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The emergence of drug resistance hinders the treatment of malignant tumors, and autophagy plays an important role in tumor chemotherapy resistance. However, its mechanism in non-small cell lung cancer (NSCLC) has not been well-researched. We aim to investigate the role of miR-101-3p in cisplatin-resistant via regulation of autophagy-related protein 4D (ATG4D) and autophagy. Cell viability, apoptosis, fluorescence intensity of GFP-LC3 and RFP-GFP-LC3 were determined using Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and Laser scanning confocal microscope analysis, respectively. The levels of LC3II/LC3I, P62 and ATG4D were detected by Western blot. The results showed that the sensitivity to cisplatin in NSCLC cells was up-regulated by miR-101-3p mimics treatment, inducing promoting cell apoptosis and inhibiting autophagy. Further mechanistic study identified that ATG4D was a direct target of miR-101-3p. Moreover, ATG4D siRNA also could reverse miR-101-3p inhibitor-induced the up-regulation of ATG4D and the ration of LC3II/LC3I, the down-regulation of p62 expression. Our findings indicated that miR-101-3p could regulate sensitivity to cisplatin of NSNCC cells by regulating autophagy mediated by ATG4D. Therefore, miR-101-3p may act as a potential therapeutic target for the treatment of NSCLC.

Atherosclerosis, a multifactorial vascular disease resulting from lipid metabolism disorders, features chronic inflammatory damage resulting from endothelial dysfunction, which usually affects multiple arteries. The carotid artery is a common site for clinical atherosclerosis evaluation. The aortic root is the standard site for quantifying atherosclerosis in mice. Due to the adverse reactions of first-line drugs, it is necessary to discover new drugs to prevent and treat atherosclerosis. Berberine (BBR) is one of the most promising natural products derived from herbal medicine Coptidis Rhizoma (Huanglian) that features significant anti-atherosclerosis properties. However, overall BBR mechanism against carotid atherosclerosis has not been clearly discovered. Our work aimed to investigate potential BBR mechanism in improving carotid atherosclerosis in ApoE knockout mice. Here, we proved that in ApoE −/− mice receiving high-fat diet for 12 weeks, BBR can reduce serum lipid levels, improve intimal hyperplasia, and antagonize carotid lipid accumulation, which may be achieved through regulating the PI3K/AKT/mTOR signaling pathway, regulating autophagy, promoting cell proliferation and inhibiting cell apoptosis. In summary, these data indicate that BBR can ameliorate carotid atherosclerosis. Therefore, it could be a promisingly therapeutic alternative for atherosclerosis.

Early brain injury, characterized by massive cell apoptosis or death, is identified as a critical pathophysiological process during subarachnoid hemorrhage (SAH). Ferroptosis, a class of autophagy-dependent cell death discovered in 2012, is induced by iron-dependent lipid peroxidation accumulation. The present study was designed to study the role of baicalin in autophagy-dependent ferroptosis in early brain injury after SAH. Neurological scores and brain water content were measured to evaluate brain injury. Measurement of iron ion, malondialdehyde (MDA), lipid reactive oxygen species was conducted for ferroptosis evaluation. Immunofluorescence staining, western blotting, and flow cytometry analysis were used to evaluate autophagy and apoptosis. First, we observed that, compared with sham rats, SAH rats had lower neurobehavioral scores. Next, baicalin was proven to decrease the Fe2+, malondialdehyde, and ROS levels in the brain tissues of rats. Also, baicalin was confirmed to suppress the beclin1, LC3-II, and LC3-I protein levels in rat brain tissues. Moreover, we found that baicalin inhibited neuronal apoptosis. Finally, the effects of baicalin on brain injury in the SAH rats were verified. Overall, our results demonstrated that baicalin suppressed autophagy-dependent ferroptosis in EBI after SAH.

Diosbulbin-B (DB) is a promising therapeutic drug for cancer treatment; however, DB-induced hepatotoxicity seriously limits its clinical utilization. Based on this, the present study investigated whether the Angelica sinensis extract, angelica sinensis polysaccharide (ASP), was effective to attenuate DB-induced cytotoxicity in hepatocytes. The primary hepatocytes were isolated from rats and cultured in vitro, which were subsequently treated with high-dose DB (100 μM) and ASP (12 μg/ml) to establish the DB-induced hepatotoxicity models. MTT assay and flow cytometry (FCM) were performed to evaluate cell viability, and the results showed that high-dose DB-induced cell apoptosis and inhibition of proliferation were reversed by co-treating cells with ASP, which were supported by our Western Blot assay data that ASP upregulated Cyclin D1 and CDK2 to abrogate high-dose DB-induced cell cycle arrest. In addition, ASP exerted its regulating effects on cell autophagy, and we found that ASP increased LC3B-II/I ratio and Atg5, but decreased p62 to activate the autophagy flux. Of note, the MEK/ERK pathway could be activated by ASP in the DB-treated hepatocytes, and the protective effects of ASP on high-dose DB-induced hepatocyte death were abolished by co-treating cells with the autophagy inhibitor (3-methyladenine, 3-MA) and MEK/ERK selective inhibitor (SCH772984). Moreover, blockage of the MEK/ERK pathway suppressed cell autophagy in the hepatocytes co-treated with ASP and high-dose DB. Taken together, this in vitro study illustrated that ASP activated the MEK/ERK pathway mediated autophagy to suppress high-dose DB-induced hepatotoxicity., graphical

Osteoarthritis (OA) is a chronic degenerative disease that significantly impacts the quality of life of the elderly population. Recently, the pathogenesis of OA has been reported to involve autophagy in chondrocytes. Intriguingly, icariin, one of the main components of epimedium, exerts multiple pharmacological effects, including a protective effect against chondrocyte damage. Thus, we aimed to investigate the therapeutic effect of icariin on OA and its potential underlying mechanism by using a rat model of OA. After treatment with icariin or an autophagy activator (rapamycin) or inhibitor (3-methyladenine), OA chondrocyte viability was measured using the CCK-8 assay, apoptosis in the chondrocytes was evaluated using the acridine orange-propidium iodide assay and flow cytometry, and OA tissue pathological state was assessed using micro-CT scanning and safranin O staining. Furthermore, immunohistochemical staining was used to measure the expression level of Beclin-1 and immunofluorescence labeling was used to visualize LC3 expression, and western blotting was used to determine the expression levels of autophagy proteins and key proteins in the PI3K signaling pathway. The apoptotic rate of OA chondrocytes was markedly elevated by 3-methyladenine and suppressed by rapamycin and icariin; autophagy genes were drastically downregulated in the 3-methyladenine group and upregulated in the rapamycin and icariin groups; and the PI3K/AKT/mTOR signaling pathway was activated by 3-methyladenine and inhibited by rapamycin and icariin. Notably, following treatment with rapamycin and icariin, the severe pathological state in OA cartilage tissues was substantially alleviated, and this was accompanied by activated autophagy and inhibited PI3K signaling in the cartilage tissues. Taken together, these findings indicate that icariin alleviates OA by regulating the autophagy of chondrocytes by mediating PI3K/AKT/mTOR signaling., Graphical abstract

Presbycusis is the cumulative effect of aging on hearing. Recent studies have shown that common mitochondrial gene deletions are closely related to deafness caused by degenerative changes in the auditory system, and some of these nuclear factors are proposed to participate in the regulation of mitochondrial function. However, the detailed mechanisms involved in age-related degeneration of the auditory systems have not yet been fully elucidated. In this study, we found that FOXG1 plays an important role in the auditory degeneration process through regulation of macroautophagy/autophagy. Inhibition of FOXG1 decreased the autophagy activity and led to the accumulation of reactive oxygen species and subsequent apoptosis of cochlear hair cells. Recent clinical studies have found that aspirin plays important roles in the prevention and treatment of various diseases by regulating autophagy and mitochondria function. In this study, we found that aspirin increased the expression of FOXG1, which further activated autophagy and reduced the production of reactive oxygen species and inhibited apoptosis, and thus promoted the survival of mimetic aging HCs and HC-like OC-1 cells. This study demonstrates the regulatory function of the FOXG1 transcription factor through the autophagy pathway during hair cell degeneration in presbycusis, and it provides a new molecular approach for the treatment of age-related hearing loss. Abbreviations: AHL: age-related hearing loss; baf: bafilomycin A1; CD: common deletion; D-gal: D-galactose; GO: glucose oxidase; HC: hair cells; mtDNA: mitochondrial DNA; RAP: rapamycin; ROS: reactive oxygen species; TMRE: tetramethylrhodamine, ethyl ester

MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12–ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation. Abbreviations: CQ: Chloroquine; EBSS: Earle’s Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1.

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type

Enhancing virus multiplication could assist in the rapid production of vaccines against viral diseases. Cold atmospheric plasma (CAP), a physical approach relying on reactive oxygen species to achieve the desirable cellular outcome, was shown to be effective in enhancing virus propagation, where bovine rhinotrachieitis virus and Madin-Darby Bovine Kidney cells were used as the modeling virus and cell line, respectively. CAP was shown to create synergies with virus infection in arresting host cells at the G2/M stage, decreasing cell membrane potential, increasing intracellular calcium level, and inducing selective autophagy. In addition, CAP was demonstrated to suppress virus-triggered immunogenic signaling as evaluated by IRF7 expression. We presented evidences on CAP-triggered maximization of host resources toward virus multiplication that is advantageous for viral vaccine production, and opened a novel regime for applying CAP in the sector of medical care and health.

Scavenger receptors are pattern recognition receptors that recognize both foreign and self-ligands, and initiate different mechanisms of cellular activation, often as co-receptors. The function of scavenger receptor CD36 in the immune system has mostly been studied in macrophages but it is also highly expressed by innate type B cells where its function is less explored. Here we report that CD36 is involved in macro-autophagy/autophagy in B cells, and in its absence, the humoral immune response is impaired. We found that CD36-deficient B cells exhibit a significantly reduced plasma cell formation, proliferation, mitochondrial mobilization and oxidative phosphorylation. These changes were accompanied by impaired initiation of autophagy, and we found that CD36 regulated autophagy and colocalized with autophagosome membrane protein MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). When we investigated T-cell-dependent immune responses, we found that mice with CD36 deficiency, specifically in B cells, exhibited attenuated germinal center responses, class switching, and antibody production as well as autophagosome formation. These findings establish a critical role for CD36 in B cell responses and may also contribute to our understanding of CD36-mediated autophagy in other cells as well as in B cell lymphomas that have been shown to express the receptor. Abbreviations: AICDA/AID: activation-induced cytidine deaminase; ATG5: autophagy related 5; ATP: adenosine triphosphate; BCR: B-cell receptor; CPG: unmethylated cytosine-guanosine; CQ: chloroquine; DC: dendritic cells; FOB: follicular B cells; GC: germinal center; Ig: immunoglobulin; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MZB: marginal zone B cells; NP-CGG: 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin; OCR: oxygen consumption rate; oxLDL: oxidized low-density lipoprotein; PC: plasma cells; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; SRBC: sheep red blood cells; Tfh: follicular helper T cells; TLR: toll-like receptor.

Macroautophagy/autophagy is an intracellular degradation process that delivers cytosolic materials and/or damaged organelles to lysosomes. De novo synthesis of the autophagosome membrane occurs within a phosphatidylinositol-3-phosphate-rich region of the endoplasmic reticulum, and subsequent expansion is critical for cargo encapsulation. This process is complex, especially in mammals, with many regulatory factors. In this study, by utilizing PRKN (parkin RBR E3 ubiquitin protein ligase)-mediated mitochondria autophagy (mitophagy)-inducing conditions in conjunction with chemical crosslinking and mass spectrometry, we identified human BCAS3 (BCAS3 microtubule associated cell migration factor) and C16orf70 (chromosome 16 open reading frame 70) as novel proteins that associate with the autophagosome formation site during both non-selective and selective autophagy. We demonstrate that BCAS3 and C16orf70 form a complex and that their association with the phagophore assembly site requires both proteins. In silico structural modeling, mutational analyses in cells and in vitro phosphoinositide-binding assays indicate that the WD40 repeat domain in human BCAS3 directly binds phosphatidylinositol-3-phosphate. Furthermore, overexpression of the BCAS3-C16orf70 complex affects the recruitment of several core autophagy proteins to the phagophore assembly site. This study demonstrates regulatory roles for human BCAS3 and C16orf70 in autophagic activity. Abbreviations: AO: antimycin A and oligomycin; Ash: assembly helper; ATG: autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; C16orf70: chromosome 16 open reading frame 70; DAPI: 4‘,6-diamidino-2-phenylindole; DKO: double knockout; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; fluoppi: fluorescent-based technology detecting protein-protein interactions; FIS1: fission, mitochondrial 1; FKBP: FKBP prolyl isomerase family member 1C; FRB: FKBP-rapamycin binding; hAG: humanized azami-green; IP: immunoprecipitation; IRES: internal ribosome entry site; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MS: mass spectrometry; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mtDNA: mitochondrial DNA; OPTN: optineurin; PFA: paraformaldehyde; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; PROPPIN: β-propellers that bind polyphosphoinositides; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like autophagy activating kinase 1; WDR45B/WIPI3: WD repeat domain 45B; WDR45/WIPI4: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1

FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1(−/−)) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1(−/−) mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1(−/−) mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis. Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type

Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2–6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo–Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing. Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel

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.

In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an nbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of HSP genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS. Abbreviations: AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein

Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of “autophagy profiles” revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates. Abbreviations: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-β: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.

Dysregulated microRNAs (miRNAs) are involved in carcinoma progression, metastasis, and poor prognosis. We demonstrated that in nasopharyngeal carcinoma (NPC), transactivated MIR106A-5p promotes a malignant phenotype by functioning as a macroautophagy/autophagy suppressor by targeting BTG3 (BTG anti-proliferation factor 3) and activating autophagy-regulating MAPK signaling. MIR106A-5p expression was markedly increased in NPC cases based on quantitative real-time PCR, miRNA microarray, and TCGA database analysis findings. Moreover, MIR106A-5p was correlated with advanced stage, recurrence, and poor clinical outcomes in NPC patients. In addition to three-dimensional cell culture assays, zebrafish and BALB/c mouse tumor models revealed that overexpressed MIR106A-5p targeted BTG3 and accelerated the NPC malignant phenotype by inhibiting autophagy. BTG3 promoted autophagy, and its expression was correlated with poor prognosis in NPC. Attenuation of autophagy, mediated by the MIR106A-5p-BTG3 axis, occurred because of MAPK pathway activation. MIR106A-5p overexpression in NPC was due to increased transactivation by EGR1 and SOX9. Our findings may lead to novel insights into the pathogenesis of NPC. Abbreviations: ACTB: actin beta; ATG: autophagy-related; ATG5: autophagy related 5; BLI: bioluminescence; BTG3: BTG anti-proliferation factor 3; CASP3: caspase 3; ChIP: chromatin immunoprecipitation; CQ: chloroquine; Ct: threshold cycle; DAPI: 4��,6-diamidino-2-phenylindole; DiL: 1,1��-dioctadecyl-3,3,3��,3��-tetramethylindocarbocyanine perchlorate; EBSS: Earle���s balanced salt solution; EGR1: early growth response 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GEO: Gene Expression Omnibus; GFP: green fluorescent protein; IF: immunofluorescence; IHC: immunohistochemistry; ISH: in situ hybridization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MIR106A-5p: microRNA 106a-5p; miRNAs: microRNAs; MKI67: marker of proliferation ki-67; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NPC: nasopharyngeal carcinoma; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SOX9: SRY-box transcription factor 9; SQSTM1: sequestosome 1; TCGA: The Cancer Genome Atlas; WB: western blot.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability. Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.

Disorders of lysosomal physiology have increasingly been found to underlie the pathology of a rapidly growing cast of neurodevelopmental disorders and sporadic diseases of aging. One cardinal aspect of lysosomal (dys)function is lysosomal acidification in which defects trigger lysosomal stress signaling and defects in proteolytic capacity. We have developed a genetically encoded ratiometric probe to measure lysosomal pH coupled with a purification tag to efficiently purify lysosomes for both proteomic and in vitro evaluation of their function. Using our probe, we showed that lysosomal pH is remarkably stable over a period of days in a variety of cell types. Additionally, this probe can be used to determine that lysosomal stress signaling via TFEB is uncoupled from gross changes in lysosomal pH. Finally, we demonstrated that while overexpression of ARL8B GTPase causes striking alkalinization of peripheral lysosomes in HEK293 T cells, peripheral lysosomes per se are no less acidic than juxtanuclear lysosomes in our cell lines. Abbreviations: ARL8B: ADP ribosylation factor like GTPase 8B; ATP: adenosine triphosphate; ATP5F1B/ATPB: ATP synthase F1 subunit beta; ATP6V1A: ATPase H+ transporting V1 subunit A; Baf: bafilomycin A1; BLOC-1: biogenesis of lysosome-related organelles complex 1; BSA: bovine serum albumin; Cos7: African green monkey kidney fibroblast-like cell line; CQ: chloroquine; CTSB: cathepsin B; CYCS: cytochrome c, somatic; DAPI: 4′,6-diamidino −2- phenylindole; DIC: differential interference contrast; DIV: days in vitro; DMEM: Dulbecco′s modified Eagle′s medium;‎ E8: embryonic day 8; EEA1: early endosome antigen 1; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GTP: guanosine triphosphate; HEK293T: human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen; HeLa: Henrietta Lacks-derived cell; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP: horseradish peroxidase; IGF2R/ciM6PR: insulin like growth factor 2 receptor; LAMP1/2: lysosomal associated membrane protein 1/2; LMAN2/VIP36: lectin, mannose binding 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PCR: polymerase chain reaction; PDL: poly-d-lysine; PGK1p: promotor from human phosphoglycerate kinase 1; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PPT1/CLN1: palmitoyl-protein thioesterase 1; RPS6KB1/p70: ribosomal protein S6 kinase B1; STAT3: signal transducer and activator of transcription 3; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGN: trans-Golgi network; TGOLN2/TGN46: trans-Golgi network protein 2; TIRF: total internal reflection fluorescence; TMEM106B: transmembrane protein 106B; TOR: target of rapamycin; TRPM2: transient receptor potential cation channel subfamily M member 2; V-ATPase: vacuolar-type proton-translocating ATPase; VPS35: VPS35 retromer complex component.

Macrophage is essential for host anti-bacterial defense by directly eliminating invading microbes and inducing a series of immune reactions. Here we identified a Streptococcus pneumoniae protein, PepO, as a TLR2/TLR4 bi-ligand. We found that PepO enhances macrophage unspecific phagocytosis and bactericidal activity, which is related to the induction of autophagy in macrophage, for the inhibition of autophagy significantly decreased the phagocytosis and bactericidal activity of PepO-treated macrophage. We confirmed that these effects of PepO are dependent on interacting with both TLR2 and TLR4. The tlr2 or tlr4 deficiency partially abolished the effect of PepO while tlr2/tlr4 deficiency abolished it completely. In vivo study demonstrated that PepO reduced the bacteria load in WT mice significantly, while the depletion of macrophage or tlr2/tlr4 deficiency abrogated the effect of PepO. Our findings suggested the therapeutic potential of PepO and provided experimental evidence for immunotherapy against infectious disease.

ATAD2 has been reported to play an important role in the processes of numerous cancers and validated to be a potential therapeutic target. This work is to discover potent ATAD2 inhibitors and elucidate the underlying mechanisms in breast cancer. A novel ATAD2 bromodomain inhibitor (AM879) was discovered by combining structure-based virtual screening with biochemical analyses. AM879 presents potent inhibitory activity towards ATAD2 bromodomain (IC50 = 3565 nM), presenting no inhibitory activity against BRD2-4. Moreover, AM879 inhibited MDA-MB-231 cells proliferation with IC50 value of 2.43 µM, suppressed the expression of c-Myc, and induced significant apoptosis. Additionally, AM978 could induce autophagy via PI3K-AKT-mTOR signalling in MDA-MB-231 cells. This study demonstrates the development of potent ATAD2 inhibitors with novel scaffolds for breast cancer therapy.

Lysosomal Ca2+ contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca2+ channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca2+ levels. The Ca2+ efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca2+ level, in which ITPR-independent regulation of Ca2+ status was observed. Further, TMBIM6 regulated the local release of Ca2+ through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca2+ efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca2+ and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved Tmbim6+/+ mice compared with that in the counter tmbim6−/- mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca2+ release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca2+ modulator that coordinates with autophagy to alleviate metabolism stress.Abbreviations: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6

Although the treatment of brain tumors by targeting kinase-regulated macroautophagy/autophagy, is under investigation, the precise mechanism underlying autophagy initiation and its significance in glioblastoma (GBM) remains to be defined. Here, we report that PAK1 (p21 [RAC1] activated kinase 1) is significantly upregulated and promotes GBM development. The Cancer Genome Atlas analysis suggests that the oncogenic role of PAK1 in GBM is mainly associated with autophagy. Subsequent experiments demonstrate that PAK1 indeed serves as a positive modulator for hypoxia-induced autophagy in GBM. Mechanistically, hypoxia induces ELP3-mediated PAK1 acetylation at K420, which suppresses the dimerization of PAK1 and enhances its activity, thereby leading to subsequent PAK1-mediated ATG5 (autophagy related 5) phosphorylation at the T101 residue. This event not only protects ATG5 from ubiquitination-dependent degradation but also increases the affinity between the ATG12–ATG5 complex and ATG16L1 (autophagy related 16 like 1). Consequently, ELP3-dependent PAK1 (K420) acetylation and PAK1-mediated ATG5 (T101) phosphorylation are required for hypoxia-induced autophagy and brain tumorigenesis by promoting autophagosome formation. Silencing PAK1 with shRNA or small molecule inhibitor FRAX597 potentially blocks autophagy and GBM growth. Furthermore, SIRT1-mediated PAK1-deacetylation at K420 hinders autophagy and GBM growth. Clinically, the levels of PAK1 (K420) acetylation significantly correlate with the expression of ATG5 (T101) phosphorylation in GBM patients. Together, this report uncovers that the acetylation modification and kinase activity of PAK1 plays an instrumental role in hypoxia-induced autophagy initiation and maintaining GBM growth. Therefore, PAK1 and its regulator in the autophagy pathway might represent potential therapeutic targets for GBM treatment. Abbreviations: 3-MA: 3-methyladenine; Ac-CoA: acetyl coenzyme A; ATG5: autophagy related 5; ATG16L1, autophagy related 16 like 1; BafA1: bafilomycin A1; CDC42: cell division cycle 42; CGGA: Chinese Glioma Genome Atlas; CHX, cycloheximide; ELP3: elongator acetyltransferase complex subunit 3; GBM, glioblastoma; HBSS: Hanks balanced salts solution; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K1: mitogen-activated protein kinase kinase 1; MAPK14, mitogen-activated protein kinase 14; PAK1: p21 (RAC1) activated kinase 1; PDK1: pyruvate dehydrogenase kinase 1; PGK1, phosphoglycerate kinase 1; PTMs: post-translational modifications; RAC1: Rac family small GTPase 1; SQSTM1: sequestosome 1; TCGA, The Cancer Genome Atlas.

Opportunistic bacterial infections amongst HIV–infected individuals contribute significantly to HIV-associated mortality. The role of HIV-mediated modulation of innate mechanisms like autophagy in promoting opportunistic infections, however, remains obscure. Here we show, HIV reactivation in or infection of macrophages inhibits autophagy and helps the survival of pathogenic Mycobacterium tuberculosis (Mtb) and nonpathogenic non-tuberculous mycobacterial strains (NTMs). The HIV-mediated impairment of xenophagy flux facilitated bacterial survival. Activation of autophagy by trehalose could induce xenophagy flux and kill intracellular Mtb or NTMs either during single or co-infections. Trehalose, we delineate, activates PIKFYVE leading to TFEB nuclear translocation in MCOLN1-dependent manner to induce autophagy. Remarkably, trehalose significantly reduced HIV-p24 levels in ex–vivo-infected PBMCs or PBMCs from treatment-naive HIV patients and also controlled mycobacterial survival within Mtb-infected animals. To conclude, we report leveraging of HIV-mediated perturbed host innate-immunity by opportunistic bacterial pathogens and show an attractive therapeutic strategy for HIV and associated co-morbidities. Abbreviations: AIDS: acquired immune deficiency syndrome; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; BafA1: bafilomycin A1; CFU: colony forming unit; CTSD: cathepsin D; CD63: CD63 molecule; EGFP: enhanced green fluorescent protein; FRET: Förster resonance energy transfer; GABARAP: gamma-aminobutyric acid receptor-associated protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GLUT: glucose transporter; HIV: human immunodeficiency virus; hMDMs: human monocyte derived macrophages; IL2: interleukin 2; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: lipidated microtubule-associated proteins 1A/1B light chain 3B; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin; mRFP: monomeric red fluorescent protein; M6PR: mannose-6-phosphate receptor; NAC: N- acetyl- L –cysteine; NTM’s: non-tuberculous mycobacteria; PBMC: Peripheral Blood Mononuclear cells; PIKFYVE: phosphoinositide kinase; FYVE-Type Zinc Finger; PHA: phytohemagglutinin; PMA: phorbol 12-myristate 13-acetate; PtdIns(3,5)P2: Phosphatidylinositol 3,5-bisphosphate; ptfLC3: pEGFP-mRFP-LC3; ROS: reactive oxygen species; SQSTM1: sequestosome1; TFEB: transcription factor EB; MCOLN1/TRPML1: mucolipin 1; PIP4P1/TMEM55B: Human trans-membrane Protein 55B; UVRAG: UV Radiation Resistance Associate; VPS35: vacuolar protein sorting associated protein 35; WDR45: WD repeat domain 45; YCAM: Yellow Chameleon.

Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition. Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4’,6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell

Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2(±), lpar1(−/−) or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2(±) and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion. Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2(±): Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1(−/−): lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

The mitochondrial-anchored deubiquitinating enzyme USP30 (ubiquitin specific peptidase 30) antagonizes PRKN/parkin-mediated mitophagy, making it a potential target for treating Parkinson disease. However, few inhibitors targeting USP30 have been reported. Here, we report a novel peptide (Q14) derived from the transmembrane (TM) domain of USP30 that can target mitochondrial-anchored USP30 directly and increase mitophagy through two intriguing and distinct mechanisms: a novel autoinhibition mechanism in USP30 and accelerated autophagosome formation via the LC3-interacting region (LIR) of the Q14 peptide. We identified the potential binding sites between the Q14 peptide and USP30 and postulated that an allosteric autoinhibition mechanism regulates USP30 activity. Furthermore, the LIR motif in the Q14 peptide offers additional binding with LC3 and accelerated autophagosome formation. The two mechanisms synergistically enhance mitophagy. Our work provides novel insight and direction to the design of inhibitors for USP30 or other deubiquitinating enzymes (DUBs). Abbreviations: 3-MA: 3-methyladenine; ATTEC: autophagosome-tethering compound; BafA1: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; FP: fluorescence polarization; FUNDC1: FUN14 domain containing 1; HCQ: hydroxychloroquine; LIR: LC3-interacting region; MST: microscale thermophoresis; mtDNA: mitochondrial DNA; mtPA-GFP: mitochondria-targeted photoactive fluorescence protein; OMM: outer mitochondrial membrane; PINK1: PTEN induced kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; Rap: rapamycin; SA: streptavidin; TM: transmembrane; Ub: ubiquitin; Ub-AMC: Ub-7-amido-4-methylcoumarin; UPS: ubiquitin-protease system; USP: ubiquitin specific peptidase; USP30: ubiquitin specific peptidase 30.

Barth syndrome (BTHS) is an X-linked genetic disorder caused by mutations in the TAFAZZIN/Taz gene which encodes a transacylase required for cardiolipin remodeling. Cardiolipin is a mitochondrial signature phospholipid that plays a pivotal role in maintaining mitochondrial membrane structure, respiration, mtDNA biogenesis, and mitophagy. Mutations in the TAFAZZIN gene deplete mature cardiolipin, leading to mitochondrial dysfunction, dilated cardiomyopathy, and premature death in BTHS patients. Currently, there is no effective treatment for this debilitating condition. In this study, we showed that TAFAZZIN deficiency caused hyperactivation of MTORC1 signaling and defective mitophagy, leading to accumulation of autophagic vacuoles and dysfunctional mitochondria in the heart of Tafazzin knockdown mice, a rodent model of BTHS. Consequently, treatment of TAFAZZIN knockdown mice with rapamycin, a potent inhibitor of MTORC1, not only restored mitophagy, but also mitigated mitochondrial dysfunction and dilated cardiomyopathy. Taken together, these findings identify MTORC1 as a novel therapeutic target for BTHS, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for BTHS. Abbreviations: BTHS: Barth syndrome; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CL: cardiolipin; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; KD: knockdown; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LV: left ventricle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; OCR: oxygen consumption rate; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; qRT-PCR: quantitative real-time polymerase chain reaction; RPS6KB/S6K: ribosomal protein S6 kinase beta; SQSTM1/p62: sequestosome 1; TLCL: tetralinoleoyl cardiolipin; WT: wild-type.

Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H2S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H2S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2+ cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68+ and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = −0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H2S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H2S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H2S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H2S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1. Conclusively, CTH-H2S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H2S may act as a warning biomarker for vulnerable plaque. Abbreviations ATG9A: autophagy related 9A; CTH: cystathionine gamma-lyase; CQ: chloroquine; HASMCs: human aortic smooth muscle cells; H2S: hydrogen sulfide; LAMP1: lysosomal associated membrane protein 1; LAPTM5: lysosomal protein transmembrane 5; NaHS: sodium hydrosulfide hydrate; ox-LDL: oxidized-low density lipoprotein; PPG: DL- propagylglycine; TFEB: transcription factor EB; 3-MA: 3-methyladenine; VSMCs: vascular smooth muscle cells.

Centrosome amplification is a phenomenon frequently observed in human cancers, so centrosome depletion has been proposed as a therapeutic strategy. However, despite being afflicted with a lack of centrosomes, many cancer cells can still proliferate, implying there are impediments to adopting centrosome depletion as a treatment strategy. Here, we show that TFEB- and TFE3-dependent autophagy activation contributes to acentrosomal cancer proliferation. Our biochemical analyses uncover that both TFEB and TFE3 are novel PLK4 (polo like kinase 4) substrates. Centrosome depletion inactivates PLK4, resulting in TFEB and TFE3 dephosphorylation and subsequent promotion of TFEB and TFE3 nuclear translocation and transcriptional activation of autophagy- and lysosome-related genes. A combination of centrosome depletion and inhibition of the TFEB-TFE3 autophagy-lysosome pathway induced strongly anti-proliferative effects in cancer cells. Thus, our findings point to a new strategy for combating cancer. Abbreviations: AdCre: adenoviral Cre recombinase; AdLuc: adenoviral luciferase; ATG5: autophagy related 5; CQ: chloroquine; DAPI: 4’,6-diamidino-2-phenylindole; DKO: double knockout; GFP: green fluorescent protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LTR: LysoTracker Red; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MITF: melanocyte inducing transcription factor; PLK4: polo like kinase 4; RFP: red fluorescent protein; SASS6: SAS-6 centriolar assembly protein; STIL: STIL centriolar assembly protein; TFEB: transcription factor EB; TFEBΔNLS: TFEB lacking a nuclear localization signal; TFE3: transcription factor binding to IGHM enhancer 3; TP53/p53: tumor protein p53

SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Here, using a genetically non-redundant Drosophila model, we show that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. We find that accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response. Abbreviations: Atg8a: Autophagy-related 8a; cnc: cap-n-collar; IFM: indirect flight muscle; KEAP1: kelch like ECH associated protein 1; LIR: LC3-interacting region; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1: Phox and Bem1; ref(2)P: refractory to sigma P; SAR: selective autophagy receptor; UBA: ubiquitin-associated.

Maintenance of bone integrity is mediated by the balanced actions of osteoblasts and osteoclasts. Because macroautophagy/autophagy regulates osteoblast mineralization, osteoclast differentiation, and their secretion from osteoclast cells, autophagy deficiency in osteoblasts or osteoclasts can disrupt this balance. However, it remains unclear whether upregulation of autophagy becomes beneficial for suppression of bone-associated diseases. In this study, we found that genetic upregulation of autophagy in osteoblasts facilitated bone formation. We generated mice in which autophagy was specifically upregulated in osteoblasts by deleting the gene encoding RUBCN/Rubicon, a negative regulator of autophagy. The rubcnflox/flox;Sp7/Osterix-Cre mice showed progressive skeletal abnormalities in femur bones. Consistent with this, RUBCN deficiency in osteoblasts resulted in elevated differentiation and mineralization, as well as an increase in the elevated expression of key transcription factors involved in osteoblast function such as Runx2 and Bglap/Osteocalcin. Furthermore, RUBCN deficiency in osteoblasts accelerated autophagic degradation of NOTCH intracellular domain (NICD) and downregulated the NOTCH signaling pathway, which negatively regulates osteoblast differentiation. Notably, osteoblast-specific deletion of RUBCN alleviated the phenotype in a mouse model of osteoporosis. We conclude that RUBCN is a key regulator of bone homeostasis. On the basis of these findings, we propose that medications targeting RUBCN or autophagic degradation of NICD could be used to treat age-related osteoporosis and bone fracture. Abbreviations: ALPL: alkaline phosphatase, liver/bone/kidney; BCIP/NBT: 5-bromo-4-chloro-3ʹ-indolyl phosphate/nitro blue tetrazolium; BMD: bone mineral density; BV/TV: bone volume/total bone volume; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NICD: NOTCH intracellular domain; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein; SERM: selective estrogen receptor modulator; TNFRSF11B/OCIF: tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin)

The N-degron pathway is a proteolytic system in which the N-terminal degrons (N-degrons) of proteins, such as arginine (Nt-Arg), induce the degradation of proteins and subcellular organelles via the ubiquitin-proteasome system (UPS) or macroautophagy/autophagy-lysosome system (hereafter autophagy). Here, we developed the chemical mimics of the N-degron Nt-Arg as a pharmaceutical means to induce targeted degradation of intracellular bacteria via autophagy, such as Salmonella enterica serovar Typhimurium (S. Typhimurium), Escherichia coli, and Streptococcus pyogenes as well as Mycobacterium tuberculosis (Mtb). Upon binding the ZZ domain of the autophagic cargo receptor SQSTM1/p62 (sequestosome 1), these chemicals induced the biogenesis and recruitment of autophagic membranes to intracellular bacteria via SQSTM1, leading to lysosomal degradation. The antimicrobial efficacy was independent of rapamycin-modulated core autophagic pathways and synergistic with the reduced production of inflammatory cytokines. In mice, these drugs exhibited antimicrobial efficacy for S. Typhimurium, Bacillus Calmette–Guérin (BCG), and Mtb as well as multidrug-resistant Mtb and inhibited the production of inflammatory cytokines. This dual mode of action in xenophagy and inflammation significantly protected mice from inflammatory lesions in the lungs and other tissues caused by all the tested bacterial strains. Our results suggest that the N-degron pathway provides a therapeutic target in host-directed therapeutics for a broad range of drug-resistant intracellular pathogens. Abbreviations: ATG: autophagy-related gene; BCG: Bacillus Calmette–Guérin; BMDMs: bone marrow-derived macrophages; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CFUs: colony-forming units; CXCL: C-X-C motif chemokine ligand; EGFP: enhanced green fluorescent protein; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; LIR: MAP1LC3/LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PB1: Phox and Bem1; SQSTM1/p62: sequestosome 1; S. Typhimurium: Salmonella enterica serovar Typhimurium; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; UBA: ubiquitin-associated.

Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions. We also identify a KFERQ-like motif in the TPPP/p25A sequence that enables its effective degradation via chaperone-mediated autophagy (CMA) in an in vitro system of rat brain lysosomes. Furthermore, in a MSA-like setting established by addition of human recombinant SNCA pre-formed fibrils (PFFs) as seeds of pathological SNCA, we thoroughly characterize the contribution of CMA and macroautophagy in particular, in the removal of the exogenously added and the seeded oligodendroglial SNCA pathological assemblies. We also show that PFF treatment impairs autophagic flux and that TPPP/p25A exerts an inhibitory effect on macroautophagy, while at the same time CMA is upregulated to remove the pathological SNCA species formed within oligodendrocytes. Finally, augmentation of CMA or macroautophagy accelerates the removal of the engendered pathological SNCA conformations further suggesting that autophagy targeting may represent a successful approach for the clearance of pathological SNCA and/or TPPP/p25A in the context of MSA. Abbreviations: 3MA: 3-methyladenine; ACTB: actin, beta; ALP: autophagy-lysosome pathway; ATG5: autophagy related 5; AR7: atypical retinoid 7; CMA: chaperone-mediated autophagy; CMV: cytomegalovirus; CTSD: cathepsin D; DAPI: 4′,6-diamidino-2-phenylindole; DMEM: Dulbecco’s modified Eagle’s medium; Epox: epoxomicin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCIs: glial cytoplasmic inclusions; GFP: green fluorescent protein; HMW: high molecular weight; h: hours; HSPA8/HSC70: heat shock protein 8; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mcherry: monomeric cherry; MFI: mean fluorescence intensity; mRFP: monomeric red fluorescent protein; MSA: multiple system atrophy; OLN: oligodendrocytes; OPCs: oligodendroglial progenitor cells; PBS: phosphate-buffered saline; PC12: pheochromocytoma cell line; PD: Parkinson disease; PFFs: pre-formed fibrils; PIs: protease inhibitors; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; Rap: rapamycin; RFP: red fluorescent protein; Scr: scrambled; SDS: sodium dodecyl sulfate; SE: standard error; siRNAs: small interfering RNAs; SNCA: synuclein, alpha; SQSTM1: sequestosome 1; TPPP: tubulin polymerization promoting protein; TUBA: tubulin, alpha; UPS: ubiquitin-proteasome system; WT: wild type

Increased bone resorption by osteoclasts after estrogen deficiency is the main cause of postmenopausal osteoporosis. TET2 (tet methylcytosine dioxygenase 2) is a DNA demethylase that regulates cellular function and differentiation potential. Macroautophagy/autophagy maintains cellular homeostasis by recycling unnecessary and damaged organelles. This study revealed that TET2 promoted bone loss in oophorectomized (OVX) mice and that TET2 promoted osteoclast differentiation by regulating autophagy. Tet2 knockdown inhibited autophagy and osteoclast differentiation in vitro. Mechanistically, Tet2 knockdown increased BCL2 (B cell leukemia/lymphoma 2) expression and BCL2 exhibited increased binding to BECN1 and negatively regulated autophagy. Small interfering RNA specific to Bcl2 interfered with BCL2 expression in Tet2-knockdown bone marrow cells/precursors, partially reversing autophagy dysregulation and promoting osteoclast differentiation. Moreover, the LV-shTet2 lentivirus prevented bone loss in OVX mice. In summary, our findings provide evidence that TET2 promotes osteoclast differentiation by inhibiting BCL2 expression and positively regulating BECN1-dependent autophagy. Abbreviations: ACP5/TRAP: acid phosphatase 5, tartrate resistant; ATP6V0D2: ATPase, H+ transporting, lysosomal V0 subunit D2; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BMs: bone marrow cells; CTSK: cathepsin K; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MMP9: matrix metallopeptidase 9; OVX: oophorectomy; RUNX1: runt related transcription factor 1; SOCS3: suppressor of cytokine signaling 3; SPI1/PU.1: Spi-1 proto-oncogene; TNFSF11/RANKL: tumor necrosis factor (ligand) superfamily, member 11; TET2: tet methylcytosine dioxygenase 2.

SARS-CoV-2 infections have resulted in a very large number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, pathogenesis and therapy remains limited, emphasizing the urgent need for fundamental studies and drug development. Studies have shown that induction of macroautophagy/autophagy and hijacking of the autophagic machinery are essential for the infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and the function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3a as an inducer of autophagy (in particular reticulophagy) and revealed that ORF3a localizes to the ER and induces RETREG1/FAM134B-related reticulophagy through the HMGB1-BECN1 (beclin 1) pathway. As a consequence, ORF3a induces ER stress and inflammatory responses through reticulophagy and then sensitizes cells to the acquisition of an ER stress-related early apoptotic phenotype and facilitates SARS-CoV-2 infection, suggesting that SARS-CoV-2 ORF3a hijacks reticulophagy and then disrupts ER homeostasis to induce ER stress and inflammatory responses during SARS-CoV-2 infection. These findings reveal the sequential induction of reticulophagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and imply the therapeutic potential of reticulophagy and ER stress-related drugs for COVID-19. Abbreviations: CQ: chloroquine; DEGs: differentially expressed genes; ER: endoplasmic reticulum; GSEA: gene set enrichment analysis; HMGB1: high mobility group box 1; HMOX1: heme oxygenase 1; MERS-CoV: Middle East respiratory syndrome coronavirus; RETREG1/FAM134B: reticulophagy regulator 1; RTN4: reticulon 4; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TN: tunicamycin

Cerebral infarction induces angiogenesis in the thalamus and influences functional recovery. The mechanisms underlying angiogenesis remain unclear. This study aimed to investigate the role of RTN4/Nogo-A in mediating macroautophagy/autophagy and angiogenesis in the thalamus following middle cerebral artery occlusion (MCAO). We assessed secondary neuronal damage, angiogenesis, vascular autophagy, RTN4 and S1PR2 signaling in the thalamus. The effects of RTN4-S1PR2 on vascular autophagy and angiogenesis were evaluated using lentiviral and pharmacological approaches. The results showed that RTN4 and S1PR2 signaling molecules were upregulated in parallel with angiogenesis in the ipsilateral thalamus after MCAO. Knockdown of Rtn4 by siRNA markedly reduced MAP1LC3B-II conversion and levels of BECN1 and SQSTM1 in vessels, coinciding with enhanced angiogenesis in the ipsilateral thalamus. This effect coincided with rescued neuronal loss of the thalamus and improved cognitive function. Conversely, activating S1PR2 augmented vascular autophagy, along with suppressed angiogenesis and aggravated neuronal damage of the thalamus. Further inhibition of autophagic initiation with 3-methyladenine or spautin-1 enhanced angiogenesis while blockade of lysosomal degradation by bafilomycin A1 suppressed angiogenesis in the ipsilateral thalamus. The control of autophagic flux by RTN4-S1PR2 was verified in vitro. Additionally, ROCK1-BECN1 interaction along with phosphorylation of BECN1 (Thr119) was identified in the thalamic vessels after MCAO. Knockdown of Rtn4 markedly reduced BECN1 phosphorylation whereas activating S1PR2 increased its phosphorylation in vessels. These results suggest that blockade of RTN4-S1PR2 interaction promotes angiogenesis and secondary neural repair in the thalamus by suppressing autophagic activation and alleviating dysfunction of lysosomal degradation in vessels after cerebral infarction. Abbreviations: 3-MA: 3-methyladenine; ACTA2/ɑ-SMA: actin alpha 2, smooth muscle, aorta; AIF1/Iba1: allograft inflammatory factor 1; BafA1: bafilomycin A1; BMVECs: brain microvascular endothelial cells; BrdU: 5-bromo-2’-deoxyuridine; CLDN11/OSP: claudin 11; GFAP: glial fibrillary acidic protein; HUVECs: human umbilical vein endothelial cells; LAMA1: laminin, alpha 1; MAP2: microtubule-associated protein 2; MBP2: myelin basic protein 2; MCAO: middle cerebral artery occlusion; PDGFRB/PDGFRβ: platelet derived growth factor receptor, beta polypeptide; RECA-1: rat endothelial cell antigen-1; RHOA: ras homolog family member A; RHRSP: stroke-prone renovascular hypertensive rats; ROCK1: Rho-associated coiled-coil containing protein kinase 1; RTN4/Nogo-A: reticulon 4; RTN4R/NgR1: reticulon 4 receptor; S1PR2: sphingosine-1-phosphate receptor 2; SQSTM1: sequestosome 1.

Following acute kidney injury (AKI), renal tubular cells may stimulate fibroblasts in a paracrine fashion leading to interstitial fibrosis, but the paracrine factors and their regulation under this condition remain elusive. Here we identify a macroautophagy/autophagy-dependent FGF2 (fibroblast growth factor 2) production in tubular cells. Upon induction, FGF2 acts as a key paracrine factor to activate fibroblasts for renal fibrosis. After ischemic AKI in mice, autophagy activation persisted for weeks in renal tubular cells. In inducible, renal tubule-specific atg7 (autophagy related 7) knockout (iRT-atg7-KO) mice, autophagy deficiency induced after AKI suppressed the pro-fibrotic phenotype in tubular cells and reduced fibrosis. Among the major cytokines, tubular autophagy deficiency in iRT-atg7-KO mice specifically diminished FGF2. Autophagy inhibition also attenuated FGF2 expression in TGFB1/TGF-β1 (transforming growth factor, beta 1)-treated renal tubular cells. Consistent with a paracrine action, the culture medium of TGFB1-treated tubular cells stimulated renal fibroblasts, and this effect was suppressed by FGF2 neutralizing antibody and also by fgf2- or atg7-deletion in tubular cells. In human, compared with non-AKI, the renal biopsies from post-AKI patients had higher levels of autophagy and FGF2 in tubular cells, which showed significant correlations with renal fibrosis. These results indicate that persistent autophagy after AKI induces pro-fibrotic phenotype transformation in tubular cells leading to the expression and secretion of FGF2, which activates fibroblasts for renal fibrosis during maladaptive kidney repair. Abbreviations: 3-MA: 3-methyladnine; ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/β-actin: actin, beta; AKI: acute kidney injury; ATG/Atg: autophagy related; BUN: blood urea nitrogen; CCN2/CTGF: cellular communication network factor 2; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CKD: chronic kidney disease; CM: conditioned medium; COL1A1: collagen, type I, alpha 1; COL4A1: collagen, type IV, alpha 1; CQ: chloroquine; ECM: extracellular matrix; eGFR: estimated glomerular filtration rate; ELISA: enzyme-linked immunosorbent assay; FGF2: fibroblast growth factor 2; FN1: fibronectin 1; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAVCR1/KIM-1: hepatitis A virus cellular receptor 1; IHC: immunohistochemistry; IRI: ischemia-reperfusion injury; ISH: in situ hybridization; LTL: lotus tetragonolobus lectin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PDGFB: platelet derived growth factor, B polypeptide; PPIB/cyclophilin B: peptidylprolyl isomerase B; RT-qPCR: real time-quantitative PCR; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; sCr: serum creatinine; SQSTM1/p62: sequestosome 1; TASCC: TOR-autophagy spatial coupling compartment; TGFB1/TGF-β1: transforming growth factor, beta 1; VIM: vimentin

The intestinal epithelial tight junctions (TJs) provide barrier against paracellular permeation of lumenal antigens. Defects in TJ barrier such as increased levels of pore-forming TJ protein CLDN2 (claudin-2) is associated with inflammatory bowel disease. We have previously reported that starvation-induced macroautophagy/autophagy enhances the TJ barrier by degrading pore-forming CLDN2. In this study, we examined the molecular mechanism underlying autophagy-induced CLDN2 degradation. CLDN2 degradation was persistent in multiple modes of autophagy induction. Immunolocalization, membrane fractionation, and pharmacological inhibition studies showed increased clathrin-mediated CLDN2 endocytosis upon starvation. Inhibition of clathrin-mediated endocytosis negated autophagy-induced CLDN2 degradation and enhancement of the TJ barrier. The co-immunoprecipitation studies showed increased association of CLDN2 with clathrin and adaptor protein AP2 (AP2A1 and AP2M1 subunits) as well as LC3 and lysosomes upon starvation, signifying the role of clathrin-mediated endocytosis in autophagy-induced CLDN2 degradation. The expression and phosphorylation of AP2M1 was increased upon starvation. In-vitro, in-vivo (mouse colon), and ex-vivo (human colon) inhibition of AP2M1 activation prevented CLDN2 degradation. AP2M1 knockout prevented autophagy-induced CLDN2 degradation via reduced CLDN2-LC3 interaction. Site-directed mutagenesis revealed that AP2M1 binds to CLDN2 tyrosine motifs (YXXФ) (67–70 and 148–151). Increased baseline expression of CLDN2 and TJ permeability along with reduced CLDN2-AP2M1-LC3 interactions in ATG7 knockout cells validated the role of autophagy in modulation of CLDN2 levels. Acute deletion of Atg7 in mice increased CLDN2 levels and the susceptibility to experimental colitis. The autophagy-regulated molecular mechanisms linking CLDN2, AP2M1, and LC3 may provide therapeutic tools against intestinal inflammation. Abbreviations: Amil: amiloride; AP2: adaptor protein complex 2; AP2A1: adaptor related protein complex 2 subunit alpha 1; AP2M1: adaptor related protein complex 2 subunit mu 1; ATG7: autophagy related 7; CAL: calcitriol; Cas9: CRISPR-associated protein 9; Con: control; CPZ: chlorpromazine; DSS: dextran sodium sulfate; EBSS: Earle’s balanced salt solution; IBD: inflammatory bowel disease; TER: trans-epithelial resistance; KD: knockdown; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MβCD: Methyl-β-cyclodextrin; MET: metformin; MG132: carbobenzoxy-Leu-Leu-leucinal; MTOR: mechanistic target of rapamycin kinase; NT: non target; RAPA: rapamycin; RES: resveratrol; SMER: small-molecule enhancer 28; SQSTM1: sequestosome 1; ST: starvation; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Deficient bone regeneration causes bone defects or nonunion in a substantial proportion of trauma patients that urges for novel therapies. To develop a reliable therapy, we investigated the effect of negative pressure wound therapy (NPWT) on bone regeneration in vivo in a rat calvarial defect model. Negative pressure (NP) treatment in vitro was mimicked to test its effect on osteoblast differentiation in rat mesenchymal stem cells (MSCs) and MC3T3-E1 cells. Transcriptomic analyses, pharmaceutical interventions, and shRNA knockdowns were conducted to explore the underlying mechanism and their clinical relevance was investigated in samples from patients with nonunion. The potential application of a combined therapy of MSCs in hydrogels with negative pressure was tested in the rat critical-size calvarial defect model. We found that NPWT promoted bone regeneration in vivo and NP treatment induced osteoblast differentiation in vitro. NP induced osteogenesis via activating macroautophagy/autophagy by AMPK-ULK1 signaling that was impaired in clinical samples from patients with nonunion. More importantly, the combined therapy involving MSCs in hydrogels with negative pressure significantly improved bone regeneration in rat critical-size calvarial defect model. Thus, our study identifies a novel AMPK-ULK1-autophagy axis by which negative pressure promotes osteoblast differentiation of MSCs and bone regeneration. NPWT treatment can potentially be adopted for therapy of bone defects. Abbreviations: ADP, adenosine diphosphate; AICAR/Aic, acadesine; ALP, alkaline phosphatase; ALPL, alkaline phosphatase, biomineralization associated; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; ARS, alizarin red S staining; ATG7, autophagy related 7; ATP, adenosine triphosphate; BA1, bafilomycin A(1); BGLAP/OCN, bone gamma-carboxyglutamate protein; BL, BL-918; BS, bone surface; BS/TV, bone surface per tissue volume; BV/TV, bone volume per tissue volume; C.C, compound C; CCN1, cellular communication network factor 1; COL1A1, collagen type I alpha 1 chain; COL4A3, collagen type IV alpha 3 chain; COL4A4, collagen type IV alpha 4 chain; COL18A1, collagen type XVIII alpha 1 chain; CQ, chloroquine; GelMA, gelatin methacryloyl hydrogel; GO, Gene Ontology; GSEA, gene set enrichment analysis; HIF1A, hypoxia inducible factor 1 subunit alpha; HPLC, high-performance liquid chromatography; ITGAM/CD11B, integrin subunit alpha M; ITGAX/CD11C, integrin subunit alpha X; ITGB1/CdD9, integrin subunit beta 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; micro-CT, microcomputed tomography; MSCs, mesenchymal stem cells; MTOR, mechanistic target of rapamycin kinase; NP, negative pressure; NPWT, negative pressure wound therapy; PRKAA1/AMPKα1, protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2, protein kinase AMP-activated catalytic subunit alpha 2; PTPRC/CD45, protein tyrosine phosphatase receptor type C; ROS, reactive oxygen species; RUNX2, RUNX family transcription factor 2; SBI, SBI-0206965; SPP1/OPN, secreted phosphoprotein 1; THY1/CD90, Thy-1 cell surface antigen; SQSTM1, sequestosome 1; TGFB3, transforming growth factor beta 3; ULK1/Atg1, unc-51 like autophagy activating kinase 1.

By promoting anabolism, MTORC1 is critical for muscle growth and maintenance. However, genetic MTORC1 upregulation promotes muscle aging and produces age-associated myopathy. Whether MTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effects of muscular MTORC1 hyperactivation, produced by simultaneous depletion of TSC1 and DEPDC5 (CKM-TD). CKM-TD mice produced myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac failure and bradypnea. These pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicated that genes mediating proteasomal and macroautophagic/autophagic pathways were highly upregulated in CKM-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. In CKM-TD muscle, autophagosome levels were increased, and the AMPK and ULK1 pathways were activated; in addition, autophagy induction was not completely blocked in CKM-TD myotubes. Despite the upregulation of autolysosomal markers, CKM-TD myofibers exhibited accumulation of autophagy substrates, such as SQSTM1/p62 and ubiquitinated proteins, suggesting that the autophagic activities were insufficient. Administration of a superoxide scavenger, tempol, normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. However, CKM-TD autophagy alterations were not normalized by rapamycin or tempol, suggesting that they may involve non-canonical targets other than MTORC1. These results collectively indicate that the concomitant muscle deficiency of TSC1 and DEPDC5 can produce early-onset myopathy through accumulation of oxidative stress, which dysregulates myocellular homeostasis.bAbbreviations:/bAMPK: AMP-activated protein kinase; CKM: creatine kinase, M-type; COX: cytochrome oxidase; DEPDC5: DEP domain containing 5, GATOR1 subcomplex subunit; DHE: dihydroethidium; EDL: extensor digitorum longus; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase-activating protein; GTN: gastrocnemius; MTORC1: mechanistic target of rapamycin kinase complex 1; PLA: plantaris; QUAD: quadriceps; RPS6KB/S6K: ribosomal protein S6 kinase beta; SDH: succinate dehydrogenase; SOL: soleus; SQSTM1: sequestosome 1; TA: tibialis anterior; TSC1: TSC complex subunit 1; ULK1: unc-51 like autophagy activating kinase 1.

The phagophore expands into autophagosomes in close proximity to endoplasmic reticulum (ER) exit sites (ERESs). Here, we propose that a single-pass ER transmembrane protein, SHISA5/SCOTIN, acts as an autophagy suppressor under basal condition by blocking the contact between the phagophore and ERES. HeLa cells lacking SHISA5 displayed higher levels of macroautophagy/autophagy. The enhanced autophagy in SHISA5 KO cells requires class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) activity and functional assembly of ERES, but not ULK1 activity. A proximity ligation assay (PLA) of SEC16A (Sec16 homolog A, endoplasmic reticulum export factor)-WIPI2 (WD repeat domain, phosphoinositide interacting 2) and SEC31A (Sec31 homolog A, COPII coat complex component)-MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) demonstrated that contact between the ERES and phagophore increased in SHISA5 KO cells, and the cytosolic domain of SHISA5 was sufficient to rescue this phenotype. Close proximity between ERES and phagophore in SHISA5 KO cells was also visualized by performing an ultrastructure correlative image analysis of SEC31A associated with LC3-positive membranes. Furthermore, we observed that SHISA5 was located near ERES under basal conditions, but displaced away from ERES under autophagy-inducing conditions. These data suggest that SHISA5 functions to block spontaneous contact between ERES and phagophore, and the blockage effect of SHISA5 should be relieved for the proper induction of autophagy. ABBREVIATIONS: ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle’s balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type

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

TBK1 (TANK-binding kinase 1) is an essential receptor protein required for the innate immune response, but the mechanisms underlying TBK1 stability, especially those regulated via autophagy, remain poorly understood. Here, we demonstrate that USP19 (ubiquitin specific peptidase 19) interacts with and promotes TBK1 lysosomal degradation via chaperone-mediated autophagy (CMA). We observed that TBK1 had a canonical CMA motif, knocking down key proteins involved in CMA (HSPA8/HSC70 or LAMP2A) or inhibiting CMA-prevented USP19-mediated TBK1 degradation. Furthermore, USP19 deficiency in macrophages caused an elevation of TBK1 and the activation of the type-I interferon signaling pathway after vesicular stomatitis virus (VSV) infection. Consistently, macrophage-specific usp19 knockout in mice resulted in attenuated VSV replication and resistance to VSV infection in vivo. Altogether, our results suggest that USP19 is a key regulator of TBK1 and uncovers a previously uncharacterized role for USP19 in CMA-mediated TBK1 degradation and infectious diseases.

Background Ketamine-related cystitis (KC) has been researched in many clinical studies, but its exact mechanism is ambiguous and needs further research. Methods We established a KC rat model and analyzed physiological, biochemical, and urodynamic parameters of ketamine (KET)-related bladder injury. Bladder histologic feature, reactive oxygen species (ROS), autophagy-, apoptosis-, and endoplasmic reticulum stress (ERS)-related markers were examined by hematoxylin and eosin staining, Masson staining, ROS kit, quantitative real-time polymerase chain reaction, and western blot. In vitro, effects of 0.01, 0.1, and 1 mM KET on cell vitality, apoptosis, ROS level, autophagy-, apoptosis-, and ERS-related markers were examined again. Effects of KET-1 and salubrinal on complex formation, autophagy-, apoptosis-, and ERS-related markers were examined by Co-Immunoprecipitation and western blot. After transfection with shIRE1, complex formation, cell biological behaviors, ROS level, autophagy-, apoptosis-, and ERS-related markers were examined again. Results KET induced bladder hyperactivity and injury. KET facilitated urinary frequency, ROS production, and induced bladder histologic injury by activating autophagy-, apoptosis-, and ERS-related markers in rats. In vitro, KET (0.01, 0.1, and 1 mM) restrained cell vitality and elevated apoptosis and ROS level via activating autophagy-, apoptosis-, and ERS-related markers. Moreover, salubrinal reversed the promotion of KET-1 on complex formation, autophagy-, apoptosis-, and ERS-related marker expressions. After transfection with shIRE1, shIRE1 weakened complex formation induced by KET-1, and the effects of KET-1 on cells were offset by shIRE1. Conclusion KET enhanced autophagy and ERS in vivo and in vitro via restraining IRE1-TRAF2-ASK1-JNK pathway.

While the functions of STING1 (stimulator of interferon response cGAMP interactor 1) during DNA virus infection had been well documented, the roles STING1 plays during RNA viruses infection is obscure. Infection with foot-and-mouth disease virus (FMDV), a well-known picornavirus, induces endoplasmic reticulum (ER) stress response and autophagy. Here, we found that the FMDV-induced integrated stress response originates from the cellular pattern recognition receptor DDX58/RIG-I (DExD/H-box helicase 58). DDX58 transmits signals to the ER-anchored adaptor protein STING1, which specifically activates the EIF2AK3/PERK (eukaryotic translation initiation factor 2A)-dependent integrated stress response and finally leads to reticulophagy and degradation of STING1 itself. Knockdown/knockout of STING1 or EIF2AK3 suppresses FMDV genome replication and viral protein expression. Reticulophagy induction by STING1 does not require its translocation to the Golgi or IFN response activation. However, STING1 polymerization is necessary for the FMDV-induced integrated stress response and reticulophagy. Our work illustrated the signaling cascades that mediate the cellular stress response to FMDV infection and indicated that induction of autophagy in response to both DNA and RNA virus infection may be an evolutionarily conserved function of STING1. Abbreviations: ATF6: activating transcription factor 6; CGAS: cyclic GMP-AMP synthase; DDX58/RIG-I: DExD/H-box helicase 58; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 2; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FMD: foot-and-mouth disease; FMDV: foot-and-mouth disease virus; IFIH1/MDA5: interferon induced with helicase C domain 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; RETREG1/FAM134B: reticulophagy regulator 1; STING1: stimulator of interferon response cGAMP interactor 1; TCID50: 50% tissue culture infectious dose; XBP1: X-box binding protein 1.

The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we show that autophagic fluxes were highly activated during the appressorial formation of M. robertsii. Genome-wide deletion of the autophagy-related genes and insect bioassays identified 10 of 23 encoded MrATG genes with requirements for topical fungal infection of insect hosts. Besides the defect in forming appressoria on insects (two null mutants), these virulence-reduced mutants were largely impaired in penetrating cellophane membrane and insect cuticles, suggesting their failures in generating proper appressorium turgor. We found that the conidial storage of lipid droplets (LDs) had no obvious difference between strains, but autophagic LD degradation was impaired in different mutants. After induction of cell autophagy by nitrogen starvation, we found that LD entry into vacuoles was unaffected in the selected mutant cells with potential failures in forming autophagosomes. The finding therefore reveals a microlipophagy machinery employed in this fungus and that the direct engulfment of LDs occurs without inhibition by the downstream defective lipolysis. Our data first unveil the activation and contribution of microlipophagy to fungal infection biology. The obtained technique may benefit future detection of microlipophagy in different organisms by examining vacuolar or lysosomal engulfment of LDs in core autophagic gene deletion mutants. Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy-related; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; LD, lipid droplet; MM: minimum medium; MM-N: minimum medium without nitrogen source; PDA: potato dextrose agar; PMSF: phenylmethylsulfonyl fluoride; RFP: red fluorescent protein; SDB: Sabouraud dextrose broth; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAG: triacylglycerol; TEM: transmission electron microscopy; WT, wild type.

Macroautophagy/autophagy is emerging as a major pathway that regulates both aging and stem cell function. Previous studies have demonstrated a positive correlation of autophagy with longevity; however, these studies did not directly address the consequence of altered autophagy in stem cells during aging. In this study, we used Becn1(F121A/F121A) knockin mice (designated as Becn1 KI mice) with the F121A allele in the autophagy gene Becn1 to investigate the consequences of enhanced autophagy in postnatal neural stem cells (NSCs) during aging. We found that increased autophagy protected NSCs from exhaustion and promoted neurogenesis in old (≥18-months-old) mice compared with age-matched wild-type (WT) mice, although it did not affect NSCs in young (3-months-old) mice. After pharmacologically-induced elimination of proliferative cells in the subventricular zone (SVZ), there was enhanced re-activation of quiescent NSCs in old Becn1 KI mice as compared to those in WT mice, with more efficient exit from quiescent status to generate proliferative cells and neuroblasts. Moreover, there was also improved maintenance and increased neuronal differentiation of NSCs isolated from the SVZ of old Becn1 KI mice in in vitro assays. Lastly, the increased neurogenesis in Becn1 KI mice was associated with better olfactory function in aged animals. Together, our results suggest a protective role of increased autophagy in aging NSCs, which may help the development of novel strategies to treat age-related neurodegeneration. Abbreviations: ATG: autophagy related; Baf A(1): bafilomycin A(1); Becn1: beclin 1; BrdU: bromodeoxyuridine/5-bromo-2ʹ-deoxyuridine; DCX: doublecortin; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSCs: hematopoietic stem cells; KI: knockin; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mo: month; NSCs: neural stem cells; OB: olfactory bulb; RB1CC1: RB1-inducible coiled-coil 1; ROS: reactive oxygen species; SOX2: SRY (sex determining region Y)-box 2; SGZ: subgranular zone; SVZ: subventricular zone; TMZ: temozolomide; WT: wild type.

Atrial fibrillation (AF) is the common arrhythmias. Myocardial fibrosis (MF) is closely related to atrial remodeling and leads to AF. MF is the main cause of cardiovascular diseases and a pathological basis of AF. Thus, the underlying mechanism in MF and AF development should be fully elucidated for AF therapeutic innovation. Autophagy is a highly conserved lysosomal degradation pathway, and the relationship between autophagy and MF has been previously shown. Moreover, research reported that quercetin (Que) could ameliorate MF. The current study aimed to explore the mechanism of Que in MF. The results in this study showed that in clinical AF patients and in aged rats, miR-223-3p was high-expressed, while FOXO3 and autophagy pathway related proteins, such as ATG7, p62/SQSTM1 and the ratio of LC3B-II/LC3B-I were significantly inhibited. In vivo and in vitro studies, we found that Que can effectively inhibit the expression of miR-223-3p in AF model cells and rats myocardial tissues, and meanwhile enhance the expression of FOXO3 and activate the autophagy pathway, and significantly inhibit myocardial fibrosis, and improve myocardial remodeling in atrial fibrillation. All in all, in this study, we found that Que prevents isoprenaline-induced MF by increasing autophagy via regulating miR-223-3p/FOXO3.