted miR-30a-mimic-induced miR-30a expression. Suppressing miR-30a resulted in increased expression of LC3 and Beclin1. However, upregulation of miR-26a reduced ERK/P38 phosphorylation, and inhibiting ERK/P38 signaling promoted Beclin1 and LC3 while reducing p62 expression. Treatment with miR-26a-mimic, autophagy inducer or ERK/P38 signaling inhibitors reduced but treatment with autophagy inhibitor or miR-30a-mimic increased parasite number.ConclusionsThe study found that C. parvum could regulate autophagy by inhibiting miR-26a and promoting miR-30a expression to facilitate the proliferation of parasites. These results revealed a new mechanism for the interaction of C. parvum with host cells.Graphical

Nano-selenium Alleviates Cadmium-Induced Mouse Leydig Cell Injury, via the Inhibition of Reactive Oxygen Species and the Restoration of Autophagic Flux.

Cadmium (Cd) is a well-known environmental pollutant that can contribute to male reproductive toxicity through oxidative stress. Nano-selenium (Nano-se) is an active single body of selenium with strong antioxidant properties and low toxicity. Some studies have addressed the potential ameliorative effect of Nano-se against Cd-induced testicular toxicity; however, the underlying mechanisms remain to be investigated. This study aimed to explore the protective effect of Nano-se on Cd-induced mouse testicular TM3 cell toxicity by regulating autophagy process. We showed that cadmium exposure to TM3 cells inhibited cell viability and elevated the level of reactive oxygen species (ROS) generation. Morphology observation by transmission electron microscope and the presence of mRFP-GFP-LC3 fluorescence puncta demonstrated that cadmium increased autophagosome formation and accumulation in TM3 cells, resulting in blocking the autophagic flux of TM3 cells. Meanwhile, cadmium remarkably increased the ratio of LC3-II to LC3-I protein expression (2.07 ± 0.31) and the Beclin-1 protein expression (1.97 ± 0.40) in TM3 cells (P < 0.01). Pretreatment with Nano-se significantly reduced Cd-induced TM3 cell toxicity (P < 0.01). Furthermore, Nano-se treatment reversed Cd-induced ROS production and autophagosome accumulation, and autophagy as evidenced by the ratio of LC3-II to LC3-I and Beclin-1 expression. In addition, ROS scavenger, N-acetyl-L-cysteine (NAC) or autophagy inhibitor, 3-methyladenine (3-MA) reversed cadmium-induced ROS generation, autophagosome accumulation, and autophagy-related protein expression levels, which confirmed that cadmium induced TM3 cell injury via ROS signal pathway and blockage of autophagic flux. Collectively, our results reveal that Nano-se attenuates Cd-induced TM3 cell toxicity through the inhibition of ROS production and the amelioration of autophagy disruption.

Excessive Protein Accumulation and Impaired Autophagy in the Hippocampus of Angelman Syndrome Modeled in Mice

BackgroundAngelman syndrome (AS), a neurodevelopmental disorder caused by abnormalities of the 15q11.2-q13.1 chromosome region, is characterized by impairment of cognitive and motor functions, sleep problems, and seizures. How the genetic defects of AS produce these neurological symptoms is unclear. Mice modeling AS (AS mice) accumulate activity-regulated cytoskeleton-associated protein (ARC/ARG3.1), a neuronal immediate early gene (IEG) critical for synaptic plasticity. This accumulation suggests an altered protein metabolism. MethodsFocusing on the dorsal hippocampus (dHC), a brain region critical for memory formation and cognitive functions, we assessed levels and tissue distribution of IEGs, de novo protein synthesis, and markers of protein synthesis, endosomes, autophagy, and synaptic functions in AS mice at baseline and following learning. We also tested autophagic flux and memory retention following autophagy-promoting treatment. ResultsAS dHC exhibited accumulation of IEGs ARC, FOS, and EGR1; autophagy proteins MLP3B, SQSTM1, and LAMP1; and reduction of the endosomal protein RAB5A. AS dHC also had increased levels of de novo protein synthesis, impaired autophagic flux with accumulation of autophagosome, and altered synaptic protein levels. Contextual fear conditioning significantly increased levels of IEGs and autophagy proteins, de novo protein synthesis, and autophagic flux in the dHC of normal mice, but not in AS mice. Enhancing autophagy in the dHC alleviated AS-related memory and autophagic flux impairments. ConclusionsA major biological deficit of AS brain is a defective protein metabolism, particularly that dynamically regulated by learning, resulting in stalled autophagy and accumulation of neuronal proteins. Activating autophagy ameliorates AS cognitive impairments and dHC protein accumulation.

Silica Nanoparticles Trigger Chaperone HSPB8-Assisted Selective Autophagy via TFEB Activation in Hepatocytes.

Silica nanoparticles (SiNPs) are one of the most common inorganic nanomaterials. Autophagy is the predominant biological response to nanoparticles and transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome pathway. Previous studies show that SiNPs induce autophagosome accumulation, yet the precise underlying mechanisms remain uncertain. The present study investigates the role of TFEB during SiNP-induced autophagy. SiNP-induced TFEB nuclear translocation is verified using immunofluorescence and western blot assay. The regulation of TFEB is proved to be via EIF2AK3 pathway. A TFEB knockout (KO) cell line is constructed to validate the TFEB involvement in SiNP-induced autophagy. The transcriptomes of wild-type and TFEB KO cells are compared using RNA-sequencing to identify genes of the TFEB-mediated autophagy and lysosome pathways affected by SiNPs. Based on these data and the Human Autophagy Database, four candidate autophagic genes are identified, including HSPB8, ATG4D, CTSB and CTSD. Specifically, that the chaperone HSPB8 is upregulated through SiNP-mediated TFEB activation and forms a chaperone-assisted selective autophagy (CASA) complex with BAG3 and HSC70, triggering HSPB8-assisted selective autophagy, is found. Thus, this study characterizes a novel mechanism underlying SiNP-induced autophagy that helps pave the way for further research on the toxicity and risk assessment of SiNPs.

Loss of FoxOs in muscle increases strength and mitochondrial function during aging.

Muscle mitochondrial decline is associated with aging-related muscle weakness and insulin resistance. FoxO transcription factors are targets of insulin action and deletion of FoxOs improves mitochondrial function in diabetes. However, disruptions in proteostasis and autophagy are hallmarks of aging and the effect of chronic inhibition of FoxOs in aged muscle is unknown. This study investigatedthe role of FoxOs in regulating muscle strength and mitochondrial function with age. We measured muscle strength, cross-sectional area, muscle fibre-type, markers of protein synthesis/degradation, central nuclei, glucose/insulin tolerance, and mitochondrial bioenergetics in 4.5-month (Young) and 22-24-month-old (Aged) muscle-specific FoxO1/3/4 triple KO (TKO) and littermate control (Ctrl) mice. Lean mass was increased in Aged TKO compared with both Aged Ctrl and younger groups by 26-33% (P<0.01). Muscle strength, measured by max force of tibialis anterior (TA) contraction, was 20% lower in Aged Ctrl compared with Young Ctrls (P<0.01) but was not decreased in Aged TKOs. Increased muscle strength in Young and Aged TKO was associated with 18-48% increased muscle weights compared with Ctrls (P<0.01). Muscle cross-sectional analysis of TA, soleus, and plantaris revealed increases in fibre size distribution and a 2.5-10-fold increase in central nuclei in Young and Aged TKO mice, without histologic signs of muscle damage. Age-dependent increases in Gadd45a and Ube4a expression as well accumulation of K48 polyubiquitinated proteins were observed in quad and TA but were prevented by FoxO deletion. Young and Aged TKO muscle showed minimal changes in autophagy flux and no accumulation of autophagosomes compared with Ctrl groups. Increased strength in Young and Aged TKO was associated with a 10-20% increase in muscle mitochondrial respiration using glutamate/malate/succinate compared with controls (P<0.05). OXPHOS subunit expression and complex I activity were decreased 16-34% in Aged Ctrl compared with Young Ctrl but were prevented in Aged TKO. Both Aged Ctrl and Aged TKO showed impairedglucose tolerance by 33% compared to young groups (P<0.05) indicating improved strength and mitochondrial respiration are not due to improved glycemia. FoxO deletion increases muscle strength even during aging. Deletion of FoxOs maintains muscle strength in part by mild suppression of atrophic pathways, including inhibition of Gadd45a and Ube4a expression, without accumulation of autophagosomes in muscle. Deletion of FoxOs also improved mitochondrial function by maintenance of OXPHOS in both young and aged TKO.

Activation of FMS-like tyrosine kinase 3 protects against isoprenaline-induced cardiac hypertrophy by improving autophagy and mitochondrial dynamics.

FMS-like receptor tyrosine kinase 3 (Flt3) expression was reported to increase in the heart in response to pathological stress, but the role of Flt3 activation and its underlying mechanisms remain poorly elucidated. This study was designed to investigate the role of Flt3 activation in sympathetic hyperactivity-induced cardiac hypertrophy and its mechanisms through autophagy and mitochondrial dynamics. In vivo, cardiac hypertrophy was established by subcutaneous injection of isoprenaline (6mg/kg·day) in C57BL/6 mice for 7 consecutive days. The Flt3-ligand intervention was launched 2 h prior to isoprenaline each day. In vitro, experiments of cardiomyocyte hypertrophy, autophagy, and mitochondrial dynamics were performed in neonatal rat cardiomyocytes (NRCMs). Our results revealed that the expression level of Flt3 protein was significantly increased in the hypertrophic myocardium provoked by isoprenaline administration. Flt3-ligand intervention alleviated isoprenaline-induced cardiac oxidative stress, hypertrophy, fibrosis, and contractile dysfunction. Isoprenaline stimulation impaired autophagic flux in hypertrophic mouse hearts, supported by the accumulation of LC3II and P62 proteins, while Flt3-ligand restored the impairment of autophagic flux. Flt3 activation normalized the imbalance of mitochondrial fission and fusion in the hearts of mice evoked by isoprenaline as evidenced by the neutralization of elevated mitochondrial fission markers and reduced mitochondrial fusion markers. In NRCMs, Flt3-ligand treatment attenuated isoprenaline-stimulated hypertrophy, which was abolished by a Flt3-specific blocker AC220. Activating Flt3 reversed isoprenaline-induced autophagosome accumulation and impairment of autophagic flux probably by enhancing SIRT1 expression and consequently TFEB nuclear translocation. Flt3 activation improved the imbalance of mitochondrial dynamics induced by isoprenaline in NRCMs through the SIRT1/P53 pathway. Activation of Flt3 mitigated ISO-stimulated hypertrophy probably involves the restoration of autophagic flux and balance of mitochondrial dynamics. Therefore, activation of Flt3 attenuates isoprenaline-induced cardiac hypertrophy in vivo and in vitro, the potential mechanism probably attributes to SIRT1/TFEB-mediated autophagy promotion and SIRT1/P53-mediated mitochondrial dynamics balance. These findings suggest that activation of Flt3 may be a novel target for protection against cardiac remodeling and heart failure during sympathetic hyperactivity.

Dieckol Inhibits Autophagic Flux and Induces Apoptotic Cell Death in A375 Human Melanoma Cells via Lysosomal Dysfunction and Mitochondrial Membrane Impairment.

Dieckol is a natural brown algal-derived polyphenol and its cytotoxic potential against various types of cancer cells has been studied. However, the effects of dieckol on autophagy in cancer cells remain unknown. Here, we show that dieckol inhibits the growth of A375 human melanoma cells by inducing apoptotic cell death, which is associated with lysosomal dysfunction and the inhibition of autophagic flux. Dieckol induces autophagosome accumulation by inhibiting autophagosome-lysosome fusion. Moreover, dieckol not only triggers lysosomal membrane permeabilization, followed by an increase in lysosomal pH and the inactivation of cathepsin B and D, but also causes the loss of mitochondrial membrane potential. Importantly, a cathepsin D inhibitor partially relieved dieckol-induced mitochondrial membrane impairment and caspase-mediated apoptosis. Collectively, our findings indicate that dieckol is a novel autophagy inhibitor that induces apoptosis-mediated cell death via lysosomal dysfunction and mitochondrial membrane impairment in A375 human melanoma cells. This suggests the novel potential value of dieckol as a chemotherapeutic drug candidate for melanoma treatment.

The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes.

Cell competition, the elimination of cells surrounded by more fit neighbors, is proposed to suppress tumorigenesis. Mahjong (Mahj), a ubiquitin E3 ligase substrate receptor, has been thought to mediate competition of cells mutated for lethal giant larvae (lgl), a neoplastic tumor suppressor that defines apical-basal polarity of epithelial cells. Here, we show that Drosophila cells mutated for mahjong, but not for lgl [l(2)gl], are competed because they express the bZip-domain transcription factor Xrp1, already known to eliminate cells heterozygous for ribosomal protein gene mutations (Rp/+ cells). Xrp1 expression in mahj mutant cells results in activation of JNK signaling, autophagosome accumulation, eIF2α phosphorylation and lower translation, just as in Rp/+ cells. Cells mutated for damage DNA binding-protein 1 (ddb1; pic) or cullin 4 (cul4), which encode E3 ligase partners of Mahj, also display Xrp1-dependent phenotypes, as does knockdown of proteasome subunits. Our data suggest a new model of mahj-mediated cell competition that is independent of apical-basal polarity and couples Xrp1 to protein turnover.

DDDR-30. UNRAVELING THE NOVEL ANTI-TUMOR MECHANISM OF ST-401, A BRAIN PENETRANT MICROTUBULE TARGETING AGENT

Abstract Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of cancers. While evidence suggests that glioblastoma (GBM) is sensitive to disruptions in microtubule (MT) functions; most MTAs do not reach the brain. To address this limitation, we developed a brain-penetrant MTA, ST-401, that kills patient-derived GBM in culture at nanomolar concentrations, binds to colchicine sites through a unique interaction mode, gently and reversibly inhibits MT assembly and kills cancer cells through a novel anti-tumor mechanism as determined by analysis of its activity in the NCI 60 cancer cell panel compared to the Approved Oncology Agents library (Horne et al. (2020) Neuro-Onc Adv. 3(1): vdaa165).We used flow cytometry to confirm that nocodazole (10-100 nM) treatment of HCT116 cells triggered apoptosis, necrosis, and accumulation of autophagosomes, as well as increase in p-P53. By sharp contrast, ST-401 did not trigger these responses. Live cell imaging showed that NOC preferentially kills HCT116 cells in mitosis whereas ST-401 preferentially kills these cells in interphase. ScRNAseq analysis of NOC and ST-401 treatment of HCT-116 cells showed that ST-401 down-regulates MYC mRNA expression, suggesting that ST-401 might affect MYC function. MYC protein levels are regulated by multiple mechanisms, including phosphorylation at residue Thr58 that promotes its ubiquitination and proteasome degradation. We discovered that ST-401 increases MYC phosphorylation on Thr58 and down-regulates MYC protein expression in both HCT116 and the GBM cell line, U251.Our result suggests that the novel anti-tumor mechanism of ST-401 might involve cell death in interphase and reduced MYC expression. Funded by NIH (CA244213 to NS and NS106924 to NS).

Abstract 15660: Impaired Mitochondrial Quality Control Drives the Development of Cardiomyopathy in Anderson-Fabry Disease

Introduction: Anderson-Fabry disease (AFD) is a systemic disorder caused by impaired alpha-galactosidase (GLA) activity, leading to a progressive cardiomyopathy, with hypertrophy and diastolic dysfunction. However, the molecular mechanisms underlying these features are unknown. Aim: To explore the role of mitochondria as a pathogenic mechanism of cardiac dysfunction in AFD. Methods: We tested a transgenic mouse that lacks endogenous GLA and expresses human R301Q-GLA [Tg/GLA knockout]. We performed serial ecochardiographic, histological, and biochemical analyses on cardiac tissue and single cell contractility on isolated adult cardiomyocytes (CM). Moreover, we assessed mitochondrial morphology/function in mouse embryonic fibroblasts (MEFs). Results: AFD-MEFs display an impaired mitochondrial respiration and reduced ATP synthesis. Increased mitochondrial levels of DRP1, LC3II, and MFN-ubiquitination along with accumulation of fragmented mitochondria and autophagosomes indicate the presence of mitophagy-targeted mitochondria that were not removed. Reduced PGC-1a and mitochondrial-DNA synthesis in AFD-MEFs indicate impaired mitochondrial biogenesis as well. Altogether, our data indicate impaired mitophagy and quality control of AFD-mitochondria. At echocardiography, AFD mice showed diastolic dysfunction and hypertrophy starting from 9 months. Isolated AFD-CMs exhibit reduced inotropic response to isoproterenol. Remarkably, alterations of cardiac mitochondrial function were observed starting from 3 months, including increased levels of MFN2-ubiquitination, DRP1 and LC3II, decreased levels of PGC-1a along with accumulation of disarranged mitochondria and autophagosomes. Frank impairment of mitochondrial respiration in AFD-CMs and reduced myocardial ATP-content was detected from 6 months, together with increased release of Cytochrome-C in the cytosol. Strikingly, mitochondrial dysfunction was also detected in fibroblasts isolated from AFD patients, which have a significantly reduced electron transport chain activity. Conclusions: In AFD, energetic alterations associated with impaired autophagy/mitophagy, are evident at 3 months and progressively lead to cardiac hypertrophy and dysfunction.

Enterovirus 71-induced autophagosome fusion with multivesicular bodies facilitates viral RNA packaging into exosomes

Exosomes have been shown to release from cells infected by viruses and deliver viral particles, genomes, and other viral genetic elements to neighboring cells resulting in modulating host immune response. Our previous study demonstrated that exosomes released from Enterovirus 71 (EV71)-infected cells contained replication-competent EV71 RNA in complex with miR-146a, Ago2, and GW182, which can be successfully transferred to recipient/target cells to establish productive infection. However, the molecular mechanisms that control viral genome package into exosomes are still unclear. In this study, we showed that the EV71-induced autophagy response contributed to viral genome package into exosomes rather than process of exosomes biogenesis. Further study showed that the autophagosomes accumulation facilitated their fusion with MVBs, which resulted in EV71 RNA package into exosome vesicles. Moreover, prevention of autophagosomes-MVBs fusion could abolish this sorting of viral RNA into exosomes. Knockdown of GW182 or Ago2 could weaken the replication ability of exosomal EV71 RNA in recipient cells through decreasing the amount of miR-146a in exosomes, but did not affect the package of viral RNA into exosomes. Our findings strongly suggested that the accumulation of autophagosomes that were induced by EV71 infection play a key role on viral spreading through exosome vesicles.

Expression of transforming growth factor-β1 and autophagy markers in the bladder of rats with neurogenic lower urinary tract injury.

This study was conducted to explore the expression of transforming growth factor-beta 1 (TGF-β1) and its correlation with autophagy markers in the bladder of rats with neurogenic lower urinary tract dysfunction (NLUTD) post spinal cord injury (SCI). A total of 36 male Wistar rats were randomly divided into the SCI group and control group. Rats in the SCI group were subjected to T10-T11 spinal cord transection. At day 1, 4, and 7, 6 rats were euthanized daily and the Basso, Beattie and Bresnahan score (BBB score), post-void residual (PVR), urinary bladder function score (UBFS) and bladder weight were assessed. The expression TGF-β1 and autophagy markers were evaluated by immunofluorescence staining, Western bolt, and qRT-PCR. A total of 36 male Wistar rats were randomly divided into the SCI group and control group, with three time points in each group. Not applicable. SCI modeling impaired the motor function of the hind limbs and the bladder function of rats. NLUTD muscle exhibited a punctated immunostaining pattern for LC3, suggesting the accumulation of autophagosomes. Our results further indicated that compared with the control group, the expression levels of TGF-β1 and LC3 were increased, while the level of P62 was decreased after SCI modeling. TGF-β1 was significantly increased in SCI rats with NLUTD and was correlated with autophagy markers LC3 and p62.

Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.

Hydrogen sulfide inhibits human T-cell leukemia virus type-1 (HTLV-1) protein expression via regulation of ATG4B.

Hydrogen sulfide (H2 S) is a redox gasotransmitter. It has been shown that H2 S has a key role in host antiviral defense by inhibiting interleukin production and S-sulfhydrating Keap1 lead to Nrf2/ARE pathway activation. However, it is yet unclear whether H2 S can play an antiviral role by regulating autophagy. In this study, we found that exogenous H2 S decreased the expression of human T-cell leukemia virus type-1(HTLV-1)protein and HTLV-1 induced autophagosomes accumulation. Transmission electron microscope assays indicated that autophagosomes accumulation decreased after H2 S administration. HTLV-1-transformed T-cell lines had a high level of CSE (H2 S endogenous enzyme) which could be induced in Hela by HTLV-1 infection. Immunoblot demonstrated that overexpression of CSE inhibited HTLV-1 protein expression and autophagy. And we got the opposite after CSE knockdown. Meanwhile, H2 S could not restrain the autophagy when ATG4B had a mutant at its site of 89. In a word, these results suggested that H2 S modulated HTLV-1 protein expression via ATG4B. Therefore, our findings suggested a new mechanism by which H2 S defended against virus infection.

Diesel exhaust particles induce human umbilical vein endothelial cells apoptosis by accumulation of autophagosomes and caspase-8 activation

Diesel exhaust particles (DEP) are risk factors for endothelial cells (ECs) dysfunction. However, the mechanism by which DEP induce ECs apoptosis remains unclear. Here, we investigated how DEP induce death of human umbilical vein ECs (HUVECs), with a focus on the autophagy-mediated apoptotic pathway. DEP induced dose-dependent HUVECs death and exposure to the IC50 concentration of DEP (70 µg/ml) led to apoptosis. DEP phosphorylated Beclin-1 (Ser93) and increased protein levels of p62 and LC3BII and the number of LC3B puncta, indicating autophagy initiation. DEP increased expression of pro- and mature forms of cathepsin D, which increases lysosomal activity. However, DEP suppressed expression of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (STX17, VAMP8, SNAP29, YKT6, and STX7) to inhibit autolysosome formation, resulting in accumulation of autophagosomes. LC3B, p62, and caspase-8 form a tertiary complex in accumulated autophagosomes, which is known to serve as a platform for caspase-8 activation. Indeed, DEP activates caspase-8 and pretreatment with a caspase-8 inhibitor suppressed DEP-induced apoptosis. Furthermore, depletion of p62 decreased caspase-8 and caspase-3 activation and inhibited the DEP-induced apoptosis. Taken together, these findings demonstrated that DEP induced HUVECs apoptosis by inhibiting autophagosome maturation and identified caspase-8 as a novel mediator of DEP-induced ECs apoptosis.

FYCO1 ameliorates adverse cardiac remodeling in response to myocardial ischemia by enhancing autophagic flux

Abstract Background Imbalances in autophagy are known to have either protective or detrimental effects on cardiac function. Although there are significant insights into the early steps of the autophagy pathway, the mechanisms of autophagosome-lysosome transport and fusion in cardiomyocytes are less understood. Fyco1 is highly expressed in the heart and facilitates transport of autophagosomes in cardiomyocytes, enhancing global autophagic flux. Purpose The aim of our study was to investigate the effects of FYCO1 overexpression post myocardial infarction. Our hypothesis is that FYCO1 overexpression leads to enhanced autophagic flux and hence blunted cardiac remodeling and improved heart function. Methods FYCO1-Tg mice were crossbred with RFP-EGFP-LC3-Tg reporter mice (FYCO1-TGxRGF) to enable the analysis of autophagic flux using confocal microscopy. Myocardial ischemia was induced by permanent ligation of left anterior descending artery (LAD). Mice were analyzed 3 days and 30 days post LAD ligation. Cardiac function (ejection fraction, EF) and left ventricular (LV) mass were measured by echocardiography. Myocardial fibrosis, Midline Infarct size (%) and LV circumference were assessed in Masson's Trichrome stainings. Autophagy activaty was analyzed by Western blot for LC3 II and LC3 II/LC3 I. Furthermore, Evans blue/TTC staining was performed to assess the area at risk and to define perfused, ischemic and necrotic area post LAD. Results In FYCO1-TGxRGFP mice 3 days post LAD we observed an overall increased autophagic flux demonstrated by significantly increased LC3 II protein levels (WT sham: 1±0,3; WT LAD: 1,3±0,2; TG sham: 10,8±1,2; TG LAD: 8,8±0,9) and LC3 II/LC3 I ratios (WT sham: 1±0,3; WT LAD: 0,99±0,1; TG sham: 10,5±1,2; TG LAD: 5,6±1) post LAD. Moreover, using confocal microscopy revealed a shift in autophagosomal transport towards plus-end-directed transport, shown by peripheral accumulation of autophagosomes (WT: 1,4±0,8; TG: 46,6±5,8) and increased levels of autolysosomes (WT: 79,2±19,2; TG: 181,1±36,7). In Evans blue/TTC staining, FYCO1-TGxRGFP mice demonstrated comparable ischemic but markedly decreased necrotic area (ischemic: 1,9±1,8%; necrotic: 37,1±11,5%) within the LV as compared to WT (ischemic: 2,1±1,9%; necrotic: 53,4±31,4%). Fibrosis in LV (WT: 19,6±1,8%; TG: 10,6±1,6%) and infarct size (WT: 34,5±3,9%; TG: 5,5±2%) were found significantly decreased in FYCO1-TGxRGFP-mice. FYCO1-TGxRGFP mice presented with increased LV mass (WT: 106,2±25,5 mg; TG: 114,2±38,3mg) but comparable LV circumference/tibia length (WT: 11,1±0,9; TG: 11,4±1,5). Importantly, global heart function of FYCO1-TGxRGFP-mice was significantly improved post LAD compared to WT (EF WT: 36,7±6,3%; TG: 46,3±18,3%). Conclusion Our study demonstrates that the activation of autophagy via FYCO1 overexpression protects against adverse cardiac remodeling and improves cardiac function post myocardial ischemia. These findings may thus offer translational potential in the treatment of myocardial infarction. Funding Acknowledgement Type of funding sources: None.

DSTYK inhibition increases the sensitivity of lung cancer cells to T cell-mediated cytotoxicity.

Lung cancer remains the leading cause of cancer-related death worldwide. We identify DSTYK, a dual serine/threonine and tyrosine non-receptor protein kinase, as a novel actionable target altered in non-small cell lung cancer (NSCLC). We also show DSTYK's association with a lower overall survival (OS) and poorer progression-free survival (PFS) in multiple patient cohorts. Abrogation of DSTYK in lung cancer experimental systems prevents mTOR-dependent cytoprotective autophagy, impairs lysosomal biogenesis and maturation, and induces accumulation of autophagosomes. Moreover, DSTYK inhibition severely affects mitochondrial fitness. We demonstrate in vivo that inhibition of DSTYK sensitizes lung cancer cells to TNF-α-mediated CD8+-killing and immune-resistant lung tumors to anti-PD-1 treatment. Finally, in a series of lung cancer patients, DSTYK copy number gain predicts lack of response to the immunotherapy. In summary, we have uncovered DSTYK as new therapeutic target in lung cancer. Prioritization of this novel target for drug development and clinical testing may expand the percentage of NSCLC patients benefiting from immune-based treatments.

The phospholipid flippase ATP8B1 is required for lysosomal fusion in macrophages.

ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases(P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.

Peli1 contributes to myocardial ischemia/reperfusion injury by impairing autophagy flux via its E3 ligase mediated ubiquitination of P62

Autophagy flux is impaired during myocardial ischemia/reperfusion (M-I/R) via the accumulation of autophagosome and insufficient clearance, which exacerbates cardiomyocyte death. Peli1 (Pellion1) is a RING finger domain-containing ubiquitin E3 ligase that could catalyze the polyubiquitination of substrate proteins. Peli1 has been demonstrated to play an important role in ischemic cardiac diseases. However, little is known about whether Peli1 is involved in the regulation of autophagy flux during M-I/R. The present study investigated whether M-I/R induced impaired autophagy flux could be mediated through Peli1 dependent mechanisms. We induced M-I/R injury in wild type (WT) and Peli1 knockout mice and observed that M-I/R significantly decreased cardiac function that was associated with increased cardiac Peli1 expression and upregulated autophagy-associated protein LC3II and P62. In contrast, Peli1 knockout mice exhibited significant improvement of M-I/R induced cardiac dysfunction and decreased LC3II and P62 expression. Besides, inhibitors of autophagy also increased the infarct size in Peli1 knockout mice after 24 h of reperfusion. Mechanistic studies demonstrated that in vivo I/R or in vitro hypoxia/reoxygenation (H/R) markedly increased the Peli1 E3 ligase activity which directly promoted the ubiquitination of P62 at lysine(K)7 via K63-linkage to inhibit its dimerization and autophagic degradation. Co-immunoprecipitation and GST-pull down assay indicated that Peli1 interacted with P62 via the Ring domain. In addition, Peli1 deficiency also decreased cardiomyocyte apoptosis. Together, our work demonstrated a critical link between increased expression and activity of Peli1 and autophagy flux blockage in M-I/R injury, providing insight into a promising strategy for treating myocardium M-I/R injury.