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Lung disease development involves multiple cellular processes, including inflammation, cell death, and proliferation. Research increasingly indicates that autophagy and its regulatory proteins can influence inflammation, programmed cell death, cell proliferation, and innate immune responses. Autophagy plays a vital role in the maintenance of homeostasis and the adaptation of eukaryotic cells to stress by enabling the chelation, transport, and degradation of subcellular components, including proteins and organelles. This process is essential for sustaining cellular balance and ensuring the health of the mitochondrial population. Recent studies have begun to explore the connection between autophagy and the development of different lung diseases. This article reviews the latest findings on the molecular regulatory mechanisms of autophagy in lung diseases, with an emphasis on potential targeted therapies for autophagy. [ABSTRACT FROM AUTHOR]

Autophagosome (AP)–lysosome/vacuole fusion is one of the hallmarks of macroautophagy. Membrane features and changes during the fusion process have mostly been described using two-dimensional (2D) models with one AP and one lysosome/vacuole. The outer membrane (OM) of a closed mature AP has been suggested to fuse with the lysosomal/vacuolar membrane. However, the descriptions in some studies for fusion-related issues are questionable or incomplete. The correct membrane features of APs and lysosomes/vacuoles are the prerequisite for describing the fusion process. We searched the literature for representative membrane features of AP-related structures based on electron microscopy (EM) graphs of both animal and yeast cells and re-evaluated the findings. We also summarized the main 2D models describing the membrane changes during AP–lysosome/vacuole fusion in the literature. We used three-dimensional (3D) models to characterize the known and unknown membrane changes during and after fusion of the most plausible 2D models. The actual situation is more complex, since multiple lysosomes may fuse with the same AP in mammalian cells, multiple APs may fuse with the same vacuole in yeast cells, and in some mutant cells, phagophores (unclosed APs) fuse with lysosomes/vacuoles. This review discusses the membrane features and highly dynamic changes during AP (phagophore)–lysosome/vacuole fusion. The resulting information will improve the understanding of AP–lysosome/vacuole fusion and direct the future research on AP–lysosome/vacuole fusion and regeneration. [ABSTRACT FROM AUTHOR]

Autophagy is a preserved process in eukaryotes that allows large material degeneration and nutrient recovery via vacuoles or lysosomes in cytoplasm. Autophagy starts from the moment of induction during the formation of a phagophore. Degradation may occur in the autophagosomes even without fusion with lysosome or vacuole, particularly in microautophagosomes. This process is arbitrated by the conserved machinery of basic autophagy‐related genes (ATGs). In selective autophagy, specific materials are recruited by autophagosomes via receptors. Selective autophagy targets a vast variety of cellular components for degradation, i.e., old or damaged organelles, aggregates, and inactive or misfolded proteins. In optimal conditions, autophagy in plants ensures cellular homeostasis, proper plant growth, and fitness. Moreover, autophagy is essential during stress responses in plants and aids in survival of plants. Several biotic and abiotic stresses, i.e., pathogen infection, nutrient deficiency, plant senescence, heat stress, drought, osmotic stress, and hypoxia induce autophagy in plants. Cell death is not a stress, which induces autophagy but in contrast, sometimes it is a consequence of autophagy. In this way, autophagy plays a vital role in plant survival during harsh environmental conditions by maintaining nutrient concentration through elimination of useless cellular components. This review discussed the recent advances regarding regulatory functions of autophagy under normal and stressful conditions in plants and suggests future prospects in mitigating climate change. Autophagy in plants offers a viable way to increase plant resilience to climate change by increasing stress tolerance and nutrient usage efficiency. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii, an opportunistic pathogen has shown an upsurge in its multi-drug resistant isolates. OmpA of A. baumannii induces incomplete autophagy and apoptosis in host cells. Various therapeutic alternatives are under investigation against A. baumannii. Here, the major emphasis has been laid on comparing the efficacy of AgNP with different capping agents. OmpA targeted lead, Ivermectin capped AgNP (IVM-AgNP) has been compared with the antibacterial polyvinylpyrrolidone capped AgNP (PVP-AgNP) for their role in the modulations of host autophagy. Upregulation of p62 and LC3B confirmed by real-time PCR analysis indicated an increased autophagic flux upon the treatment with AgNPs. The elongation and closure of autophagic vacuoles was also supported by upregulated Atg genes (Atg4, Atg3, Atg5) in A. baumannii infected cells after treatment with AgNP. Autophagic flux increased on treatment with PVP-AgNP as suggested by the rise in mcherryLC3B fluorescence in A549 cells treated with PVP-AgNP as compared to the GFP-LC3B of IVM-AgNP. This suggests that PVP-AgNP treatment more effectively promotes the elongation and maturation stages of autophagy by increasing autophagic flux. These results indicate that capped AgNPs have the efficiency to revert the incomplete autophagy induced by A. baumannii back to normal autophagic levels. [ABSTRACT FROM AUTHOR]

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP–ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z -direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple At ATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP– At ATG8e marker, that most probably does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume positively correlated with the intensity of autophagy induction. Compared with the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP–ATG8 markers. [ABSTRACT FROM AUTHOR]

One of the key events in autophagy is the formation of a double-membrane phagophore, and many regulatory mechanisms underpinning this remain under investigation. WIPI2b is among the first proteins to be recruited to the phagophore and is essential for stimulating autophagy flux by recruiting the ATG12–ATG5–ATG16L1 complex, driving LC3 and GABARAP lipidation. Here, we set out to investigate how WIPI2b function is regulated by phosphorylation. We studied two phosphorylation sites on WIPI2b, S68 and S284. Phosphorylation at these sites plays distinct roles, regulating WIPI2b's association with ATG16L1 and the phagophore, respectively. We confirm WIPI2b is a novel ULK1 substrate, validated by the detection of endogenous phosphorylation at S284. Notably, S284 is situated within an 18-amino acid stretch, which, when in contact with liposomes, forms an amphipathic helix. Phosphorylation at S284 disrupts the formation of the amphipathic helix, hindering the association of WIPI2b with membranes and autophagosome formation. Understanding these intricacies in the regulatory mechanisms governing WIPI2b's association with its interacting partners and membranes, holds the potential to shed light on these complex processes, integral to phagophore biogenesis. Synopsis: WIPI2b is a novel ULK1 substrate which is phosphorylated at two key sites, S68 and S284. The phosphorylation at S68 and S284 of WIPI2b regulates its function at the phagophore disrupting the association with ATG16L1 and membranes. These results sheds light on the complex process of autophagosome biogenesis. WIPI2b is a novel ULK1 substrate, phosphorylated at S284 and S68. Phosphorylation at S68 reduces WIPI2b-ATG16L1 association. Phosphorylation at S284 prevents the formation of WIPI2bs amphipathic helix in the 6CD loop, likely disrupting membrane association. WIPI2b is a novel ULK1 substrate which is phosphorylated at two key sites, S68 and S284. The phosphorylation at S68 and S284 of WIPI2b regulates its function at the phagophore disrupting the association with ATG16L1 and membranes. These results sheds light on the complex process of autophagosome biogenesis. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome characterized by excessive fat deposition in hepatocytes and a major cause of end-stage liver disease. Autophagy is a metabolic pathway responsible for degrading cytoplasmic products and damaged organelles, playing a pivotal role in maintaining the homeostasis and functionality of hepatocytes. Recent studies have shown that pharmacological intervention to activate or restore autophagy provides benefits for liver function recovery by promoting the clearance of lipid droplets (LDs) in hepatocytes, decreasing the production of pro-inflammatory factors, and inhibiting activated hepatic stellate cells (HSCs), thus improving liver fibrosis and slowing down the progression of NAFLD. This article summarizes the physiological process of autophagy, elucidates the close relationship between NAFLD and autophagy, and discusses the effects of drugs on autophagy and signaling pathways from the perspectives of hepatocytes, kupffer cells (KCs), and HSCs to provide assistance in the clinical management of NAFLD. [ABSTRACT FROM AUTHOR]

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12–ATG5 conjugate to form an ATG12–ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.Abbreviation: ABE: acyl-biotin exchange; ATG: autophagy related; Baf-A1: bafilomycin A1; 2-BP: 2-bromopalmitate; CCD: coiled‐coil domain; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle’s balanced salt solution; HAM: hydroxylamine; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NP-40: Nonidet P-40; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex I; PTM: post-translational modification; RAB33B: RAB33B, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; WD: tryptophan and aspartic acid; WIPI2B: WD repeat domain, phosphoinositide interacting 2B; WT: wild-type; ZDHHC: zinc finger DHHC-type palmitoyltransferase. [ABSTRACT FROM AUTHOR]

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations: ABLV: Australian bat lyssavirus; ATG5: autophagy related 5; Baf A1:bafilomycin A1;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red fluorescent protein; MOI: multiplicity of infection; NC: negativecontrol; MVB: multivesicular body; NEDD4: neural precursorcell-expressed developmentally down-regulated 4; RABV: rabies virus;SQSTM1/p62: sequestosome 1; VLP: virus-like particle; VPS4B: vacuolarprotein sorting 4B; TEM: transmission electron microscopy; WB:western blotting; WT: wild-type; μm: micrometer; μM: micromole. [ABSTRACT FROM AUTHOR]

The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation.Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A1; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4’,6-diamidino-2’-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; μm: micrometer; μM: micromole. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis (CT) is the leading cause of bacterial sexually transmitted diseases worldwide, which can cause diseases such as pelvic inflammatory disease, and cervical and fallopian tube inflammation, and poses a threat to human health. Rosmarinic acid (RosA) is an active ingredient of natural products with anti-inflammatory and immunomodulatory effects. This study aimed to investigate the role of RosA in inhibiting autophagy-regulated immune cells-CD8+ T cells via the Ras/Raf/MEK/ERK signaling pathway in a CT-infected mouse model. Mice were inoculated with CT infection solution vaginally, and the mechanistic basis of RosA treatment was established using H&E staining, flow cytometry, immunofluorescence, transmission electron microscopy, and western blot. The key factors involved in RosA treatment were further validated using the MEK inhibitor cobimetinib. Experimental results showed that both RosA and the reference drug azithromycin could attenuate the pathological damage to the endometrium caused by CT infection; flow cytometry showed that peripheral blood CD8+ T cells increased after CT infection and decreased after treatment with RosA and the positive drug azithromycin (positive control); immunofluorescence showed that endometrial CD8 and LC3 increased after CT infection and decreased after RosA and positive drug treatment; the results of transmission electron microscopy showed that RosA and the positive drug azithromycin inhibited the accumulation of autophagosomes; western bolt experiments confirmed the activation of autophagy proteins LC3Ⅱ/Ⅰ, ATG5, Beclin-1, and p62 after CT infection, as well as the inhibition of Ras/Raf/MEK/ERK signaling. RosA and azithromycin inhibition of autophagy proteins activates Ras/Raf/MEK/ERK signaling. In addition, the MEK inhibitor cobimetinib attenuated RosA's protective effect on endometrium by further activating CD8+ T cells on a CT-induced basis, while transmission electron microscopy, immunofluorescence, and western blots showed that cobimetinib blocked ERK signals activation and further induced phagocytosis on a CT-induced basis. These data indicated that RosA can activate the Ras/Raf/MEK/ERK signaling pathway to inhibit autophagy, and RosA could also regulate the activation of immune cells-CD8+T cells to protect the reproductive tract of CT-infected mice. • RosA has a therapeutic effect on CT-induced reproductive tract infection in mice. • RosA can activate the Ras/Raf/MEK/ERK signaling pathway to inhibit autophagy, and can also regulate the activation of immune cells-CD8+T cells to protect the reproductive tract of CT-infected mice. • Natural compound RosA could inhibit inflammation caused by reproductive tract infection. [ABSTRACT FROM AUTHOR]

Autophagy is a cytoplasmic defense mechanism that cells use to break and reprocess their intracellular components. This utilization of autophagy is regarded as a savior in nutrient-deficient and other stressful conditions. Hence, autophagy keeps contact with and responds to miscellaneous cellular tensions and diverse pathways of signal transductions, such as growth signaling and cellular death. Importantly, autophagy is regarded as an effective tumor suppressor because regular autophagic breakdown is essential for cellular maintenance and minimizing cellular damage. However, paradoxically, autophagy has also been observed to promote the events of malignancies. This review discussed the dual role of autophagy in cancer, emphasizing its influence on tumor survival and progression. Possessing such a dual contribution to the malignant establishment, the prevention of autophagy can potentially advocate for the advancement of malignant transformation. In contrast, for the context of the instituted tumor, the agents of preventing autophagy potently inhibit the advancement of the tumor. Key regulators, including calpain 1, mTORC1, and AMPK, modulate autophagy in response to nutritional conditions and stress. Oncogenic mutations like RAS and B-RAF underscore autophagy's pivotal role in cancer development. The review also delves into autophagy's context-dependent roles in tumorigenesis, metastasis, and the tumor microenvironment (TME). It also discusses the therapeutic effectiveness of autophagy for several cancers. The recent implication of autophagy in the control of both innate and antibody-mediated immune systems made it a center of attention to evaluating its role concerning tumor antigens and treatments of cancer. [ABSTRACT FROM AUTHOR]

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion. Abbreviations: Cardiolipin: 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17. [ABSTRACT FROM AUTHOR]

In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome–lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication. [ABSTRACT FROM AUTHOR]

Synapse maintenance is essential for generating functional circuitry, and decrement in this process is a hallmark of neurodegenerative disease. Yet, little is known about synapse maintenance in vivo. Cysteine string protein a (CSPa), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single-nucleus transcriptomics on the cortex of young CSPa knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in the CSPa KO brain. Significantly, all neuronal classes in CSPa KO brains show strong signatures of repression in synaptic pathways, while up-regulating autophagy-related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. Glial responses varied by cell type, with microglia exhibiting activation. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPa KO mice. This was mediated by synaptogenic adhesion molecules, with the classical Neurexin1-Neuroligin 1 pair being the most prominent, suggesting that communication of glial cells with neurons is strengthened in CSPa KO mice to preserve synapse maintenance. Together, this study provides a rich dataset of transcriptional changes in the CSPa KO cortex and reveals insights into synapse maintenance and neurodegeneration. [ABSTRACT FROM AUTHOR]

Objective: Multiple myeloma, which affects plasma cells, is the second most common hematological malignancy. Despite the development of new drugs and treatment protocols, patient survival has not reached the desired level. In this study, we investigated the effects of Myxoma virus (MYXV), an oncolytic virus, on autophagy in myeloma cells. Materials and Methods: We analyzed protein expressions of ATG-5, p62, Beclin-1, LC3B, and the apoptosis marker Bcl-2 as autophagy markers in human U-266 and mouse MOPC-315 myeloma cell lines subjected to different doses of MYXV. In addition, autophagic images of myeloma cells were investigated using transmission electron microscopy (TEM). Results: In the first 24 h, which is the early stage of autophagy, ATG-5 and Beclin-1 expression levels were increased in the U-266 and MOPC- 315 cell lines in the groups that had received MYXV at a multiplicity of infection of 15. At 48 h, a significant increase was detected in the expression of LC3B, which is a late indicator. Autophagosomes were observed in myeloma cells by TEM. Conclusion: MYXV shows an antimyeloma effect by increasing autophagy in myeloma cells.

In the last two decades, alpha-synuclein (alpha-syn) assumed a prominent role as a major component and seeding structure of Lewy bodies (LBs). This concept is driving ongoing research on the pathophysiology of Parkinson's disease (PD). In line with this, alpha-syn is considered to be the guilty protein in the disease process, and it may be targeted through precision medicine to modify disease progression. Therefore, designing specific tools to block the aggregation and spreading of alpha-syn represents a major effort in the development of disease-modifying therapies in PD. The present article analyzes concrete evidence about the significance of alpha-syn within LBs. In this effort, some dogmas are challenged. This concerns the question of whether alpha-syn is more abundant compared with other proteins within LBs. Again, the occurrence of alpha-syn compared with non-protein constituents is scrutinized. Finally, the prominent role of alpha-syn in seeding LBs as the guilty structure causing PD is questioned. These revisited concepts may be helpful in the process of validating which proteins, organelles, and pathways are likely to be involved in the damage to meso-striatal dopamine neurons and other brain regions involved in PD. [ABSTRACT FROM AUTHOR]

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway that functions in nutrient recycling and as a mechanism of innate immunity. Previously, we reported a novel host–bacteria interaction between cariogenic S. mutans and bitter taste receptor (T2R14) in gingival epithelial cells (GECs), leading to an innate immune response. Further, S. mutans might be using the host immune system to inhibit other Gram-positive bacteria, such as S. aureus. To determine whether these bacteria exploit the autophagic machinery of GEC, it is first necessary to evaluate the role of T2R14 in modulating autophagic flux. So far, the role of T2R14 in the regulation of autophagy is not well characterized. Therefore, in this study, for the first time, we report that T2R14 downregulates autophagy flux in GECs, and T2R14 knockout increases acidic vacuoles. However, the treatments of GEC WT with a T2R14 agonist and antagonist did not lead to a significant change in acidic vacuole formation. Transmission electron microscopy morphometric results also suggested an increased number of autophagic vesicles in T2R14-knockout GEC. Further, our results suggest that S. mutans competence stimulating peptide CSP-1 showed robust intracellular calcium release and this effect is both T2R14- and autophagy protein 7-dependent. In this study, we provide the first evidence that T2R14 modulates autophagy flux in GEC. The results of the current study could help in identifying the impact of T2R in regulation of the immuno-microenvironment of GEC and subsequently oral health. [ABSTRACT FROM AUTHOR]

In eukaryotes, targeting intracellular components for lysosomal degradation by autophagy represents a catabolic process that evolutionarily regulates cellular homeostasis. The successful completion of autophagy initiates the engulfment of cytoplasmic materials within double-membrane autophagosomes and subsequent delivery to autolysosomes for degradation by acidic proteases. The formation of autolysosomes relies on the precise fusion of autophagosomes with lysosomes. In recent decades, numerous studies have provided insights into the molecular regulation of autophagosome–lysosome fusion. In this review, an overview of the molecules that function in the fusion of autophagosomes with lysosomes is provided. Moreover, the molecular mechanism underlying how these functional molecules regulate autophagosome–lysosome fusion is summarized. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a finely regulated process that involves the interaction of several cellular mechanisms to ensure the proper development and maturation of germ cells. This study assessed autophagy contribution and its relation to apoptosis in fish spermatogenesis during starvation. To that end, Nile tilapia males were subjected to 0 (control), 7, 14, 21, and 28 days of starvation to induce autophagy. Testes samples were obtained for analyses of spermatogenesis by histology, electron microscopy, immunohistochemistry, and western blotting. Sperm quality was assessed using a computer-assisted sperm analysis (CASA) system. Data indicated a significant reduction in gonadosomatic index, seminiferous tubule area, and spermatozoa proportion in fish subject to starvation compared to the control group. Immunoblotting revealed a reduction of Bcl2 and Beclin 1 associated with increased Bax and Caspase-3, mainly after 21 and 28 days of starvation. LC3 and P62 indicated reduced autophagic flux in these starvation times. Immunolabeling for autophagic and apoptotic proteins occurred in all development stages of the germ cells, but protein expression varied throughout starvation. Beclin 1 and Cathepsin D decreased while Bax and Caspase-3 increased in spermatocytes, spermatids, and spermatozoa after 21 and 28 days. Autophagic and lysosomal proteins colocalization indicated the fusion of autophagosomes with lysosomes and lysosomal degradation in spermatogenic cells. The CASA system indicated reduced sperm motility and velocity in animals subjected to 21 and 28 days of starvation. Altogether, the data support autophagy acting at different spermatogenesis stages in Nile tilapia, with decreased autophagy and increased apoptosis after 21 and 28 days of starvation, which results in a decrease in the spermatozoa number and sperm quality. [Display omitted] • Starvation induces autophagy during spermatogenesis in Nile tilapia. • LC3, Beclin-1, and Cathepsin D are expressed in germ and somatic cells. • Prolonged starvation decreases autophagy and increases apoptosis in the testis. • Sperm motility and velocity are reduced with 21 and 28 days of starvation. [ABSTRACT FROM AUTHOR]

Autophagy is a cellular process by which proteins and organelles are degraded inside the lysosome. Exercise is known to influence the regulation of autophagy in skeletal muscle. However, as gold standard techniques to assess autophagy flux in vivo are restricted to animal research, important gaps remain in our understanding of how exercise influences autophagy activity in humans. Using available datasets, we show how the gene expression profile of autophagy receptors and ATG8 family members differ between human and mouse skeletal muscle, providing a potential explanation for their differing exercise-induced autophagy responses. Furthermore, we provide a comprehensive view of autophagy regulation following exercise in humans by summarizing human transcriptomic and phosphoproteomic datasets that provide novel targets of potential relevance. These newly identified phosphorylation sites may provide an explanation as to why both endurance and resistance exercise lead to an exercise-induced reduction in LC3B-II, while possibly divergently regulating autophagy receptors, and, potentially, autophagy flux. We also provide recommendations to use ex vivo autophagy flux assays to better understand the influence of exercise, and other stimuli, on autophagy regulation in humans. This review provides a critical overview of the field and directs researchers towards novel research areas that will improve our understanding of autophagy regulation following exercise in humans. [ABSTRACT FROM AUTHOR]

Autophagy plays a fundamental role in maintaining cellular homeostasis by eliminating intracellular components via lysosomes. Successful degradation through autophagy relies on the fusion of autophagosomes to lysosomes, which leads to the formation of autolysosomes containing acidic proteases that degrade the sequestered materials. Viral infections can exploit autophagy in infected cells to balance virus–host cell interactions by degrading the invading virus or promoting viral growth. In recent years, cumulative studies have indicated that viral infections may interfere with the fusion of autophagosomes and lysosomes, thus benefiting viral replication and associated pathogenesis. In this review, I provide an overview of the current understanding of the molecular mechanism by which human viral infections deregulate autophagosome–lysosome fusion and summarize the physiological significance in the virus life cycle and host cell damage. [ABSTRACT FROM AUTHOR]

A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

Abstract Background Autophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several stages of autophagy, including autophagosome-late endosome/lysosome fusion. However, the role of Stx17 and potential mechanisms in autophagic defects induced by Meth remain poorly understood. Methods To address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the “cargos” mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays. Results Overexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aβ42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice. Conclusion Stx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury.

Abstract Background Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells. Results Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death. Conclusions Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress.

Abstract TRIM proteins are characterized by their conserved N-terminal RING, B-box, and coiled-coil domains. These proteins are efficient regulators of autophagy, apoptosis, and innate immune responses and confer immunity against viruses and bacteria. TRIMs function as receptors or scaffold proteins that target substrates for autophagy-mediated degradation. Most TRIMs interact with the BECN1-ULK1 complex to form TRIMosomes, thereby efficiently targeting substrates to autophagosomes. They regulate the functions of ATG proteins through physical interactions or ubiquitination. TRIMs affect the lipidation of MAP1LC3B1 to form MAP1LC3B2, which is a prerequisite for phagophore and autophagosome formation. In addition, they regulate MTOR kinase and TFEB, thereby regulating the expression of ATG genes. TRIM proteins are efficient regulators of apoptosis and are crucial for regulating cell proliferation and tumor formation. Many TRIM proteins regulate intrinsic and extrinsic apoptosis via the cell surface receptors TGFBR2, TNFRSF1A, and FAS. Mitochondria modulate the anti- and proapoptotic functions of BCL2, BAX, BAK1, and CYCS. These proteins use a multipronged approach to regulate the intrinsic and extrinsic apoptotic pathways, culminating in coordinated activation or inhibition of the initiator and executor CASPs. Furthermore, TRIMs can have a dual effect in determining cell fate and are therefore crucial for cellular homeostasis. In this review, we discuss mechanistic insights into the role of TRIM proteins in regulating autophagy and apoptosis, which can be used to better understand cellular physiology. These findings can be used to develop therapeutic interventions to prevent or treat multiple genetic and infectious diseases. Graphical Abstract

Macroautophagy/autophagy is a highly conserved metabolic process that degrades intracellular components and recycles bioenergetic substrates. SQSTM1/p62 (sequestosome 1) is a classical autophagy receptor that participates in selective autophagy to eliminate abnormal intracellular components and recycle bioenergetic substrates. In autophagy, SQSTM1 recruits ubiquitinated substrates to form SQSTM1 droplets and delivers these cargoes to phagophores, the precursors to autophagosomes. Recently, we reported a previously unidentified SQSTM1 S-acylation, which is catalyzed by S-acyltransferase ZDHHC19 and reversed by LYPLA1/APT1. S-acylation of SQSTM1 enhances the affinity of SQSTM1 droplets with the phagophore membrane, thereby promoting efficient autophagic degradation of ubiquitinated substrates. Our study uncovers the role of the S-acylation-deacylation cycle in regulating SQSTM1-mediated selective autophagy. [ABSTRACT FROM AUTHOR]

AbstractContext Current chemotherapeutic drugs cannot meet the treatment needs of patients with nasopharyngeal carcinoma (NPC), so urgent action is needed to discover novel chemotherapeutic agents. Our previous study revealed that garcinone E (GE) inhibited the proliferation and metastasis of NPC, suggesting that the compound might display promising anticancer activity.Objective To examine the mechanism underlying the anti-NPC activity of GE for the first time.Materials and methods For MTS assay, NPC cells were treated with 2.5-20 μmol/L GE or dimethyl sulfoxide for 24, 48, and 72 h. Colony formation capacity, cell cycle distribution, and in vivo xenograft experiment of GE were assessed. MDC staining, StubRFP-sensGFP-LC3 observation, LysoBrite Blue staining, and immunofluorescence examined the autophagy of NPC cells after GE exposure. Western blotting, RNA-sequencing, and RT-qPCR measured protein and mRNA levels.Results GE suppressed cell viability with an IC50 of 7.64, 8.83 and 4.65 μmol/L for HK1, HONE1 and S18 cells. GE inhibited colony formation and cell cycle, increased autophagosome number, and inhibited the autophagic flux partially by blocking lysosome-autophagosome fusion, and repressed S18 xenograft growth. GE dysregulated the expression of autophagy- and cell cycle-related proteins such as Beclin-1, SQSTM1/p62, LC3, CDKs, and Cyclins. Bioinformatics GO and KEGG pathway enrichment analysis of RNA-seq showed that autophagy was enriched in differentially expressed genes upon GE treatment.Discussion and conclusion GE acts as an autophagic flux inhibitor, which may have potential chemotherapeutic use for NPC treatment and may have an application in basic research to explore the mechanisms of autophagy.

Key message: Comprehensive analysis of Gossypium ATG8 family indicates that GhATG8f could improve salt tolerance of cotton by increasing SOD, POD and CAT activity and proline accumulation. In plants, autophagy is regulated by several genes that play important roles in initiating and controlling the process. ATG8, functioning as a protein similar to ubiquitin, is involved in crucial tasks throughout the autophagosome formation process. In this research, we conducted an extensive and all-encompassing investigation of 64 ATG8 genes across four varieties of cotton. According to the subcellular localization prediction results, 49 genes were found in the cytoplasm, 6 genes in the chloroplast, 1 gene in the peroxisome, 5 genes in the nucleus, and 3 genes in the extracellular region. Phylogenetic analysis categorized a total of 5 subfamilies containing sixty-four ATG8 genes. The expression of the majority of GhATG8 genes was induced by salt, drought, cold, and heat stresses, as revealed by RNA-seq and real-time PCR. Analysis of cis-elements in the promoters of GhATG8 genes revealed the predominant presence of responsive elements for plant hormones and abiotic stress, suggesting that GhATG8 genes might have significant functions in abiotic stress response. Furthermore, we additionally performed a gene interaction network analysis for the GhATG8 proteins. The salt stress resistance of cotton was reduced due to the downregulation of GhATG8f expression, resulting in decreased activity of CAT, SOD, and POD enzymes, as well as decreased fresh weight and proline accumulation. In summary, our research is the initial exploration of ATG8 gene components in cotton, providing a basis for future investigations into the regulatory mechanisms of ATG8 genes in autophagy and their response to abiotic stress. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved intracellular degradation mechanism. The most distinctive feature of autophagy is the formation of double-membrane structures called autophagosomes, which compartmentalize portions of the cytoplasm. The outer membrane of the autophagosome fuses with the vacuolar/lysosomal membrane, leading to the degradation of the contents of the autophagosome. Approximately 30 years have passed since the identification of autophagy-related (ATG) genes and Atg proteins essential for autophagosome formation, and the primary functions of these Atg proteins have been elucidated. These achievements have significantly advanced our understanding of the mechanism of autophagosome formation. This article summarizes our current knowledge on how the autophagosome precursor is generated, and how the membrane expands and seals to complete the autophagosome. [ABSTRACT FROM AUTHOR]

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

Background: Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells. Results: Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death. Conclusions: Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress. [ABSTRACT FROM AUTHOR]

TRIM proteins are characterized by their conserved N-terminal RING, B-box, and coiled-coil domains. These proteins are efficient regulators of autophagy, apoptosis, and innate immune responses and confer immunity against viruses and bacteria. TRIMs function as receptors or scaffold proteins that target substrates for autophagy-mediated degradation. Most TRIMs interact with the BECN1-ULK1 complex to form TRIMosomes, thereby efficiently targeting substrates to autophagosomes. They regulate the functions of ATG proteins through physical interactions or ubiquitination. TRIMs affect the lipidation of MAP1LC3B1 to form MAP1LC3B2, which is a prerequisite for phagophore and autophagosome formation. In addition, they regulate MTOR kinase and TFEB, thereby regulating the expression of ATG genes. TRIM proteins are efficient regulators of apoptosis and are crucial for regulating cell proliferation and tumor formation. Many TRIM proteins regulate intrinsic and extrinsic apoptosis via the cell surface receptors TGFBR2, TNFRSF1A, and FAS. Mitochondria modulate the anti- and proapoptotic functions of BCL2, BAX, BAK1, and CYCS. These proteins use a multipronged approach to regulate the intrinsic and extrinsic apoptotic pathways, culminating in coordinated activation or inhibition of the initiator and executor CASPs. Furthermore, TRIMs can have a dual effect in determining cell fate and are therefore crucial for cellular homeostasis. In this review, we discuss mechanistic insights into the role of TRIM proteins in regulating autophagy and apoptosis, which can be used to better understand cellular physiology. These findings can be used to develop therapeutic interventions to prevent or treat multiple genetic and infectious diseases. [ABSTRACT FROM AUTHOR]

Background: Autophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several stages of autophagy, including autophagosome-late endosome/lysosome fusion. However, the role of Stx17 and potential mechanisms in autophagic defects induced by Meth remain poorly understood. Methods: To address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the "cargos" mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays. Results: Overexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aβ42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice. Conclusion: Stx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury. [ABSTRACT FROM AUTHOR]

Simple Summary: This article focuses on the impairment of lysosomes in the context of lysosomal storage disorders. Lysosomal storage disorders are a group of rare diseases with different causes united by the malfunctioning of the lysosomes. Lysosomes are intracellular vesicles, and their main function is to decompose intracellular waste in a process known as autophagy. Besides this function, lysosomes participate in a wide range of essential mechanisms aimed to keep the internal balance within our cells, which is called homeostasis. Maintaining homeostasis is a fundamental goal of all biological systems, from the simplest to the most complex, and is essential for proper functioning. Many of these disorders are originated from single-gene mutations. This provides a valuable starting point for scientists to trace the path from a single molecule to disease symptoms across the complexity of living organisms. The purpose of this paper is to provide insights from the molecular to the clinical level on this group of diseases, focusing on changes in autophagy and the latest therapeutic approaches in the field. Lysosomes are the main organelles responsible for the degradation of macromolecules in eukaryotic cells. Beyond their fundamental role in degradation, lysosomes are involved in different physiological processes such as autophagy, nutrient sensing, and intracellular signaling. In some circumstances, lysosomal abnormalities underlie several human pathologies with different etiologies known as known as lysosomal storage disorders (LSDs). These disorders can result from deficiencies in primary lysosomal enzymes, dysfunction of lysosomal enzyme activators, alterations in modifiers that impact lysosomal function, or changes in membrane-associated proteins, among other factors. The clinical phenotype observed in affected patients hinges on the type and location of the accumulating substrate, influenced by genetic mutations and residual enzyme activity. In this context, the scientific community is dedicated to exploring potential therapeutic approaches, striving not only to extend lifespan but also to enhance the overall quality of life for individuals afflicted with LSDs. This review provides insights into lysosomal dysfunction from a molecular perspective, particularly in the context of human diseases, and highlights recent advancements and breakthroughs in this field. [ABSTRACT FROM AUTHOR]

Objective: Multiple myeloma, which affects plasma cells, is the second most common hematological malignancy. Despite the development of new drugs and treatment protocols, patient survival has not reached the desired level. In this study, we investigated the effects of Myxoma virus (MYXV), an oncolytic virus, on autophagy in myeloma cells. Materials and Methods: We analyzed protein expressions of ATG-5, p62, Beclin-1, LC3B, and the apoptosis marker Bcl-2 as autophagy markers in human U-266 and mouse MOPC-315 myeloma cell lines subjected to different doses of MYXV. In addition, autophagic images of myeloma cells were investigated using transmission electron microscopy (TEM). Results: In the first 24 h, which is the early stage of autophagy, ATG-5 and Beclin-1 expression levels were increased in the U-266 and MOPC-315 cell lines in the groups that had received MYXV at a multiplicity of infection of 15. At 48 h, a significant increase was detected in the expression of LC3B, which is a late indicator. Autophagosomes were observed in myeloma cells by TEM. Conclusion: MYXV shows an antimyeloma effect by increasing autophagy in myeloma cells. [ABSTRACT FROM AUTHOR]

Autophagosome biogenesis, from the appearance of the phagophore to elongation and closure into an autophagosome, is one of the long‐lasting open questions in the autophagy field. Recent studies utilising cryo‐electron tomography and detailed analysis of the image data have revealed new information on the membrane dynamics of these events, including the shape and dimensions of omegasomes, phagophores and autophagosomes, and their relationships with the organelles around them. One of the important predictions from the new results is that 60–80% of the autophagosome membrane area is delivered by direct lipid transfer or lipid synthesis. Cryo‐electron tomography can be expected to provide new directions for autophagy research in the near future. [ABSTRACT FROM AUTHOR]

In the past decade, tumor treatment strategies have shifted from tumors to the immune system of tumor patients. NIR laser-induced photothermal therapy can not only generate heat or reactive oxygen species to kill tumors but also induce immunogenic cell death and activate the overall antitumor immune response. The ICD process is usually accompanied by autophagosome formation, which contains tumor-associated antigens. However, the fusion of autophagosome and lysosome impairs antigens, weakening the antitumor immune response. Thus, administration with autophagy terminal inhibitors could be a compensatory strategy. In this work, we prepared a thermally sensitive liposome drug delivery system containing the photothermal agent IR780 and autophagy inhibitor HCQ (IR780/HCQ-Lip). IR780/HCQ-Lip has a uniform particle size distribution and good photodynamic and photothermal properties. The inhibition of autophagy enhanced the phototherapy effect. Compared with the survival rate of 4T1 cells being treated with IR780-Lip phototherapy alone (36.87%), the survival rate of the IR780/HCQ-Lip group decreased to 3.71%. IR780/HCQ-Lip caused a massive accumulation of autophagosomes, promoted the maturation of dendritic cells (DCs), and enhanced the infiltration of intratumoral T cells. In addition, IR780/HCQ-Lip produced a systemic immune response and also showed good inhibition of distal tumor growth under laser action in distal tumor experiments. Thus, IR780/HCQ-Lip exhibited a promising combination therapy for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Current chemotherapeutic drugs cannot meet the treatment needs of patients with nasopharyngeal carcinoma (NPC), so urgent action is needed to discover novel chemotherapeutic agents. Our previous study revealed that garcinone E (GE) inhibited the proliferation and metastasis of NPC, suggesting that the compound might display promising anticancer activity. To examine the mechanism underlying the anti-NPC activity of GE for the first time. For MTS assay, NPC cells were treated with 2.5-20 μmol/L GE or dimethyl sulfoxide for 24, 48, and 72 h. Colony formation capacity, cell cycle distribution, and in vivo xenograft experiment of GE were assessed. MDC staining, StubRFP-sensGFP-LC3 observation, LysoBrite Blue staining, and immunofluorescence examined the autophagy of NPC cells after GE exposure. Western blotting, RNA-sequencing, and RT-qPCR measured protein and mRNA levels. GE suppressed cell viability with an IC50 of 7.64, 8.83 and 4.65 μmol/L for HK1, HONE1 and S18 cells. GE inhibited colony formation and cell cycle, increased autophagosome number, and inhibited the autophagic flux partially by blocking lysosome-autophagosome fusion, and repressed S18 xenograft growth. GE dysregulated the expression of autophagy- and cell cycle-related proteins such as Beclin-1, SQSTM1/p62, LC3, CDKs, and Cyclins. Bioinformatics GO and KEGG pathway enrichment analysis of RNA-seq showed that autophagy was enriched in differentially expressed genes upon GE treatment. GE acts as an autophagic flux inhibitor, which may have potential chemotherapeutic use for NPC treatment and may have an application in basic research to explore the mechanisms of autophagy. [ABSTRACT FROM AUTHOR]

Over the past decade, accumulated studies have reported the presence of non-canonical macroautophagy/autophagy characterized by the shared usage of the autophagy machinery and distinct components that function in multiple scenarios but do not involve lysosomal degradation. One type of non-canonical autophagy is secretory autophagy, which facilitates the secretion of various cargoes. In a recent work from Gao et al. the ER-membrane protein STING1 has been identified as a novel substrate of secretory autophagy. The secretion of activated STING1 is mediated by its packing into the rafeesome, a newly identified organelle formed upon the fusion of RAB22A-mediated non-canonical autophagosome with an early endosome. Moreover, extracellular vesicles containing activated STING1 induce antitumor immunity in recipient cells, a process potentially promoted by RAB22A. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of lidocaine on mitophagy in model rat of acute lung injury(ALI). Methods The rats were divided into the control group, acute lung injury group (group ALI:intraperitoneal injection of 10 mg/kg of lipopolysaccharide(LPS) and lidocaine intervention group (group lidocaine:after intraperitoneal injection of LPS,receiving a loading dose of lidocaine of 10 mg/kg and continued for 3 h with dosage of 10 mg/kg per hour). The rats were sacrificed 24 hrs after LPS, blood was collected for blood gas analysis, and calculated for oxygenation index (OI).Thoracotomy was performed immediately after blood collection, the samples of left lung tissue were collected to determine the expression of mitophagy-related protein E3 ubiquitin ligase (Parkin), microtubule-associated protein 1 light chain 3 (LC3) and p62 by Western blot, and then LC3-Ⅱ/LC3-Ⅰ was calculated. After-wards, right-upper lung tissue structure was microscopy and the mitochondrial autophagy in the right lower lung tissue was examined by transmission electron microscopy. Results Compared with control group, the level of Parkin, LC3-Ⅱ/LC3-Ⅰ and PaCO2 was increased in both group ALI and group lidocaine(P＜0.05),while PaO2,OI,pH and p62 were decreased(P＜0.05). Compared with group ALI,the level of Parkin and LC3-Ⅱ/LC3-Ⅰ was decreased in group of lidocaine intervention (P＜0.05),while PaO2,OI,pH and p62 were increased(P＜0.05).The results of light microscopy showed intact alveolar structure and normal cell morphology in group control, inflammatory cell infiltration, interval thickening and alveolar collapse were found in group ALI, and less degree in group lidocaine than in group ALI. Transmission electron microscopy showed that the lung tissue and mitochondrial structure were normal in group control; in group ALI, the lung tissue cell was found to be swollen and formed with physalides companying by mitochondrial autophagosomes; The degree of injury in group lidocaine was decreased as compared with group ALI. Conclusions Lidocaine attenuates acute lung injury and the mechanism may be related to inhibition of mitophagy hyperactivation.

Autophagy plays a vital role in cell homeostasis by eliminating nonfunctional components and promoting cell survival. Here, we examined the levels of autophagy signaling proteins after 7 days of overload hypertrophy in the extensor digitorum longus (EDL) and soleus muscles of control and diabetic rats. We compared control and 3‐day streptozotocin‐induced diabetic rats, an experimental model for type 1 diabetes mellitus (T1DM). EDL muscles showed increased levels of basal autophagy signaling proteins. The diabetic state did not affect the extent of overload‐induced hypertrophy or the levels of autophagy signaling proteins (p‐ULK1, Beclin‐1, Atg5, Atg12‐5, Atg7, Atg3, LC3‐I and II, and p62) in either muscle. The p‐ULK‐1, Beclin‐1, and p62 protein expression levels were higher in the EDL muscle than in the soleus before the hypertrophic stimulus. On the contrary, the soleus muscle exhibited increased autophagic signaling after overload‐induced hypertrophy, with increases in Beclin‐1, Atg5, Atg12‐5, Atg7, Atg3, and LC3‐I expression in the control and diabetic groups, in addition to p‐ULK‐1 in the control groups. After hypertrophy, Beclin‐1 and Atg5 levels increased in the EDL muscle of both groups, while p‐ULK1 and LC3‐I increased in the control group. In conclusion, the baseline EDL muscle exhibited higher autophagy than the soleus muscle. Although TDM1 promotes skeletal muscle mass loss and strength reduction, it did not significantly alter the extent of overload‐induced hypertrophy and autophagy signaling proteins in EDL and soleus muscles, with the two groups exhibiting different patterns of autophagy activation.

BackgroundAutophagosome, the central organelle in autophagy process, can assemble via canonical pathway mediated by LC3-II, the lipidated form of autophagy-related protein LC3/ATG8, or noncanonical pathway mediated by the small GTPase Rab9. Canonical autophagy is essential for exocrine pancreas homeostasis, and its disordering initiates and drives pancreatitis. The involvement of noncanonical autophagy has not been explored. We examine the role of Rab9 in pancreatic autophagy and pancreatitis severity.MethodsWe measured the effect of Rab9 on parameters of autophagy and pancreatitis responses using transgenic mice overexpressing Rab9 (Rab9TG) and adenoviral transduction of acinar cells. Effect of canonical autophagy on Rab9 was assessed in ATG5-deficient acinar cells.ResultsPancreatic levels of Rab9 and its membrane-bound (active) form decreased in rodent pancreatitis models and in human disease. Rab9 overexpression stimulated noncanonical and inhibited canonical/LC3-mediated autophagosome formation in acinar cells through up-regulation of ATG4B, the cysteine protease that delipidates LC3-II. Conversely, ATG5 deficiency caused Rab9 increase in acinar cells. Inhibition of canonical autophagy in Rab9TG pancreas was associated with accumulation of Rab9-positive vacuoles containing markers of mitochondria, protein aggregates, and trans-Golgi. The shift to the noncanonical pathway caused pancreatitis-like damage in acinar cells and aggravated experimental pancreatitis.ConclusionsThe results show that Rab9 regulates pancreatic autophagy and indicate a mutually antagonistic relationship between the canonical/LC3-mediated and noncanonical/Rab9-mediated autophagy pathways in pancreatitis. Noncanonical autophagy fails to substitute for its canonical counterpart in protecting against pancreatitis. Thus, Rab9 decrease in experimental and human pancreatitis is a protective response to sustain canonical autophagy and alleviate disease severity.

The recruitment of ATG12–ATG5-ATG16L1 complex to phagophore mediated by the specific interaction between ATG16L1 and WIPI2, is pivotal to the formation of autophagosomes during macroautophagy. Recently, we reported that ATG16L1 contains two distinct WIPI2-binding sites, the previously reported WIPI2-binding site (WBS1), and the newly identified site (WBS2). By determining the crystal structures of WIPI2 with ATG16L1 WBS1 and WBS2 respectively, we uncovered that, unlike ATG16L1 WBS1, ATG16L1 WBS2 and its binding mechanism to WIPI2 are conserved from yeast to mammals. Using cell-based functional assays, we further demonstrated that the integrity of two WIPI2-binding sites of ATG16L1 is essential for normal autophagic flux. In summary, our study provided mechanistic insights into the interaction of two key autophagic proteins, ATG16L1 and WIPI2, and revealed a dual-binding-site mode adopted by ATG16L1 to associate with WIPI2. Abbreviations: ATG: autophagy-related protein; CCD: coiled-coil domain; ITC: isothermal titration calorimetry; PI3KC3-C1: class III phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; ULK: Unc-51-like kinase; WBS: WIPI2-binding site; WIPI: WD repeat domain phosphoinositide-interacting protein. [ABSTRACT FROM AUTHOR]

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.