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Macroautophagy/autophagy is a cellular catabolic process that results in lysosome-mediated recycling of organelles and protein aggregates, as well as the destruction of intracellular pathogens. Its role in the maintenance of the intestinal epithelium is of particular interest, as several autophagy-related genes have been associated with intestinal disease. Autophagy and its regulatory mechanisms are involved in both homeostasis and repair of the intestine, supporting intestinal barrier function in response to cellular stress through tight junction regulation and protection from cell death. Furthermore, a clear role has emerged for autophagy not only in secretory cells but also in intestinal stem cells, where it affects their metabolism, as well as their proliferative and regenerative capacity. Here, we review the physiological role of autophagy in the context of intestinal epithelial maintenance and how genetic mutations affecting autophagy contribute to the development of intestinal disease. Abbreviations: AKT1S1: AKT1 substrate 1; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; APC: APC regulator of WNT signaling pathway; ATF6: activating transcription factor 6; ATG: autophagy related; atg16l1[ΔIEC] mice: mice with a specific deletion of Atg16l1 in intestinal epithelial cells; ATP: adenosine triphosphate; BECN1: beclin 1; bsk/Jnk: basket; CADPR: cyclic ADP ribose; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CD: Crohn disease; CDH1/E-cadherin: cadherin 1; CF: cystic fibrosis; CFTR: CF transmembrane conductance regulator; CGAS: cyclic GMP-AMP synthase; CLDN2: claudin 2; CoPEC: colibactin-producing E. coli; CRC: colorectal cancer; CYP1A1: cytochrome P450 family 1 subfamily A member 1; DC: dendritic cell; DDIT3: DNA damage inducible transcript 3; DEPTOR: DEP domain containing MTOR interacting protein; DSS: dextran sulfate sodium; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; HMGB1: high mobility group box 1; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; IBD: inflammatory bowel disease; IEC: intestinal epithelial cell; IFN: interferon; IFNG/IFNγ:interferon gamma; IL: interleukin; IRGM: immunity related GTPase M; ISC: intestinal stem cell; LGR5: leucine rich repeat containing G protein-coupled receptor 5; LRRK2: leucine rich repeat kinase 2; MAP1LC3A/LC3: microtubule associated protein 1 light chain 3 alpha; MAPK/JNK: mitogen-activated protein kinase; MAPK14/p38 MAPK: mitogen-activated protein kinase 14; MAPKAP1: MAPK associated protein 1; MAVS: mitochondrial antiviral signaling protein; miRNA: microRNA; MLKL: mixed lineage kinase domain like pseudokinase; MLST8: MTOR associated protein, LST8 homolog; MNV: murine norovirus; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NLRP: NLR family pyrin domain containing; NOD: nucleotide binding oligomerization domain containing; NRBF2: nuclear receptor binding factor 2; OPTN: optineurin; OXPHOS: oxidative phosphorylation; P: phosphorylation; Patj: PATJ crumbs cell polarity complex component; PE: phosphatidyl-ethanolamine; PI3K: phosphoinositide 3-kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PPARG: peroxisome proliferator activated receptor gamma; PRR5: proline rich 5; PRR5L: proline rich 5 like; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RER: rough endoplasmic reticulum; RHEB: Ras homolog, MTORC1 binding; RICTOR: RPTOR independent companion of MTOR complex 2; RIPK1: receptor interacting serine/threonine kinase 1; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SH3GLB1: SH3 domain containing GRB2 like, endophilin B1; SNP: single-nucleotide polymorphism; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; STING1: stimulator of interferon response cGAMP interactor 1; TA: transit-amplifying; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; TGM2: transglutaminase 2; TJ: tight junction; TJP1/ZO1: tight junction protein 1; TNBS: 2,4,6-trinitrobenzene sulfonic acid; TNF/TNFα: tumor necrosis factor; Tor: target of rapamycin; TRAF: TNF receptor associated factor; TRIM11: tripartite motif containing 11; TRP53: transformation related protein 53; TSC: TSC complex subunit; Ub: ubiquitin; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; USO1/p115: USO1 vesicle transport factor; UVRAG: UV radiation resistance associated; WIPI: WD repeat domain, phosphoinositide interacting; WNT: WNT family member; XBP1: X-box binding protein 1; ZFYVE1/DFCP1: zinc finger FYVE–type containing 1.

Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions. Abbreviations: ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2’-deoxycytidine; BafA1: bafilomycin A1; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle’s balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.

Synaptosomal associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), is a ubiquitously expressed protein that is generally involved in fusion of the plasma membrane and secretory or endosomal recycling vesicles during several types of exocytosis. SNAP23 is expressed in phagocytes, such as neutrophils, macrophages, and dendritic cells, and functions in both exocytosis and phagocytosis. This review focuses on the function of SNAP23 in immunoglobulin G Fc receptor-mediated phagocytosis by macrophages. SNAP23 and its partner SNAREs mediate fusion of the plasma membrane with intracellular organelles or vesicles to form phagosomes as well as the fusion of phagosomes with endosomes or lysosomes to induce phagosome maturation, characterized by reactive oxygen species production and acidification. During these processes, SNAP23 function is regulated by phosphorylation. In addition, microtubule-associated protein 1A/1B light chain 3 (LC3)-associated phagocytosis, which tightly promotes or suppresses phagosome maturation depending on the foreign target, requires SNAP23 function. SNAP23 that is enriched on the phagosome membrane during LC3-associated phagocytosis may be phosphorylated or dephosphorylated, thereby enhancing or inhibiting subsequent phagosome maturation, respectively. These findings have increased our understanding of the SNAP23-associated membrane trafficking mechanism in phagocytes, which has important implications for microbial pathogenesis and innate and adaptive immune responses.

Neural tube defects (NTDs) are severe congenital malformations caused by failed neural tube closure. Recently, autophagy is revealed to play a vital role in neuroepithelium development and neurulation. Autophagy and beclin 1 regulator 1 (Ambra1) is a crucial regulator of autophagy initiation, and its deficiency in mice leads to exencephaly and/or spina bifida. However, the genetic contribution of AMBRA1 to the etiology of human NTDs remains unknown. In this study, we identified five rare missense mutations of AMBRA1 in 352 NTDs cases, which were absent in 224 matched controls. Western blotting and fluorescence puncta counting for MAP1LC3A/LC3 in HEK293T cells suggested that four of the mutations (AMBRA1 p.Thr80Met, p.Leu274Phe, p.Ser743Phe, and p.Met884Val) affected autophagy initiation to various extents. Furthermore, these four mutations also displayed loss-of-function effects compared with wild-type AMBRA1 when we injected messenger RNA (mRNA) to overexpress or rescue ambra1a-morpholino oligos (MO) knockdown in zebrafish. It is intriguing that trehalose, a natural disaccharide, could rescue ambra1a-MO knockdown in a dose-dependent manner independently or together with AMBRA1 mRNA. Taken together, our findings suggest that rare mutations of the autophagy regulator gene AMBRA1 may contribute to the etiology of human neural tube defects, and trehalose is a promising treatment for a subset of NTDs caused by autophagy impairment.

Cancer cell plasticity generates heterogeneous oncogenic subpopulations in tumors. How macroautophagy/autophagy, a catabolic system required for sustaining cell homeostasis, affects cancer cell plasticity, remains elusive. In this study, we report that MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha), a key molecule in autophagy, is negatively associated with histological grade and distant metastasis of lung cancer. This is achieved in part, if not all, by maintaining the mitochondria and energy homeostasis to meet the proliferation demand of lung cancer cells driven by SOX2 (SRY-box transcription factor 2) signaling. Basal autophagy is preferentially active in SOX2-positive lung cancer cells with high-proliferative and low-invasive properties. The high-proliferative cancer cells exhibit higher oxygen consumption rate (OCR), elevated reactive oxygen species (ROS), and profound fragmented mitochondrial patterns compared to their high-invasive counterparts. SOX2 expression promotes LC3A expression and enhances proliferation but attenuates invasion in lung cancer cells. LC3A silencing enriches cells harboring low-proliferative and high-invasive features, concomitant with decreased OCR and ROS levels and reduced expression of SOX2. Our findings provide novel insights into how basal autophagy cross talks with SOX2 proliferation signaling to regulate mitochondrial metabolism and determines cancer cell plasticity with an impact on lung tumor progression. ATG14: autophagy related 14; CDH2: cadherin 2; ChIP-qPCR: chromatin immunoprecipitation quantitative polymerase chain reaction; CQ: chloroquine; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; EPCAM: epithelial cell adhesion molecule; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NDUFV2: NADH:ubiquinone oxidoreductase core subunit V2; OCR: oxygen consumption rate; ROS: reactive oxygen species; RT-qPCR: reverse-transcriptase quantitative polymerase chain reaction; SC: scrambled control; shRNA: short hairpin RNA; SNAI2: snail family transcriptional repressor 2; SOX2: SRY-box transcription factor 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-β: transforming growth factor beta; TOMM20: translocase of outer mitochondrial membrane 20; ZEB1: zinc finger E-box binding homeobox 1

Selective degradation of protein aggregates by macroautophagy/autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like proteins, NEDD8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3-interacting region which bind to NEDD8 and LC3 respectively. Both NEDD8 and HYPK are positive modulators of basal and proteotoxicity-induced autophagy, leading to protection of cells from protein aggregates, such as aggregates of mutant HTT exon 1. Thus, we propose an indispensable and additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress. Abbreviations: ATG5, autophagy related 5; ATG12, autophagy related 12; ATG14, autophagy related 14; BECN1, beclin 1; CBL, casitas B-lineage lymphoma; CBLB, Cbl proto-oncogene B; GABARAP, GABA type A receptor-associated protein; GABARAPL1, GABA type A receptor associated protein like 1; GABARAPL2, GABA type A receptor associated protein like 2; GFP, green fluorescent protein; HTT, huntingtin; HTT97Q exon 1, huntingtin 97-glutamine exon 1; HUWE1, HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; HYPK, huntingtin interacting protein K; IgG, immunoglobulin G; IMR-32, Institute for Medical Research-32; KD, knockdown; Kd, dissociation constant; LAMP1, lysosomal associated membrane protein 1; LIR, LC3 interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAP1LC3A/LC3A, microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MARK1, microtubule affinity regulating kinase 1; MARK2, microtubule affinity regulating kinase 2; MARK3, microtubule affinity regulating kinase 3; MARK4, microtubule affinity regulating kinase 4; MCF7, Michigan Cancer Foundation-7; MTOR, mechanistic target of rapamycin kinase; NAE1, NEDD8 activating enzyme E1 subunit 1; NBR1, NBR1 autophagy cargo receptor; NEDD8, NEDD8 ubiquitin like modifier; Ni-NTA, nickel-nitrilotriacetic acid; NUB1, negative regulator of ubiquitin like proteins 1; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; PolyQ, poly-glutamine; PSMD8, proteasome 26S subunit, non-ATPase 8; RAD23A, RAD23 homolog A, nucleotide excision repair protein; RAD23B, RAD23 homolog B, nucleotide excision repair protein; RFP, red fluorescent protein; RPS27A, ribosomal protein S27a; RSC1A1, regulator of solute carriers 1; SNCA, synuclein alpha; SIK1, salt inducible kinase 1; siRNA, small interfering ribonucleic acid; SOD1, superoxide dismutase 1; SPR, surface plasmon resonance; SQSTM1, sequestosome 1; SUMO1, small ubiquitin like modifier 1; TAX1BP1, Tax1 binding protein 1; TDRD3, tudor domain containing 3; TNRC6C, trinucleotide repeat containing adaptor 6C; TOLLIP, toll interacting protein; TUBA, tubulin alpha; TUBB, tubulin beta class I; UBA, ubiquitin-associated; UBA1, ubiquitin like modifier activating enzyme 1; UBA5, ubiquitin like modifier activating enzyme 5; UBAC1, UBA domain containing 1; UBAC2, UBA domain containing 2; UBAP1, ubiquitin associated protein 1; UBAP2, ubiquitin associated protein 2; UBASH3B, ubiquitin associated and SH3 domain containing B; UBD/FAT10, ubiquitin D; UBE2K, ubiquitin conjugating enzyme E2 K; UBLs, ubiquitin-like proteins; UBL7, ubiquitin like 7; UBQLN1, ubiquilin 1; UBQLN2, ubiquilin 2; UBQLN3, ubiquilin 3; UBQLN4, ubiquilin 4; UBXN1, UBX domain protein 1; ULK1, unc-51 like autophagy activating kinase 1; URM1, ubiquitin related modifier 1; USP5, ubiquitin specific peptidase 5; USP13, ubiquitin specific peptidase 13; VPS13D, vacuolar protein sorting 13 homolog D.

Background Autophagy is closely related to the progression of breast cancer. The aim at this study is to establish a prognostic-related model comprised of hub autophagy genes (AGs) to assess patient prognosis. Simultaneously, the model can guide clinicians to make up individualized strategies and stratify patients aged 40–60 years based on risk level. Methods The hub AGs were identified with univariate COX regression and LASSO regression. The functions and alterations of these selected AGs were analyzed as well. Moreover, the multivariate COX regression and correlation analysis between hub AGs and clinicopathological parameters were done. Results Totally, 33 prognostic-related AGs were obtained from the univariate COX regression (P P P P P Conclusions Our prognostic model had the relatively robust predictive ability on prognosis for patients aged 40–60 years. If the stage was added into the prognostic model, the predictive ability would be more powerful.

Background: Autophagy is reported as a survival or death-promoting pathway that is highly debatable in different kinds of cancer. Here, we examined the co-effect of cold atmospheric plasma (CAP) and silymarin nanoemulsion (SN) treatment on G-361 human melanoma cells via autophagy induction. Methods: The temperature and pH of the media, along with the cell number, were evaluated. The intracellular glucose level and PI3K/mTOR and EGFR downstream pathways were assessed. Autophagy-related genes, related transcriptional factors, and autophagy induction were estimated using confocal microscopy, flow cytometry, and ELISA. Results: CAP treatment increased the temperature and pH of the media, while its combination with SN resulted in a decrease in intracellular ATP with the downregulation of PI3K/AKT/mTOR survival and RAS/MEK transcriptional pathways. Co-treatment blocked downstream paths of survival pathways and reduced PI3K (2 times), mTOR (10 times), EGFR (5 times), HRAS (5 times), and MEK (10 times). CAP and SN co-treated treatment modulates transcriptional factor expressions (ZKSCAN3, TFEB, FOXO1, CRTC2, and CREBBP) and specific genes (BECN-1, AMBRA-1, MAP1LC3A, and SQSTM) related to autophagy induction. Conclusion: CAP and SN together activate autophagy in G-361 cells by activating PI3K/mTOR and EGFR pathways, expressing autophagy-related transcription factors and genes.

Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4B(C74A)). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4B(C74A), compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment. Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short

Atrazine (ATR), a bio accumulative herbicide is frequently used in agriculture to control unwanted weeds. Due to continuous application, atrazine persists in the environment and causes deleterious impacts including neurotoxicity, hepatotoxicity, and gut microbiota disorders. Therefore, this study for the first time reports the variation in the gut microbiota, induction of process of apoptosis and autophagy in mice induced by ATR. Results indicated that TUNEL-positive hepatocytes suggestive of apoptosis were increased in livers of different experimental mice. Results on metabolic analysis in liver tissues indicated an overall change in seventy-six metabolites particularly Uridine 5'-diphosphate, Propenoylcarnitine and Chinenoside V resulting in generation of energy-related metabolic disorders and imbalance of oxidation/autoxidation status. Results on gut microbiome inquisition showed that ATR changed the richness and diversity of gut microbiota of mice and number of Firmicutes. Moreover, results also revealed that ATR induced apoptosis via disruption of apoptotic (Bax, Bcl2, and Casp3) and autophagy (LC3/Map1lc3a, Beclin 1/Becn1 and P62/Sqstm1) genes. Results of our experimental study confirmed that changes in gut microbiota play a significant role in process of gut immune regulation and inflammation via different metabolites. In conclusion, the findings of our study provide a new idea for the involvement of mechanisms of detoxification in liver and inquisition of gut microbiota plays crucial role in regulation of physiological activities through liver-gut axis to mitigate toxic effects in animals.

Background: Autophagy is a cell survival mechanism, which has been implicated in atherogenesis in mouse models by studying core autophagy machinery proteins using knock-out models. MAP1LC3A and MAP1LC3B play a key role in autophagy activity and have been implicated as prognostic factors in several human cancers. However, data on the involvement of autophagy in human atherosclerotic disease and plaque vulnerability are still sparse and completely lacking with regards to the involvement of MAP1LC3. Approach and Results: Using two independent biobanks of human carotid atherosclerotic plaques, we observe that MAP1LC3A mRNA and protein levels are decreased in plaques from patients with symptomatic disease compared to asymptomatic. Notably, MAP1LC3A mRNA levels strongly correlate with vascular smooth muscle cell markers, while MAP1LC3B does not. In in vivo models, we show that MAP1LC3A mRNA is downregulated during the progression of atherosclerosis in hypercholesterolemic mice as well as upon hyperplasia induced by balloon-injury in rats. In vitro, we show that ablation of MAP1LC3A in human carotid VSMC induces a transient compensatory increase in myocardin, a master regulator of vascular smooth muscle cell phenotypic switch. Conclusions: Taken together, these results demonstrate that reduced MAP1LC3A expression is a relevant marker of vulnerable plaque phenotype, suggesting an impact on vascular smooth muscle cell biology in the context of atherogenesis.

Effects of Taraxaci Herba on Cell Death in Breast Cancer Cells Kook-Jang Seo, Kyung-Mi Park, Sung-Hee Joh, Seung-Jeong Yang College of Korean Medicine, Graduate School of Dong-Shin University Objectives: This study was designed to investigate the effects of Taraxaci Herba (TH) on cell death in breast cancer cells. Methods: In this experiment, the effects of TH on proliferation rates, cell morphology and growth pattern, intracellular reactive oxygen species (ROS) production. In addition, the effects on nuclear condensation, fragmentation and formation of acidic vesicular organelles (AVO) in MCF-7 cells were also investigated. Finally, autophagy related with protein was observed by using western blot method. Results: TH inhibited proliferation of MCF-7 cells, TH elevated intracellular ROS levels significantly. Treatment with TH did not affect nuclear morphologies such as condensation or fragmentation. On the other hand, TH treatment effectively induced AVO. Finally, one of autophagy related with protein, Microtubule-associated proteins 1A/1B light chain 3A (MAP1LC3A, LC3) level was elevated by treatment with TH. Conclusions: These data indicate that TH is able to be used for patient with breast cancer and mechanisms are involved in autophagy through ROS generation.

Recent mass mortality outbreaks around the world in Pacific oysters, Crassostrea gigas, have seriously affected the aquaculture economy. Although the causes for these mortality outbreaks appear complex, infectious agents are involved. Two pathogens are associated with mass mortality outbreaks, the virus ostreid herpesvirus 1 (OsHV-1) and the bacterium Vibrio aestuarianus. Here we describe the interactions between these 2 pathogens and autophagy, a conserved intracellular pathway playing a key role in innate immunity. We show for the first time that autophagy pathway is present and functional in Pacific oysters and plays an important role to protect animals from infections. This study contributes to better understand the innate immune system of Pacific oysters.

Aggresomes are transient microtubule-dependent inclusion bodies that sequester misfolded proteins and are ultimately removed by autophagy. Here we report the generation of a choroid plexus carcinoma cell line; Children’s Cancer Hospital Egypt (CCHE)-45, which is characterized by the constitutive formation of aggresomes. When examining the autophagy pathway as the main route for aggresomes clearance, CCHE-45 cells displayed increased autophagy flux mediated by MAP1LC3B. MAP1LC3A-Variant1 gene expression was silenced by promoter methylation. Restoring MAP1LC3A-Variant1 expression resulted in the formation of MAP1LC3A positive autophagosmes and the disruption of the aggresomes' vimentin cage independent of MAP1LC3B positive autophagosomes. Our data supports the notion that basal quality control autophagy and vimentin cage clearance in CCHE-45 are mediated by MAP1LC3A. Hence we propose that absence of MAP1LC3A disrupts the autophagic pathway and leads to the failure of aggresome vimentin cage degradation. Consequently, this could represent a targetable pathway in autophagy-dependent cancers.

Mechanistic target of rapamycin (mTOR) functions as a key homeostatic regulator of cell growth and orchestrates whether anabolic or catabolic reactions are favoured. mTOR complex 1 (mTORC1) manages multiple biosynthetic pathways and promotes cell growth when nutrients are in plentiful supply. Many advances have been made over the last decade on nutrient sensing centred on mTORC1. Recent research reveals that mTORC1 maintains nutrient homeostasis through lysosomal biogenesis and autophagic processes. Cells utilise autophagy to recycle damaged or unwanted organelles and macromolecules and in so doing, generate energy and recover precursor building blocks necessary for normal growth. It is clear that mTOR and autophagy are closely integrated within cells, where defects in signalling through both pathways are known to drive the onset of a range of human diseases, such as cancer and neurodegenerative disease. This review focuses on the dynamic signalling interplay between mTOR and autophagy, which is governed by a core set of proteins that sense nutrients at lysosomal membranes.

Summary Autophagy promotes tumor growth by generating nutrients from the degradation of intracellular structures. Here we establish, using shRNAs, a dominant-negative mutant, and a pharmacologic inhibitor, mefenamic acid (MFA), that the Transient Receptor Potential Melastatin 3 (TRPM3) channel promotes the growth of clear cell renal cell carcinoma (ccRCC) and stimulates MAP1LC3A (LC3A) and MAP1LC3B (LC3B) autophagy. Increased expression of TRPM3 in RCC leads to Ca 2+ influx, activation of CAMKK2, AMPK, and ULK1, and phagophore formation. In addition, TRPM3 Ca 2+ and Zn 2+ fluxes inhibit miR-214, which directly targets LC3A and LC3B. The von Hippel-Lindau tumor suppressor (VHL) represses TRPM3 directly through miR-204 and indirectly through another miR-204 target, Caveolin 1 (CAV1).

The spliceosome is a large ribonucleoprotein complex that guides pre-mRNA splicing in eukaryotic cells. Here, we determine whether the spliceosome could constitute an attractive therapeutic target in cancer. Analysis of gene expression arrays from lung, breast, and ovarian cancers datasets revealed that several genes encoding components of the core spliceosome composed of a heteroheptameric Sm complex were overexpressed in malignant disease as compared with benign lesions and could also define a subset of highly aggressive breast cancers. siRNA-mediated depletion of SmE (SNRPE) or SmD1 (SNRPD1) led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells. SNRPE or SNRPD1 depletion did not lead to apoptotic cell death but autophagy, another form of cell death. Indeed, induction of autophagy was revealed by cytoplasmic accumulation of autophagic vacuoles and by an increase in both LC3 (MAP1LC3A) protein conversion and the amount of acidic autophagic vacuoles. Knockdown of SNRPE dramatically decreased mTOR mRNA and protein levels and was accompanied by a deregulation of the mTOR pathway, which, in part, explains the SNRPE-dependent induction of autophagy. These findings provide a rational to develop new therapeutic agents targeting spliceosome core components in oncology. Cancer Res; 73(7); 2247–58. ©2013 AACR.

The epidermal growth factor receptor (EGFR) is a well-studied receptor-tyrosine kinase that serves vital roles in regulation of organ development and cancer progression. EGFR not only exists on the plasma membrane, but also widely expressed in the nucleus, endosomes, and mitochondria. Most recently, several lines of evidences indicated that autophagy is regulated by EGFR in kinase-active and -independent manners. In this review, we summarized recent advances in our understanding of the functions of different subcellularly located EGFR on autophagy. Specifically, plasma membrane- and cytoplasm-located EGFR (pcEGFR) acts as a tyrosine kinase to regulate autophagy via the PI3K/AKT1/mTOR, RAS/MAPK1/3, and STAT3 signaling pathways. The kinase-independent function of pcEGFR inhibits autophagy by maintaining SLC5A1-regulated intracellular glucose level. Endosome-located EGFR phosphorylates and inhibits Beclin1 to suppress autophagy, while kinase-independent endosome-located EGFR releases Beclin1 from the Rubicon-Beclin1 complex to increase autophagy. Additionally, the nuclear EGFR activates PRKDC/PNPase/MYC signaling to inhibit autophagy. Although the role of mitochondria-located EGFR in autophagy is largely unexplored, the production of ATP and reactive oxygen species mediated by mitochondrial dynamics is most likely to influence autophagy.

FYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general.

The cellular stress response autophagy has been implicated in various diseases including neuro-degeneration and cancer. The role of autophagy in cancer is not clearly understood and both tumour promoting and tumour suppressive effects of autophagy have been reported, which complicates the design of therapeutic strategies based on targeting the autophagy pathway. Here, we have systematically analyzed gene expression data for 47 autophagy genes for deletions, amplifications and mutations in various cancers. We found that several cancer types have frequent autophagy gene amplifications, whereas deletions are more frequent in prostate adenocarcinomas. Other cancer types such as glioblastoma and thyroid carcinoma show very few alterations in any of the 47 autophagy genes. Overall, individual autophagy core genes are altered at low frequency in cancer, suggesting that cancer cells require functional autophagy. Some autophagy genes show frequent single base mutations, such as members of the ULK family of protein kinases. Furthermore, we found hotspot mutations in the arginine-rich stretch in MAP1LC3A resulting in reduced cleavage of MAP1LC3A by ATG4B both in vitro and in vivo, suggesting a functional implication of this gene mutation in cancer development.

Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy.

Autophagy is a self-degradation mechanism by which cells recycle their own cytoplasmic constituents. It has been claimed that, under certain conditions, such a process may be associated with tumor progression. In this study, the autophagic activity was investigated in a series of 99 uveal melanomas after immunohistochemical staining for the autophagy-associated proteins MAP1LC3A and BECN1, most commonly known as LC3A and Beclin 1, respectively. These were assessed in parallel with the hypoxia-inducible factor 1α (HIF1A) and its downstream protein lactate dehydrogenase 5 (composed by five LDHA subunits). Increased autophagic reactivity, detected by MAP1LC3A or BECN1, was associated with intense pigmentation and tumor hypoxia. Uveal melanomas with extensive overexpression of BECN1 or those with underexpression of this protein were associated with the worst prognosis, but the former manifested metastases much earlier than the latter; only 58% of patients with extensive BECN1 overexpression were alive at 4 years, compared with 80% of patients with underexpressed patterns. It is concluded that autophagy is commonly upregulated in uveal melanomas, and may be associated with hypoxia and intense pigmentation. There is a strong association between extensive BECN1 overexpression and early metastases/poor prognosis, and between underexpression of this protein and late metastases/better prognosis.

Research highlights: {yields} We investigated the effect 6 Gy of WBI on the autophagic machinery of normal mouse lung. {yields} Irradiation induces dysfunction of the autophagic machinery in normal lung, characterized by decreased transcription of the LC3A/Beclin-1 mRNA and accumulation of the LC3A, and p62 proteins. {yields} The membrane bound LC3A-II protein levels increased in the cytosolic fraction (not in the pellet), contrasting the patterns noted after starvation-induced autophagy. {yields} Administration of amifostine, reversed all the LC3A and p62 findings, suggesting protection of the normal autophagic function. -- Abstract: Purpose: The effect of ionizing irradiation on the autophagic response of normal tissues is largely unexplored. Abnormal autophagic function may interfere the protein quality control leading to cell degeneration and dysfunction. This study investigates its effect on the autophagic machinery of normal mouse lung. Methods and materials: Mice were exposed to 6 Gy of whole body {gamma}-radiation and sacrificed at various time points. The expression of MAP1LC3A/LC3A/Atg8, beclin-1, p62/sequestosome-1 and of the Bnip3 proteins was analyzed. Results: Following irradiation, the LC3A-I and LC3A-II protein levels increased significantly at 72 h and 7 days. Strikingly, LC3A-II protein was increased (5.6-fold at 7 days; p < 0.001) only in the cytosolic fraction, butmore » remained unchanged in the membrane fraction. The p62 protein, was significantly increased in both supernatant and pellet fraction (p < 0.001), suggesting an autophagosome turnover deregulation. These findings contrast the patterns of starvation-induced autophagy up-regulation. Beclin-1 levels remained unchanged. The Bnip3 protein was significantly increased at 8 h, but it sharply decreased at 72 h (p < 0.05). Administration of amifostine (200 mg/kg), 30 min before irradiation, reversed all the LC3A and p62 findings on blots, suggesting restoration of the normal autophagic function. The LC3A and Beclin1 mRNA levels significantly declined following irradiation (p < 0.01), whereas Bnip3 levels increased. Conclusions: It is suggested that irradiation induces dysfunction of the autophagic machinery in normal lung, characterized by decreased transcription of the LC3A/Beclin-1 mRNA and accumulation of the LC3A, and p62 proteins. Whether this is due to defective maturation or to aberrant degradation of the autophagosomes requires further investigation.« less

International audience; The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.

Huntington disease (HD) is caused by a unique mutation, an abnormal expansion of a polyglutamine (polyQ) tract in the HTT (huntingtin) protein. It has long been hypothesized that in addition to gained toxicity from the expanded polyQ, loss of HTT's normal cellular functions also contributes to HD pathogenesis. Consistently, wild-type HTT has a well-documented neuronal protective activity and is essential for postnatal neuronal survival. However, despite its relatively simple genetic cause, the etiology of HD remains unclear, due in part to the poor understanding of the physiological cellular functions of HTT. The perplexing HTT is a large ∼3,144 amino acid-long protein with ubiquitous localization, but it lacks any known functional domain that could provide clues about its cellular activities. The phenotypes of HTT knockout (KO) mice are equally puzzling: homozygote HTT KO mice die by day 7.5, but this early embryonic lethality is due to a critical role of HTT in extraembryonic membranes, not in the embryo per se, as it can be rescued if wild-type HTT is provided in extraembryonic tissues. HTT has a vast number of reported interacting partners that have been used to infer some of the growing list of cellular pathways that HTT could participate in. However, it is still uncertain how HTT achieves its well-documented neuroprotective role. Using both Drosophila and mammalian experimental models, we recently demonstrated that HTT has an essential function in selective autophagy where it serves as a scaffold by modulating the activities of the cargo receptor SQSTM1/p62 and the autophagy initiation kinase ULK1. We previously created a null deletion mutant (htt-ko) of the single HTT homolog (htt) in Drosophila and showed that in contrast to mouse, htt-ko flies, which develop ex utero, are fully viable with only mild aging-related defects. Unexpectedly, ectopic expression of a truncated form of the mammalian MAPT induced severe defects in htt-ko flies, suggesting that htt protects against pathogenic MAPT toxicity. Further, in genetic screens using this MAPT-induced phenotype as a functional readout, we detected dosage-sensitive genetic interaction between htt-ko and several components of the autophagy pathway, including atg8a (MAP1LC3A homolog), atg1 (ULK1 homolog) and ref(2)P (SQSTM1/p62 homolog), thus unveiling a functional link between HTT and autophagy. HTT is not a foreigner to the autophagy field, although the main emphasis has been on the toxic effect of mutant HTT on this cellular clearance mechanism. In fact, our earlier work in HD demonstrated that reduced autophagy in the disease context, also reported by many other groups, originated from diminished ability to “trap” cytosolic cargo. We attributed the “empty autophagosomes” phenotype in HD cells to defective cargo recognition due to the presence of mutant polyQ-HTT in the inner part of the closing autophagosomes. However, our genetic studies ablating the wild-type HTT made us reconsider this proposed gain-of-toxicity function. In this recent study, we have found that in both htt-ko flies and HTT-depleted mammalian cells, autophagic abnormalities are present but surprisingly, starvation-induced autophagy is largely normal. However, loss of HTT compromised autophagic induction when challenged by several other stresses, including proteotoxicity, lipotoxicity, and mitochondria damage. In all these instances, mammalian cells depleted of HTT also showed an “empty autophagosomes” defect, suggesting that wild-type HTT is required for efficient cargo recognition by the autophagosome. We found that HTT and SQSTM1/p62, as well as their Drosophila counterparts Htt and Ref(2)P, physically interact and that proteotoxic stress promotes this interaction. Depletion of HTT reduces the association of SQSTM1 with MAP1LC3A and also compromises the binding of SQSTM1 with proteins with lysine-63-linked ubiquitin (K63-Ub) chains, which are preferential substrates of autophagy. However, loss of HTT does not affect self-polymerization of SQSTM1 or its association with K48-Ub-modified proteins mainly cleared by the proteasome. Collectively, these findings support the idea that HTT facilitates cargo recognition by modulating the assembly of the cargo receptors and autophagy proteins. We also found that the dependence on HTT for the initiation of stress-induced selective autophagy is related to its physical interaction with ULK1, whose kinase activity is essential for autophagy initiation. HTT depletion does not affect starvation-induced activation of ULK1 kinase, but compromises ULK1 activation upon proteotoxicity challenge, revealing a contribution of HTT to differentially regulating autophagosome biogenesis under basal or stress conditions. We found that the regulation of HTT on ULK1 is at the level of its interaction with the MTORC1 complex, another main regulator of ULK1 that binds to and inactivates ULK1 under nutrient-rich conditions. We showed that (1) ULK1 exists in 2 mutually exclusive complexes, ULK1-HTT and ULK1-MTORC1; (2) HTT does not affect the kinase activity of MTORC1; (3) HTT competes with MTORC1 for binding with ULK1; (4) stresses that induce selective autophagy (e.g., proteotoxicity, lipotoxicity, or mitophagy), but not starvation, result in an increased association of ULK1 with HTT at the expense of MTOR, thus freeing ULK1 from its inhibition by MTORC1. Collectively, HTT promotes selective autophagy by activating and bringing together SQSTM1/p62 and ULK1 to assure spatial proximity between the cargo and autophagy initiation components, thereby orchestrating 2 major autophagy steps: cargo recognition and autophagy induction (Fig. 1). Figure 1. Model of HTT in promoting selective autophagy. HTT serves as a scaffolding for selective autophagy by bringing together cargo bound through SQSTM1 and an initiator of autophagy, ULK1 kinase. Basal autophagy: under basal conditions, binding of HTT to the ... Increasing evidence supports a tangled relationship between HTT, HD, and autophagy. Thus, after the first report of altered autophagy in HD by DiFiglia, the Holzbaur lab showed that HTT facilitates axonal trafficking of autophagosomes, a function that is compromised by polyQ-HTT; Hayden's group reported an autophagy-inducing domain within HTT; Steffan with Thompson and colleagues also recently reported physical interactions between HTT and several autophagy proteins including SQSTM1 and ULK1. Our finding places HTT in the core of the autophagic process, bringing together components of the cargo recognition and autophagy initiation complexes, and raising many new interesting questions. How is HTT differentially activated upon different stress challenges? Does polyQ expansion directly compromise the endogenous activities of HTT, as implicated by the very similar “empty autophagosomes” phenotypes in both HD and HTT-depleted cells, and if so, will this effect be partially responsible for the toxicity of mutant polyQ-HTT in long-lived neurons? Last, how can we exploit HTT's role in selective autophagy to modulate this process in the fight against this devastating brain disease called HD?

The molecular machinery required for autophagy is highly conserved in all eukaryotes as seen by the high degree of conservation of proteins involved in the formation of the autophagosome membranes. Recently, both yeast Apg8p and its rat homologue Map1lc3 were identified as essential constituents of autophagosome membrane as a processed form. In addition, both the yeast and human proteins exist in two modified forms produced by a series of post-translational modifications including a critical C-terminal cleavage after a conserved Gly residue, and the smaller processed form is associated with the autophagosome membranes. Herein, we report the identification and characterization of three human orthologs of the rat Map1LC3, named MAP1LC3A, MAP1LC3B, and MAP1LC3C. We show that the three isoforms of human MAP1LC3 exhibit distinct expression patterns in different human tissues. Importantly, we found that the three isoforms of MAP1LC3 differ in their post-translation modifications. Although MAP1LC3A and MAP1LC3C are produced by the proteolytic cleavage after the conserved C-terminal Gly residue, like their rat counterpart, MAP1LC3B does not undergo C-terminal cleavage and exists in a single modified form. The essential site for the distinct post-translation modification of MAP1LC3B is Lys-122 rather than the conserved Gly-120. Subcellular localization by cell fractionation and immunofluorescence revealed that three human isoforms are associated with membranes involved in the autophagic pathway. These results revealed different regulation of the three human isoforms of MAP1LC3 and implicate that the three isoforms may have different physiological functions.

Autophagy is the principle pathway within cells involved in clearing damaged proteins and organelles. Therefore autophagy is necessary to maintain the turnover balance of peptides and homoeostasis. Autophagy occurs at basal levels under normal conditions but can be upregulated by chemical inducers or stress conditions. The zebrafish (Danio rerio) serves as a versatile tool to understand the functions of genes implicated in autophagy. We report the identification of the zebrafish orthologues of mammalian genes MAP1LC3A (map1lc3a) and MAP1LC3B (map1lc3b) by phylogenetic and conserved synteny analysis and we examine their expression during embryonic development. The zebrafish map1lc3a and map1lc3b genes both show maternally contributed transcripts in early embryogenesis. However, levels of map1lc3a transcript steadily increase until at least 120h post-fertilisation while the levels of map1lc3b show a more variable pattern across developmental time. We have also validated the LC3I ratio/LC3I immunoblot autophagy assay in the presence of chloroquine (a lysosomal proteolysis inhibitor). We found that the LC3II/LC3I ratio is significantly increased in the presence of sodium azide with chloroquine supporting that hypoxia induces autophagy in zebrafish. This was supported by our qPCR assay that showed increased map1lc3a transcript levels in the presence of sodium azide. In contrast, levels of map1lc3b transcripts were reduced in the presence of rapamycin but the decrease in the presence of sodium azide did not reach statistical significance. Our study supports the use of zebrafish for analysing the interplay between hypoxia, development and autophagy.

Purpose Age-related macular degeneration pathogenesis involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome system and the autophagy pathway are major proteolytic processes in eukaryotic cells. SQSTM1/p62 has been shown as a key player linking the proteasomal and lysosomal clearance systems. The present study investigated the effects of AICAR (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) with/without MG-132 (proteasome inhibitor) on autophagy regulation in RPE cells. Methods ARPE-19 cells were treated with MG-132 (5µM) and/or AICAR (2mM). p62 and MAP1LC3A/LC3 (LC3II) were analyzed by Western blotting. LC3 lipidation was used to study autophagic flux. Transmission electron microscopy was used to detect protein aggregates and autophagosomes. pDendra2-hLC3 construct was used to detect macroautophagy in confocal microscopy analysis. Results AICAR+MG-132 co-treatment induces autophagy clearance of p62 and increased LC3 lipidation. AICAR is able to completely abolish the MG-132-induced protein aggregation after 24 h treatment; at the same time, the co-treatment increases the number of autophagic vesicles. Cells treated with MG-132+AICAR exhibit a strong reduction of the perinuclear aggregates containing both LC3II and p62 proteins. Conclusion Autophagy is emerging as a novel target of therapies aimed to counteract protein aggregation and improve cell viability. In this way, our findings indicate that AICAR could be useful in the acceleration of protein clearance in RPE cells.

Background Autophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway. The phosphatidylethanolamine (PE)-conjugation of the Atg8 protein plays an important role in the yeast autophagy process. In humans, six Atg8 homologs, including MAP1LC3A, MAP1LC3B, MAP1LC3C (refer to LC3A, LC3B, and LC3C hereafter), GABARAP, GABARAPL1, and GABARAPL2 have been reported. All of them can be conjugated to PE through a ubiquitin-like conjugation system, and be located to autophagosomes. Results In this study, we found 3 new alternative splicing isoforms in LC3B, GABARAP, and GABARAPL1, (designated as LC3B-a, GABARAP-a and GABARAPL1-a, respectively). None of them can go through the PE-conjugation process and be located to autophagosomes. Interestingly, compared with LC3B, LC3B-a has a single amino acid (Arg68) deletion due to the NAGNAG alternative splicing in intron 3. Through structural simulations, we found that the C-terminal tail of LC3B-a is less mobile than that of LC3B, thus affecting its C-terminal cleavage by human ATG4 family proteins. Furthermore, we found that Arg68 is an essential residue facilitating the interaction between human Atg8 family proteins and ATG4B by forming a salt bridge with Asp171 of ATG4B. Depletion of this salt bridge reduces autophagosomes formation and autophagic flux under both normal and nutrition starvation conditions. Conclusions These results suggest Arg68 is an essential residue for the C-terminal cleavage of Atg8 family proteins during the autophagy process.

Autophagy is a cellular catabolic process in which various cytosolic components are degraded. For example, autophagy can mediate lipolysis of neutral lipid droplets. In contrast, we here report that autophagy is required to facilitate normal levels of neutral lipids in C. elegans. Specifically, by using multiple methods to detect lipid droplets including CARS microscopy, we observed that mutants in the gene bec- 1 (VPS30/ATG6/BECN1), a key regulator of autophagy, failed to store substantial neutral lipids in their intestines during development. Moreover, loss of bec-1 resulted in a decline in lipid levels in daf-2 [insulin/IGF-1 receptor (IIR) ortholog] mutants and in germline-less glp-1/Notch animals, both previously recognized to accumulate neutral lipids and have increased autophagy levels. Similarly, inhibition of additional autophagy genes, including unc-51/ULK1/ATG1 and lgg-1/ATG8/MAP1LC3A/LC3 during development, led to a reduction in lipid content. Importantly, the decrease in fat accumulation observed in animals with reduced autophagy did not appear to be due to a change in food uptake or defecation. Taken together, these observations suggest a broader role for autophagy in lipid remodeling in C. elegans.

Background The members of the microtubule-associated protein 1 light chain (MAP1LC) family, especially those of the LC3 family (MAP1LC3A, B, C), are known to induce autophagy upon localization onto the autophagosomal membrane. In this regard, LC3 can be utilized as a marker for the formation of autophagosomes during the process of autophagy. The aims of this study are to clone porcine MAP1LC3A, and analyze the pattern of its expression in the ovarian tissues of normal and miniature pig ovary in an attempt to understand the distinct mode of apoptosis between two strains. Methods Rapid amplification of cDNA ends (RACE) were used to obtain the 5′ and 3′ ends of the porcine MAP1LC3A full length cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, and western blot analysis were performed to examine the expression of porcine MAP1LC3A. The localization of MAP1LC3A in the ovary was determined by In situ Hybridization and Immunohistochemical staining. Results We cloned the full-length cDNA of porcine MAP1LC3A and identified an open reading frame of 980 bp encoding 121 amino acids. Based on its homology to known mammalian proteins (98%) this novel cDNA was designated as porcine MAP1LC3A and registered to the GenBank (Accession No. GU272221). We compared the expression of MAP1LC3A in the Graafian follicles of normal and miniature pigs by in situ hybridization at day 15 of the estrus cycle. While normal pigs showed a stronger expression of MAP1LC3A mRNA than miniature pigs in the theca cell area, the expression was lower in the granulosa cells. Immunofluorescence analysis of the MAP1LC3A fusion reporter protein showed the subcellular localization of porcine MAP1LC3A and ATG5 as a punctate pattern in the cytoplasm of porcine granulosa cells under stress conditions. In addition, the expressions of MAP1LC3A and ATG5 were higher in normal pigs than in miniature pigs both in the presence and absence of rapamycin. Conclusions The newly cloned porcine MAP1LC3A provides a novel autophagosomal marker in both normal and miniature pig. We demonstrated that the expression of MAP1LC3A in graafian follicle is distinct in normal and miniature pig, which may explain the unique folliculogenesis of miniature pigs.

Macroautophagy is a process that leads to the bulk degradation of subcellular constituents by producing autophagosomes/autolysosomes. It is believed that Atg5 (ref. 4) and Atg7 (ref. 5) are essential genes for mammalian macroautophagy. Here we show, however, that mouse cells lacking Atg5 or Atg7 can still form autophagosomes/autolysosomes and perform autophagy-mediated protein degradation when subjected to certain stressors. Although lipidation of the microtubule-associated protein light chain 3 (LC3, also known as Map1lc3a) to form LC3-II is generally considered to be a good indicator of macroautophagy, it did not occur during the Atg5/Atg7-independent alternative process of macroautophagy. We also found that this alternative process of macroautophagy was regulated by several autophagic proteins, including Unc-51-like kinase 1 (Ulk1) and beclin 1. Unlike conventional macroautophagy, autophagosomes seemed to be generated in a Rab9-dependent manner by the fusion of isolation membranes with vesicles derived from the trans-Golgi and late endosomes. In vivo, Atg5-independent alternative macroautophagy was detected in several embryonic tissues. It also had a function in clearing mitochondria during erythroid maturation. These results indicate that mammalian macroautophagy can occur through at least two different pathways: an Atg5/Atg7-dependent conventional pathway and an Atg5/Atg7-independent alternative pathway.

Age-related macular degeneration is an eye disease that is the main cause of legal blindness in the elderly in developed countries. Despite this, its pathogenesis is not completely known, and many genetic, epigenetic, environmental and lifestyle factors may be involved. Vision loss in age-related macular degeneration (AMD) is usually consequence of the occurrence of its wet (neovascular) form that is targeted in the clinic by anti-VEGF (vascular endothelial growth factor) treatment. The wet form of AMD is associated with the accumulation of cellular waste in the retinal pigment epithelium, which is removed by autophagy and the proteosomal degradation system. In the present work, we searched for the association between genotypes and alleles of single nucleotide polymorphisms (SNPs) of autophagy-related genes and wet AMD occurrence in a cohort of Finnish patients undergoing anti-VEGF therapy and controls. Additionally, the correlation between treatment efficacy and genotypes was investigated. Overall, 225 wet AMD patients and 161 controls were enrolled in this study. Ten SNPs (rs2295080, rs11121704, rs1057079, rs1064261, rs573775, rs11246867, rs3088051, rs10902469, rs73105013, rs10277) in the mTOR (Mechanistic Target of Rapamycin), ATG5 (Autophagy Related 5), ULK1 (Unc-51-Like Autophagy Activating Kinase 1), MAP1LC3A (Microtubule Associated Protein 1 Light Chain 3 α), SQSTM1 (Sequestosome 1) were analyzed with RT-PCR-based genotyping. The genotype/alleles rs2295080-G, rs11121704-C, rs1057079-C and rs73105013-T associated with an increased, whereas rs2295080-TT, rs2295080-T, rs11121704-TT, rs1057079-TT, rs1057079-T, rs573775-AA and rs73105013-C with a decreased occurrence of wet AMD. In addition, the rs2295080-GG, rs2295080-GT, rs1057079-TT, rs11246867-AG, rs3088051-CC and rs10277-CC genotypes were a positively correlated cumulative number of anti-VEGF injections in 2 years. Therefore, variability in autophagy genes may have an impact on the risk of wet AMD occurrence and the efficacy of anti-VEGF treatment.