Biogenesis of autophagosomes from the ERGIC membrane system

Introduction Macroautophagy(hereafter referred as autophagy)is a process of cellular self-degradation.In response to nutrient deprivation or other stimuli,a nascent double-membrane autophagosome,encapsulating intracellular materials or damaged organelles,is generated.The autophagosome is transported toward and eventually fuses with the lysosome(or the vacuole in yeast and plant cells).

Protective effect of Liangxue Huayu decoction on human retinal pigment epithelial cell(ARPE-19)injury induced by hypoxia through autophagy pathway

Background:Exploring the protective mechanism of the Liangxue Huayu(LXHY)decoction on human retinal pigment epithelial(RPE)cells induced by hypoxia through the autophagy pathway.Methods:The appropriate LXHY decoction concentration was determined by CCK-8.ARPE-19 cells were divided into the normal control group(A group),CoCl_(2)group(B group),3-Methyladenine(3-MA)group(treated with 3-MA(the inhibition of autophagy pathway))(C group),blank serum(BS)group(D group),LXHY drug-contained serum(DCS)group(E group),and Rapamycin(RAP)group[treated with LXHY drug-contained serum combined with rapamycin group(the activation of autophagy pathway)](F group).Counting the number of autophagosomes and autolysosomes in each group of cells under transmission electron microscopy.After infection of cells in each group by mRFP-GFP-LC3 fusion protein adenovirus,the strength of autophagic flux was detected.The mRNA expression levels of LC3 and Beclin-1 were detected by Q-PCR.Results:CCK-8 assay results showed that LXHY DCS could inhibit the cell proliferation of ARPE-19 under hypoxia(all P<0.05).As the transmission electron microscopy assay result showed,compared with the normal control group,the number of autolysosomes was significantly increased in the CoCl_(2)group(P<0.05).Compared with CoCl_(2)group,the number of autolysosomes was significantly reduced the 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.001).As autophagic flux assay result showed,compared with the normal control group,the level of autophagosomes and autolysosomes were significantly risen in CoCl_(2)group(all P<0.001).Compared with the CoCl_(2)group,the level of autophagosomes and autolysosomes were significantly fell down in 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.05).The level of autolysosomes in the LXHY drug-contained serum group was lower than in the blank serum group(P<0.05).Compared with the LXHY drug-contained serum group,the levels of autophagosomes and autolysosomes were significantly risen in the LXHY drug-contained serum combined with the rapamycin group(all P<0.05).As the Q-PCR result showed,compared with the normal control group,the expression of LC3 and Beclin-1 mRNA were significantly reduced in the CoCl_(2)group(all P<0.001).Compared with the CoCl_(2)group,the expression of LC3 mRNA were significantly increased in the 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.001).Beclin-1 mRNA expression was increased significantly(all P<0.001)in the blank serum group and the LXHY drug-contained serum group.And Beclin-1 mRNA expression in the LXHY drug-contained serum group was statistically significant increased than blank serum group(P<0.001).In the LXHY drug-contained serum combined with the rapamycin group,the LC3 and Beclin-1 mRNA expression was reduced significantly compared with the LXHY drug-contained serum group(all P<0.001).Conclusion:The LXHY DCS has the ability to protect the human retinal pigment epithelial cell(ARPE-19)damage under hypoxia through the autophagy pathway.

Autophagy activation aggravates neuronal injury in the hippocampus of vascular dementia rats

It remains unclear whether autophagy affects hippocampal neuronal injury in vascular dementia. In the present study, we investigated the effects of autophagy blockade on hippocampal neuro- nal injury in a rat model of vascular dementia. In model rats, hippocampal CA1 neurons were severely damaged, and expression of the autophagy-related proteins beclin-1, cathepsin B and microtubule-associated protein 1 light chain 3 was elevated compared with that in sham-operated animals. These responses were suppressed in animals that received a single intraperitoneal injection of wortmannin, an autophagy inhibitor, prior to model establishment. The present results confirm that autophagy and autophagy-related proteins are involved in the pathological changes of vascular dementia, and that inhibition of autophagy has neuroprotective effects.

Schwann cells differentiated from skin-derived precursors provide neuroprotection via autophagy inhibition in a cellular model of Parkinson’s disease

Autophagy has been shown to play an important role in Parkinson’s disease.We hypothesized that skin-derived precursor cells exhibit neuroprotective effects in Parkinson’s disease through affecting autophagy.In this study,6-hydroxydopamine-damaged SH-SY5Y cells were pretreated with a culture medium containing skin-derived precursors differentiated into Schwann cells(SKP-SCs).The results showed that the SKP-SC culture medium remarkably enhanced the activity of SH-SY5Y cells damaged by 6-hydroxydopamine,reduced excessive autophagy,increased tyrosine hydroxylase expression,reducedα-synuclein expression,reduced the autophagosome number,and activated the PI3K/AKT/mTOR pathway.Autophagy activator rapamycin inhibited the effects of SKP-SCs,and autophagy inhibitor 3-methyladenine had the opposite effect.These findings confirm that SKP-SCs modulate the PI3K/AKT/mTOR pathway to inhibit autophagy,thereby exhibiting a neuroprotective effect in a cellular model of Parkinson’s disease.This study was approved by the Animal Ethics Committee of Laboratory Animal Center of Nantong University(approval No.S20181009-205)on October 9,2018.

Homeostatic cell cycle and the origin of autophagosomal vesicles

The autophagosomes were identified in the viable cycloheximide (CHX)-treated cells which had an incapacitated translational process and thus disabled synthesis of endoplasmic reticulum (ER)-derived vesicular transporters. They were found devoid of the proteins transported from ER to cell organelles, were unable to fuse with ER, Golgi or mitochondria, and displayed affinity with lysosomes. The analysis of autophagosomes, derived from the CHX cell organelles, revealed that their lipid composition resemble that of the maternal organelle. Thus, the ER-derived autophagosomes were marked with the presence of phosphatidylinositol (PI), Golgi-derived vesicles contained sphingomyelin (SM) and glycosphingolipids (GLL), and the mitochondria-derived autophagosomes contained phosphatidylglycerol (PG) and cardiolipin (CL). The incubation of the vesicles with intact lysosomes afforded their and the lysosome membrane lipids degradation. The analysis of the products derived from incubation of lysosomes and autophagosomes with radiolabeled SM, in the presence and the absence of TritonX100, allowed us to conclude that during autophagosome degradation the lysosomal enzymes are not released to cytosol, and that only lysosomes contain the enzymes degrading membrane lipids. In summary, our findings allowed us to authenticate the vesicles generated in the CHX-treated cells as organelle-specific autophagosomes and to determine that complete cycle of cell restitution and debridement includes intralysosomal degradation of the lysosomal membrane engulfing the autophagosomes vesicles.

Autophagy: a double-edged sword for neuronal survival after cerebral ischemia

Evidence suggests that autophagy may be a new therapeutic target for stroke, but whether acti- vation of autophagy increases or decreases the rate of neuronal death is still under debate. This review summarizes the potential role and possible signaling pathway of autophagy in neuronal survival after cerebral ischemia and proposes that autophagy has dual effects.

Autophagy and ethanol-induced liver injury

The majority of ethanol metabolism occurs in the liver. Consequently, this organ sustains the greatest damage from ethanol abuse. Ethanol consumption disturbs the delicate balance of protein homeostasis in the liver, causing intracellular protein accumulation due to a disruption of hepatic protein catabolism. Evidence indicates that ethanol or its metabolism impairs trafficking events in the liver, including the process of macroautophagy, which is the engulfment and degradation of cytoplasmic constituents by the lysosomal system. Autophagy is an essential, ongoing cellular process that is highly regulated by nutrients, endocrine factors and signaling pathways. A great number of the genes and gene products that govern the autophagic response have been characterized and the major metabolic and signaling pathways that activate or suppress autophagy have been identified. This review describes the process of autophagy, its regulation and the possible mechanisms by which ethanol disrupts the process of autophagic degradation. The implications of autophagic suppression are discussed in relation to the pathogenesis of alcohol-induced liver injury.

Autophagy in mammalian cells

Autophagy is a regulated process for the degradation of cellular components that has been well conserved in eukaryotic cells. The discovery of autophagy-regulating proteins in yeast has been important in understanding this process. Although many parallels exist between fungi and mammals in the regulation and execution of autophagy, there are some important differences. The preautophagosomal structure found in yeast has not been identified in mammals, and it seems that there may be multiple origins for autophagosomes, including endoplasmic reticulum, plasma membrane and mitochondrial outer membrane. The maturation of the phagophore is largely dependent on 5'-AMP activated protein kinase and other factors that lead to the dephosphorylation of mammalian target of rapamycin. Once the process is initiated, the mammalian phagophore elongates and matures into an autophagosome by processes that are similar to those in yeast. Cargo selection is dependent on the ubiquitin conjugation of protein aggregates and organelles and recognition of these conjugates by autophagosomal receptors. Lysosomal degradation of cargo produces metabolites that can be recycled during stress. Autophagy is an impor-tant cellular safeguard during starvation in all eukaryotes; however, it may have more complicated, tissue specific roles in mammals. With certain exceptions, autophagy seems to be cytoprotective, and defects in the process have been associated with human disease.

Involvement of autophagy in alcoholic liver injury and hepatitis C pathogenesis

This review describes the principal pathways of macroautophagy (i.e. autophagy), microautophagy and chaperone-mediated autophagy as they are currently known to occur in mammalian cells. Because of its crucial role as an accessory digestive organ, the liver has a particularly robust autophagic activity that is sensitive to changes in plasma and dietary components. Ethanol consumption causes major changes in hepatic protein and lipid metabolism and both are regulated by autophagy, which is significantly affected by hepatic ethanol metabolism. Ethanol exposure enhances autophagosome formation in liver cells, but suppresses lysosome function. Excessive ethanol consumption synergizes with hepatitis C virus (HCV) to exacerbate liver injury, as alcohol-consuming HCV patients frequently have a longer course of infection and more severe manifestations of chronic hepatitis than abstinent HCV patients. Alcohol-elicited exacerbation of HCV infection pathogenesis is related to modulation by ethanol metabolism of HCV replication. Additionally, as part of this mechanism, autophagic proteins have been shown to regulate viral (HCV) replication and their intracel-lular accumulation. Because ethanol induces autophagosome expression, enhanced levels of autophagic proteins may enhance HCV infectivity in liver cells of alcoholics and heavy drinkers.

Lipid rafts participate in aberrant degradative autophagic-lysosomal pathway of amyloid-beta peptide in Alzheimer's disease

Amyloid-beta peptide is the main component of amyloid plaques, which are found in Alzhei- mer＇s disease. The generation and deposition of amyloid-beta is one of the crucial factors for the onset and progression of Alzheimer＇s disease. Lipid rafts are glycolipid-rich liquid domains of the plasma membrane, where certain types of protein tend to aggregate and intercalate. Lipid rafts are involved in the generation of amyloid-beta oligomers and the formation of amyloid-beta peptides. In this paper, we review the mechanism by which lipid rafts disturb the aberrant deg- radative autophagic-lysosomal pathway of amyloid-beta, which plays an important role in the pathological process of Alzheimer＇s disease. Moreover, we describe this mechanism from the view of the Two-system Theory of fasciology and thus, suggest that lipid rafts may be a new target of Alzheimer＇s disease treatment.

Ultrastructure of human neural stem/progenitor cells and neurospheres

BACKGROUND： Biological and morphological characteristics of neural stem/progenitor cells （NSPCs） have been widely investigated. OBJECTIVE： To explore the ultrastructure of human embryo-derived NSPCs and neurospheres cultivated in vitro using electron microscopy. DESIGN, TIME AND SETTING： A cell biology experiment was performed at the Brain Tumor Laboratory of Soochow University, and Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University between August 2007 and April 2008. MATERIALS： Human fetal brain tissue was obtained from an 8-week-old aborted fetus; serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium was provided by Gibco, USA; scanning electron microscope was provided by Hitachi Instruments, Japan; transmission electron microscope was provided by JEOL, Japan. METHODS： NSPCs were isolated from human fetal brain tissue and cultivated in serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium. Cells were passaged every 5-7 days. After three passages, NSPCs were harvested and used for ultrastructural examination. MAIN OUTCOME MEASURES： Ultrastructural examination of human NSPCs and adjacent cells in neurospheres. RESULTS： Individual NSPCs were visible as spherical morphologies with rough surfaces under scanning electron microscope. Generally, they had large nuclei and little cytoplasm. Nuclei were frequently globular with large amounts of euchromatin and a small quantity of heterochromatin, and most NSPCs had only one nucleolus. The Golgi apparatus and endoplasmic reticulum were underdeveloped; however, autophagosomes were clearly visible. The neurospheres were made up of NSPCs and non-fixiform material inside. Between adjacent cells and at the cytoplasmic surface of apposed plasma membranes, there were vesicle-like structures. Some membrane boundaries with high permeabilities were observed between some contiguous NSPCs in neurospheres, possibly attributable to plasmalemmal fusion between adjacent cells. CONCLUSION： A large number of autophagosomes were observed in NSPCs and gap junctions were visible between adjacent NSPCs.

Autophagy in degenerating axons following spinal cord injury: evidence for autophagosome biogenesis in retraction bulbs

Macroautophagy （here autophagy） is a catabolic mechanism responsible for the degradation of bulk cytoplasm, long-lived proteins and organeUes. During autophagy, the cargos are engulfed by double-membrane structures named phagophores, which expand to form the autophagosomes. Subsequently, these autophagosomes fuse with lysosomes, in which the cytoplasmic cargos are degraded. Autophagy is a constitutive pro- cess, which plays an important role in cellular homeostasis. In primary neurons autophagosome formation occurs continuously and preferentially at the distal end of axons. On the other hand, autophagy is increased by different stresses, and its dysregulation or excessive induction may lead to detrimental effects. Many neurological disorders have been associated with alterations in the autophagic pathway and an increase in autophagy during axonal degeneration was described.

Effect of the Vacuolation of Helicobacter Pylori

Cytotoxic test in vitro combined with cytochemical stain, fluorescent stain, transmission electronmicrograph was used to study the vacuolated effect by helicobacter pylori (H.pylori) (Toxin+) and its pathological mechanism. 78..26 % patients with peptic ulcer associated with H.pylori was infected with H.pylori (Toxin+), while 42.86 % patients with gastritis was infected with H.pylori (Toxin+). It was positive in vacuole with acridine orange and acid phosphatase stain. Transmission electronmicrograph of vacuole revealed the presence of abounding membrane. There was a closed relationship between infection with H.pylori (Toxin+) and peptic ulcer disease. The vacuole induced by H.pylori (Toxin+) was autophagosome, which was pathological phenomenon induced by toxin.

Dual Roles of CD38 in Autophagy

CD38 is a versatile, ubiquitously expressed protein that was identified as a multifunctional enzyme. Recently, cumulating evidence has suggested that CD38 is involved in autophagy, which is an evolutionarily conserved lysosomal degradation and recycling system. Acting as a enzyme, CD38 utilizes nicotinamide adenine dinucleotide phosphate (NADP) to synthesize nicotinic acid adenine dinucleotide phosphate (NAADP), which acts as a key messenger for Ca2+-mobilizing in lysosome by targeting two-pore channels (TPCs) or transient receptor potential mucolipins (TRPMLs). Multiple studies have indicated that CD38 is involved in autophagy by modulating intracellular Ca2+ signaling. However, the control of autophagy by CD38 signaling is the subject of two contrary views. The autophagosomes trafficking and fusion with lysosomes to form autolysosomes are crucial steps in autophagy. On the one hand, the avail-able evidence indicates that lysosome trafficking and fusion to autophagosomes is positively modulated by CD38. On the other hand, overexpression of TPC2, which is positively modulated by CD38, was shown to promote the accumulation of autophagosomes, thus suppress autophagy. This review will reveal the interesting contrary dual roles of CD38 in autophagy, and critical insight into the molecular mechanisms of CD38 in autophagy regulation.

Ultrasound-activated in situ click chemistry to trigger autophagosome tracking for enhanced autophagy blockade and synergistic cancer therapy

The blockade of cytoprotective autophagy has been demonstrated to effectively enhance the efficacy of sonodynamic therapy(SDT).However,the limited recognition of antiautophagy agents for autophagosomes impedes the clinical application of autophagy inhibition.To efficiently deliver hydroxychloroquine(HCQ),an autophagy inhibitor,to autophagosomes,we utilized a strategy based on in situ click chemistry between sulfhydryl(-SH)and maleimide(Mal)groups to trigger autophagosomes tracking and suppress tumor growth synergistically.A cascade nanoreactor was synthesized by encapsulating Mal-modified HCQ(MHCQ)into a manganese porphyrin-based metal-organic framework with sonosensitizer properties,followed by poly(ethylene glycol)ylated liposomal membrane coating.After ultrasound irradiation,SDT-induced apoptotic cells released damaged proteins with free-SH groups,which MHCQ rapidly captured in situ via a Malthiol click reaction.When autophagosomes actively wrapped damaged proteins for detoxification,they simultaneously internalized HCQ anchored on proteins.In this scenario,antiautophagy drugs could actively track intracellular autophagosomes instead of undergoing passive diffusion in the cytosol.The interaction between HCQ and autophagic vesicles was greatly enhanced,which strengthened the blocking efficiency of autophagy and resulted in complete cell death.Overall,this study with smart design provides a promising strategy for improving intracellular targeted delivery to autophagosomes,thereby enhancing antitumor therapy.

Stay in touch with the endoplasmic reticulum

The endoplasmic reticulum(ER),which is composed of a continuous network of tubules and sheets,forms the most widely distributed membrane system in eukaryotic cells.As a result,it engages a variety of organelles by establishing membrane contact sites(MCSs).These contacts regulate organelle positioning and remodeling,including fusion and fission,facilitate precise lipid exchange,and couple vital signaling events.Here,we systematically review recent advances and converging themes on ER-involved organellar contact.The molecular basis,cellular influence,and potential physiological functions for ER/nuclear envelope contacts with mitochondria,Golgi,endosomes,lysosomes,lipid droplets,autophagosomes,and plasma membrane are summarized.

The late stage of autophagy: cellular events and molecular regulation

Autophagy is an intracellular degradation system that delivers cytoplasmic contents to the lysosome for degradation.It is a“self-eating”process and plays a“house-cleaner”role in cells.The complex process consists of several sequential steps-induction,autophagosome formation,fusion of lysosome and autophagosome,degradation,efflux transportation of degradation products,and autophagic lysosome reformation.In this review,the cellular and molecular regulations of late stage of autophagy,including cellular events after fusion step,are summarized.

Membrane dynamics of ATG4B and LC3 in autophagosome formation

The biogenesis of autophagosomes provides the basis for macroautophagy to capture and degrade intracellular cargoes.Binding of the autophagy-related protein ATG8/LC3 to autophagic membranes is essential to autophagosome formation,which involves the specific and dynamic processing of ATG8/LC3 by cysteine protease ATG4.However,to date,the mechanism whereby ATG4 is recruited to the membranes,the interaction of ATG4 and ATG8/LC3 on the membranes,and its role in the growth of phagophore are not completely understood.Here,we used fluorescence recovery after photobleaching to monitor the turnover of GFP-tagged ATG4B and LC3B in living animal cells.The data show that ATG4B localizes to early autophagic membranes in an LC3B-dependent manner.During autophagy,ATG4B and LC3B undergo rapid cytosol/isolation membrane exchange but not at the cytosol/completed autophagosome.In addition,ATG4B activity controls the efficiency of autophagosome formation by impacting the membrane binding/dissociation of LC3B.These data suggest that ATG4 and LC3 play interdependent roles in the formation of autophagosomes.

Hepatic COX1 loss leads to impaired autophagic flux and exacerbates nonalcoholic steatohepatitis

The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis(NASH)remain largely unknown.We aimed to elucidate the roles of hepatic cyclooxygenase 1(COX1)in autophagy and the pathogenesis of diet-induced steatohepatitis in mice.Human nonalcoholic fatty liver disease(NAFLD)liver samples were used to examine the protein expression of COX1 and the level of autophagy.Cox1^(Δhepa)mice and their wildtype littermates were generated and fed with 3 different NASH models.We found that hepatic COX1 expression was increased in patients with NASH and diet induced NASH mice models accompanied by impaired autophagy.COX1 was required for basal autophagy in hepatocytes and liver specific COX1 deletion exacerbated steatohepatitis by inhibiting autophagy.Mechanistically,COX1 directly interacted with WD repeat domain,phosphoinositide interacting 2(WIPI2),which was crucial for autophagosome maturation.Adeno-associated virus(AAV)-mediated rescue of WIPI2 reversed the impaired autophagic flux and improved NASH phenotypes in Cox1^(Δhepa)mice,indicating that COX1 deletion-mediated steatohepatitis was partially dependent on WIPI2-mediated autophagy.In conclusion,we demonstrated a novel role of COX1 in hepatic autophagy that protected against NASH by interacting with WIPI2.Targeting the COX1 WIPI2 axis may be a novel therapeutic strategy for NASH.

Cytoplasmic YAP1-mediated ESCRT-III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

Background:Nuclear Yes1-associated transcriptional regulator(YAP1)promotes tumor progression.However,the function of cytoplasmic YAP1 in breast cancer cells and its impact on the survival of breast cancer patients remain unclear.Our research aimed to explore the biological function of cytoplasmic YAP1 in breast cancer cells and the possibility of cytoplasmic YAP1 as a predictive marker of breast cancer survival.Methods:We constructed cell mutant models,including NLS-YAP15SA(nuclear localized),YAP1S94A(incapable of binding to the TEA domain transcription factor family)and YAP1S127D(cytoplasmic localized),and used Cell Counting Kit-8(CCK-8)assays,5-ethynyl-2’-deoxyuridine(EdU)incorporation assays,and Western blotting(WB)analysis to detect cell proliferation and apoptosis.The specific mechanism of cytoplasmic YAP1-mediated endosomal sorting complexes required for transport III(ESCRT-III)assembly was studied by co-immunoprecipitation,immunofluorescence staining,and WB analysis.Epigallocatechin gallate(EGCG)was used to simulate YAP1 retention in the cytoplasm in in vitro and in vivo experiments to study the function of cytoplasmic YAP1.YAP1 binding to NEDD4-like E3 ubiquitin protein ligase(NEDD4L)was identified using mass spectrometry and was verified in vitro.Breast tissue microarrays were used to analyze the relationship between cytoplasmic YAP1 expression and the survival of breast cancer patients.Results:YAP1 was mainly expressed in the cytoplasm in breast cancer cells.Cytoplasmic YAP1 promoted autophagic death of breast cancer cells.Cytoplasmic YAP1 bound to the ESCRT-III complex subunits charged multivesicular body protein 2B(CHMP2B)and vacuolar protein sorting 4 homolog B(VPS4B),promoting assembly of CHMP2B-VPS4B and activating autophagosome formation.EGCG retained YAP1 in the cytoplasm,promoting the assembly of CHMP2B-VPS4B to promote autophagic death of breast cancer cells.YAP1 bound to NEDD4L,and NEDD4L mediated ubiquitination and degradation of YAP1.Breast tissue microarrays revealed that high levels of cytoplasmic YAP1 were beneficial to the survival of breast cancer patients.Conclusions:Cytoplasmic YAP1 mediated autophagic death of breast cancer cells by promoting assembly of the ESCRT-III complex;furthermore,we established a new breast cancer survival prediction model based on cytoplasmic YAP1 expression.