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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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

BACKGROUND: Meningiomas are one of the most common benign tumors in the intracranial field, and clinical treatment is mainly surgery. However, the postoperative recurrence rate is high, and other treatment modalities do not yet have a definite effect, so the exploration of new targeted drugs is promising for meningioma treatment. At present, the pathogenesis of autophagy in benign meningiomas is not very clear, and a large number of experiments are still needed for in-depth study. OBJECTIVE: To investigate the ultrastructural features and clinical significance of autophagy in benign meningiomas. METHODS: Meningiomas were diagnosed by light microscopy with different subtyping. Diagnosis and typing of meningiomas were aided by transmission electron microscopy, and the ultrastructural morphology and number of autophagosomes in the cytoplasm of meningioma cells of different grades were observed. Western blot assay was used to detect the expression levels of autophagy-related proteins Beclin1 and LC3 in different grades of meningiomas. The altered expression of Vimentin, EMA, PR, S-100 and GFAP in meningiomas was evaluated by immunohistochemical staining. RESULTS AND CONCLUSION: 176 meningiomas were identified, including 98 males and 78 females. (1) Histopathological types CNS WHO grade 1 benign meningiomas comprised a total of 168 cases, including 62 cases of meningocortical type, 36 cases of fibrous type, 58 cases of transitional type, 12 cases of angiomatous type, 4 cases of CNS WHO grade 2 clear cell type, 2 cases of atypical type, and 2 cases of WHO grade 3 anaplastic lesions. (2) Transmission electron microscopy revealed autophagic bodies in 27 cases of WHO grade 1 meningiomas, and no autophagic bodies were detected in WHO grades 2 and 3 meningiomas. (3) LC3 and Beclin1 were highly expressed in WHO grade 1 benign meningiomas by western blot assay and were negatively expressed in WHO grades 2 and 3 meningiomas. (4) The immunohistochemical markers Vimentin, EMA, and PR, were expressed to various degrees, while GFAP and S-100 were negative. (5) A combination of electron microscopy, immunohistochemical staining and western blot assay showed that autophagy only plays an important role in the occurrence and development of WHO grade 1 benign meningiomas. [ABSTRACT FROM AUTHOR]

Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this context, it was demonstrated that autophagy plays an important role in megakaryopoiesis, the development and differentiation of hematopoietic progenitor cells into megakaryocytes. Furthermore, in recent years, autophagic proteins were detected in platelets, anucleate cells generated by megakaryocytes, responsible for hemostasis, thrombosis, and a key cell in inflammation and host immune responses. In the last decade studies have indicated the occurrence of autophagy in platelets. Moreover, autophagy in platelets was subsequently demonstrated to be involved in platelet aggregation, adhesion, and thrombus formation. Here, we review the current knowledge about autophagy in platelets, its function, and clinical implications. However, at the advent of platelet autophagy research, additional discoveries derived from evolving work will be required to precisely define the contributions of autophagy in platelets, and to expand the ever increasing physiologic and pathologic roles these remarkable and versatile blood cells play. • Autophagy plays an important role in megakaryopoiesis. • Autophagosome formation and autophagy are essential in platelet biology. • Autophagy in platelets is involved in platelet aggregation, adhesion, and thrombus formation. • The importance of platelet autophagic functions is demonstrated in clinically relevant conditions and disease states, such as diabetes, hypoxia/ischemia, ITP, and sepsis. • Elaborate methods are available to study autophagy in platelets reliably and in great detail. [ABSTRACT FROM AUTHOR]

Neuroinflammation is an early event during the pathogenesis of neurodegenerative disorders. Most studies focus on how the factors derived from pathogens or tissue damage activate the inflammation-pyroptosis cell death pathway. It is unclear whether endogenous neurotransmitters could induce inflammatory responses in neurons. Our previous reports have shown that dopamine-induced elevation of intracellular Zn2+ concentration via the D1-like receptor (D1R) is a prerequisite for autophagy and cell death in primary cultured rat embryonic neurons. Here we further examined that this D1R-Zn2+ signaling initiates the transient inflammatory response leading to cell death in cultured cortical neurons. Pretreating the cultured neurons with Zn2+ chelator and inhibitors against inflammation could enhance the cell viability in neurons treated with dopamine and dihydrexidine, an agonist of D1R. Both dopamine and dihydrexidine greatly enhanced inflammasome formation; a Zn2+ chelator, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine, suppressed this increment. Dopamine and dihydrexidine increased the expression levels of NOD-like receptor pyrin domain-containing protein 3 and enhanced the maturation of caspase-1, gasdermin D, and IL-1β; these changes were all Zn2+-dependent. Dopamine treatment did not recruit the N-terminal of the gasdermin D to the plasma membrane but enhanced its localization to the autophagosomes. Pretreating the neurons with IL-1β could increase the viability of neurons challenged with dopamine. These results demonstrate a novel D1R-Zn2+ signaling cascade activating neuroinflammation and cell death. Therefore, maintaining a balance between dopamine homeostasis and inflammatory responses is an important therapeutic target for neurodegeneration. Dopamine elicits transient inflammatory responses in cultured cortical neurons via the D1R−Zn2+ signaling pathway. Dopamine elevates [Zn2+]i to induce the formation of inflammasomes, which activates caspase-1, resulting in the maturation of IL-1β and gasdermin D (GSDMD). Therefore, the homeostasis of dopamine and Zn2+ are critical therapeutic targets for inflammation-derived neurodegeneration. [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. [ABSTRACT FROM AUTHOR]

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin–proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin–RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor-cell-therapy resistance. [ABSTRACT FROM AUTHOR]

Macroautophagy is characterized by the de novo formation of double-membrane vesicles termed autophagosomes. The precursor structure of autophagosomes is a membrane cistern called phagophore, which elongates through a massive acquisition of lipids until closure. The phagophore establishes membrane-contact sites (MCSs) with the endoplasmic reticulum (ER), where conserved ATG proteins belonging to the ATG9 lipid scramblase, ATG2 lipid transfer and Atg18/WIPI4 β-propeller families concentrate. Several recent in vivo and in vitro studies have uncovered the relevance of these proteins and MCSs in the lipid supply required for autophagosome formation. Although important conceptual advances have been reached, the functional interrelationship between ATG9, ATG2 and Atg18/WIPI4 proteins at the phagophore-ER MCSs and their role in the phagophore expansion are not completely understood. In this review, we describe the current knowledge about the structure, interactions, localizations, and molecular functions of these proteins, with a particular emphasis on the yeast Saccharomyces cerevisiae and mammalian systems. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction, causing increased reactive oxygen species (ROS) production, is a molecular feature of heart failure (HF). A defective antioxidant response and mitophagic flux were reported in circulating leucocytes of patients with chronic HF and reduced ejection fraction (HFrEF). Atrial natriuretic peptide (ANP) exerts many cardiac beneficial effects, including the ability to protect cardiomyocytes by promoting autophagy. We tested the impact of ANP on autophagy/mitophagy, altered mitochondrial structure and function and increased oxidative stress in HFrEF patients by both ex vivo and in vivo approaches. The ex vivo study included thirteen HFrEF patients whose peripheral blood mononuclear cells (PBMCs) were isolated and treated with αANP (10–11 M) for 4 h. The in vivo study included six HFrEF patients who received sacubitril/valsartan for two months. PBMCs were characterized before and after treatment. Both approaches analyzed mitochondrial structure and functionality. We found that levels of αANP increased upon sacubitril/valsartan, whereas levels of NT-proBNP decreased. Both the ex vivo direct exposure to αANP and the higher αANP level upon in vivo treatment with sacubitril/valsartan caused: (i) improvement of mitochondrial membrane potential; (ii) stimulation of the autophagic process; (iii) significant reduction of mitochondrial mass—index of mitophagy stimulation—and upregulation of mitophagy-related genes; (iv) reduction of mitochondrial damage with increased inner mitochondrial membrane (IMM)/outer mitochondrial membrane (OMM) index and reduced ROS generation. Herein we demonstrate that αANP stimulates both autophagy and mitophagy responses, counteracts mitochondrial dysfunction, and damages ultimately reducing mitochondrial oxidative stress generation in PBMCs from chronic HF patients. These properties were confirmed upon sacubitril/valsartan administration, a pivotal drug in HFrEF treatment. [ABSTRACT FROM AUTHOR]

Autophagy–the lysosomal degradation of cytoplasm–plays a central role in cellular homeostasis and protects cells from potentially harmful agents that may accumulate in the cytoplasm, including pathogens, protein aggregates, and dysfunctional organelles. This process is initiated by the formation of a phagophore membrane, which wraps around a portion of cytoplasm or cargo and closes to form a double-membrane autophagosome. Upon the fusion of the autophagosome with a lysosome, the sequestered material is degraded by lysosomal hydrolases in the resulting autolysosome. Several alternative membrane sources of autophagosomes have been proposed, including the plasma membrane, endosomes, mitochondria, endoplasmic reticulum, lipid droplets, hybrid organelles, and de novo synthesis. Here, we review recent progress in our understanding of how the autophagosome is formed and highlight the proposed role of vesicles that contain the lipid scramblase ATG9 as potential seeds for phagophore biogenesis. We also discuss how the phagophore is sealed by the action of the endosomal sorting complex required for transport (ESCRT) proteins. [ABSTRACT FROM AUTHOR]

Doxorubicin (Dox), an anthracycline antibiotic, is an anticancer drug that inhibits DNA replication and cellular metabolic processes in cancer cells with high proliferative potential. However, Dox causes severe side effects, including myocardial damage and heart failure, but the molecular mechanism underlying Dox-induced myocardial injury remains uncertain. In the present study, we evaluated the effects of Dox on the mitochondrial quality control system and regulation of mitochondrial respiration and autophagy in an in vitro rat myoblast H9c2 cell culture model using western blotting, immunohistochemistry, the Seahorse XF24 system, and flow cytometry. Our results showed that Dox did not impair the initiation of autophagic flux or the functions of lysosomes; however, Dox affected the mitochondrial quality control system, leading to a fission-dominant morphology and impaired regulation of mitochondrial respiration, thereby increasing oxidative stress and inhibited progression of autophagy, particularly the fusion of autophagosomes with lysosomes. This inhibition caused a significant decrease in the formation of autolysosomes and was responsible for the accumulation of dysfunctional mitochondria and subsequent increase in oxidative stress, eventually leading to increased myocardial cell death. [Display omitted] • Doxorubicin impaired the regulation of mitochondrial respiration. • Doxorubicin increased mitochondrial reactive oxygen species production. • Doxorubicin inhibited the progression of autophagy, which increased cell death. • Doxorubicin inhibited interaction of the SNARE molecules SNAP29, STX17, and VAMP8. [ABSTRACT FROM AUTHOR]

Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) is a very serious clinical traumatic illness with a very high disability rate. It not only causes serious functional disorders below the injured segment, but also causes unimaginable economic burden to social development. Exosomes are nano-sized cellular communication carriers that exist stably in almost all organisms and cell types. Because of their capacity to transport proteins, lipids, and nucleic acids, they affect various physiological and pathological functions of recipient cells and parental cells. Autophagy is a process that relies on the lysosomal pathway to degrade cytoplasmic proteins and organelles and involves a variety of pathophysiological processes. Exosomes and autophagy play critical roles in cellular homeostasis following spinal cord injury. Presently, the coordination mechanism of exosomes and autophagy has attracted much attention in the early efficacy of spinal cord injury. In this review, we discussed the interaction of autophagy and exosomes from the perspective of molecular mechanisms, which might provide novel insights for the early therapeutic application of spinal cord injury. [ABSTRACT FROM AUTHOR]

Glutamine is one of the most abundant amino acids in the cell. In mitochondria, glutaminases 1 and 2 (GLS1/2) hydrolyze glutamine to glutamate, which serves as the precursor of multiple metabolites. Here, we show that ammonium generated during GLS1/2-mediated glutaminolysis regulates lysosomal pH and in turn lysosomal degradation. In primary human skin fibroblasts BJ cells and mouse embryonic fibroblasts, deprivation of total amino acids for 1 h increased lysosomal degradation capacity as shown by the increased turnover of lipidated microtubule-associated proteins 1A/1B light chain 3B (LC3-II), several autophagic receptors, and endocytosed DQ-BSA. Removal of glutamine but not any other amino acids from the culture medium enhanced lysosomal degradation similarly as total amino acid starvation. The presence of glutamine in regular culture media increased lysosomal pH by >0.5 pH unit and the removal of glutamine caused lysosomal acidification. GLS1/2 knockdown, GLS1 antagonist, or ammonium scavengers reduced lysosomal pH in the presence of glutamine. The addition of glutamine or NH4Cl prevented the increase in lysosomal degradation and curtailed the extension of mTORC1 function during the early time period of amino acid starvation. Our findings suggest that glutamine tunes lysosomal pH by producing ammonium, which regulates lysosomal degradation to meet the demands of cellular activities. During the early stage of amino acid starvation, the glutamine-dependent mechanism allows more efficient use of internal reserves and endocytosed proteins to extend mTORC1 activation such that the normal anabolism is not easily interrupted by a brief disruption of the amino acid supply. [ABSTRACT FROM AUTHOR]

Eukaryotes utilize different communication strategies to coordinate processes between different cellular compartments either indirectly, through vesicular transport, or directly, via membrane contact sites (MCSs). MCSs have been implicated in lipid metabolism, calcium signaling and the regulation of organelle biogenesis in various cell types. Several studies have shown that MCSs play a crucial role in the regulation of macroautophagy, an intracellular catabolic transport route that is characterized by the delivery of cargoes (proteins, protein complexes or aggregates, organelles and pathogens) to yeast and plant vacuoles or mammalian lysosomes, for their degradation and recycling into basic metabolites. Macroautophagy is characterized by the de novo formation of double-membrane vesicles called autophagosomes, and their biogenesis requires an enormous amount of lipids. MCSs appear to have a central role in this supply, as well as in the organization of the autophagy-related (ATG) machinery. In this review, we will summarize the evidence for the participation of specific MCSs in autophagosome formation, with a focus on the budding yeast and mammalian systems. [ABSTRACT FROM AUTHOR]

Objective To assess whether 2, 4-dinitrophenol（DNP） has the ability of radio sensitization. Methods The MTT method and cell clone formation experiment were used to detect the effect of different concentrations of DNP on the proliferation of Hela and KB cells. The cell clone formation experiment was also used to detect the radio sensitizing effect of DNP on Hela and KB cells. Flow cytometry was used to detect the change of the Hela and KB cells cycle and mitochondrial membrane potential in different conditions, and the transmission electron microscope was used to observe the formation of the autophagosome. The expressing of autophagy-related proteins LC-Ⅰ, LC-Ⅱ was detected by the western blotting. Results With the concentration of DNP increased, the proliferation of Hela and KB cells was more obviously inhibited. When the concentration of DNP was 200 μmol/L, the SERD0 of Hela and KB cells were 1. 45 and 1. 18. After Hela and KB cells were irradiated by 4 Gy X-ray combined with 200 μmol/L DNP for 24h, the number of G2/M cells increased significantly and the mitochondrial membrane potential decreased significantly. DNP could promote the formation of autophagosome of Hela and KB cells. After DNP treatment, the expression of LC-Ⅱ in Hela and KB cells was significantly increased, and the ratio of LC-Ⅱ/LC-Ⅰ was also significantly higher than that in the control group. Conclusion DNP can inhibit the proliferation of tumor cells in a concentration-dependent manner and has radio sensitizing effect. DNP may be a potential radiosensitizer. [ABSTRACT FROM AUTHOR]

Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response. [ABSTRACT FROM AUTHOR]

Autophagy, an intracellular conserved degradative process, plays a central role in the renewal/recycling of a cell to maintain the homeostasis of nutrients and energy within the cell. ATG5, a key component of autophagy, regulates the formation of the autophagosome, a hallmark of autophagy. ATG5 binds with ATG12 and ATG16L1 resulting in E3 like ligase complex, which is necessary for autophagosome expansion. Available data suggest that ATG5 is indispensable for autophagy and has an imperative role in several essential biological processes. Moreover, ATG5 has also been demonstrated to possess autophagy‐independent functions that magnify its significance and therapeutic potential. ATG5 interacts with various molecules for the execution of different processes implicated during physiological and pathological conditions. Furthermore, ATG5 genetic variants are associated with various ailments. This review discusses various autophagy‐dependent and autophagy‐independent roles of ATG5, highlights its various deleterious genetic variants reported until now, and various studies supporting it as a potential drug target. Significance statement: ATG5, a candidate protein in autophagy, plays an important role during autophagosome formation. Apart from that, ATG5 also plays autophagy‐independent, significant, diverse roles in essential physiological processes and is involved in various pathological conditions. No review is available that has focused comprehensively on the roles of ATG5 in various human diseases. Therefore, the central idea of this review is to highlight the multifaceted roles of ATG5 by providing detailed coverage of available information and providing in‐depth knowledge about its various interacting partners and molecules. This further can provide several ways by which its interactions could be regulated and therapeutically targeted to restrict deleterious pathways and prevent disease development. [ABSTRACT FROM AUTHOR]

Physiological effects mediated by melatonin are attributable to its potent antioxidant activity as well as its role as a signaling molecule in inducing a vast array of melatonin-mediated genes. Here, we propose melatonin as a signaling molecule essential for protein quality control (PQC) in plants. PQC occurs by the coordinated activities of three systems: the chaperone network, autophagy, and the ubiquitin–proteasome system. With regard to the melatonin-mediated chaperone pathway, melatonin increases thermotolerance by induction of heat shock proteins and confers endoplasmic reticulum stress tolerance by increasing endoplasmic reticulum chaperone proteins. In chloroplasts, melatonin-induced chaperones, including Clps and CpHSP70s, play key roles in the PQC of chloroplast-localized proteins, such as Lhcb1, Lhcb4, and RBCL, during growth. Melatonin regulates PQC by autophagy processes, in which melatonin induces many autophagy (ATG) genes and autophagosome formation under stress conditions. Finally, melatonin-mediated plant stress tolerance is associated with up-regulation of stress-induced transcription factors, which are regulated by the ubiquitin–proteasome system. In this review, we propose that melatonin plays a pivotal role in PQC and consequently functions as a pleiotropic molecule under non-stress and adverse conditions in plants. [ABSTRACT FROM AUTHOR]

Our previous study has demonstrated that porcine pulmonary microvascular endothelial cells (MVECs) are susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). The innate immune response of MVECs infected with HP-PRRSV would play important roles in controlling virus proliferation, resisting cellular injury, and preventing the virus from spreading to other tissues and organs. Type I interferon is one of the most effective antiviral cytokines in the innate immune response, and interferon-induced proteins with tetratricopeptide repeats (IFITs) are members of interferon-stimulated genes induced by viruses and other pathogens, which are crucial in inhibiting virus proliferation and regulating the innate immune response. However, their effects on HP-PRRSV-induced innate immunity in porcine pulmonary MVECs remain unclear. Here, the roles of IFITs in porcine pulmonary MVECs infected with the HP-PRRSV HN strain were investigated, and the effects of astragalus polysaccharides (APS), a widely used traditional Chinese herbal ingredient with the immunopotentiating effect, on them were studied. The results showed that more autophagosomes were observed in HP-PRRSV-infected MVECs, and the expression of IFN-α, IFIT3, and IFIT5 decreased or increased at different time points after infection. When silencing the genes of IFIT3 or IFIT5, the HP-PRRSV replication in MVECs was significantly increased. The expression of IFIT3 and IFIT5 could be upregulated by APS, whose inhibitory effects on the HP-PRRSV replication significantly declined when the genes of IFIT3 or IFIT5 were silenced. The results suggest that IFIT3 and IFIT5 play an important role in inhibiting the HP-PRRSV replication in porcine pulmonary MVECs, and APS suppress the multiplication of HP-PRRSV by upregulating their expression. [ABSTRACT FROM AUTHOR]

Environmental noise is a common hazard in military operations. Military service members during long operations are often exposed to around-the-clock noise and suffer massive emotional and cognitive dysfunction related to an Alzheimer's disease (AD)-like neuropathology. It is essential to clarify the mechanisms underlying the effects of around-the-clock noise exposure on the central nervous system. Here, Wistar rats were continuously exposed to white noise (95 dB during the on-duty phase [8:00–16:00] and 75 dB during the off-duty phase (16:00–8:00 the next day)) for 40 days. The levels of phosphorylated tau, amyloid-β (Aβ), and neuroinflammation in the cortex and hippocampus were assessed and autophagosome (AP) aggregation was observed by transmission electron microscopy. Dyshomeostasis of autophagic flux resulting from around-the-clock noise exposure was assessed at different stages to investigate the potential pathological mechanisms. Around-the-clock noise significantly increased Aβ peptide, tau phosphorylation at Ser396 and Ser404, and neuroinflammation. Moreover, the AMPK-mTOR signaling pathway was depressed in the cortex and the hippocampus of rats exposed to around-the-clock noise. Consequently, autophagosome–lysosome fusion was deterred and resulted in AP accumulation. Our results indicate that around-the-clock noise exposure has detrimental influences on autophagic flux homeostasis and may be associated with AD-like neuropathology in the cortex and the hippocampus. [ABSTRACT FROM AUTHOR]

Autophagy is an evolutionally conserved degradation mechanism for maintaining cell homeostasis whereby cytoplasmic components are wrapped in autophagosomes and subsequently delivered to lysosomes for degradation. This process requires the concerted actions of multiple autophagy-related proteins and accessory regulators. In neurons, autophagy is dynamically regulated in different compartments including soma, axons, and dendrites. It determines the turnover of selected materials in a spatiotemporal control manner, which facilitates the formation of specialized neuronal functions. It is not surprising, therefore, that dysfunctional autophagy occurs in epilepsy, mainly caused by an imbalance between excitation and inhibition in the brain. In recent years, much attention has been focused on how autophagy may cause the development of epilepsy. In this article, we overview the historical landmarks and distinct types of autophagy, recent progress in the core machinery and regulation of autophagy, and biological roles of autophagy in homeostatic maintenance of neuronal structures and functions, with a particular focus on synaptic plasticity. We also discuss the relevance of autophagy mechanisms to the pathophysiology of epileptogenesis. [ABSTRACT FROM AUTHOR]

Helicobacter pylori-mediated gastric carcinogenesis involves upregulation of the E3 ubiquitin ligase Siah2 and its phosphorylation-mediated stabilization. This study elucidates a novel mechanism of oxidative stress regulation by phosphorylated Siah2 in H. pylori-infected gastric epithelial cancer cells (GECs). We identify that H. pylori-mediated Siah2 phosphorylation at the 6th serine residue (P-S6-Siah2) enhances proteasomal degradation of the 78-kDa glucose-regulated protein (GRP78) possessing antioxidant functions. S6 phosphorylation stabilizes Siah2 and P-S6-Siah2 potentiates H. pylori-mediated reactive oxygen species (ROS) generation. However, infected S6A phospho-null Siah2-expressing cells have decreased cellular GRP78 level as surprisingly these cells release GRP78 to a higher extent and accumulate significantly higher ROS than the wild type (WT) Siah2 construct-expressing cells. Ectopic expression of GRP78 prevents the loss of mitochondrial membrane potential and cellular ROS accumulation caused by H. pylori. H. pylori-induced mitochondrial damage and mitochondrial membrane potential loss are potentiated in Siah2-overexpressing cells but these effects are further enhanced in S6A-expressing cells. This study also confirms that while phosphorylation-mediated Siah2 stabilization optimally upregulates aggresome accumulation, it suppresses autophagosome formation, thus decreasing the dependency on the latter mechanism in regulating cellular protein abundance. Disruption of the phospho-Siah2-mediated aggresome formation impairs proliferation of infected GECs. Thus, Siah2 phosphorylation has diagnostic and therapeutic significance in H. pylori-mediated gastric cancer (GC). [ABSTRACT FROM AUTHOR]

Autophagy is an evolutionarily conserved multistep degradation mechanism in eukaryotes, that maintains cellular homoeostasis by replenishing cells with nutrients through catabolic lysis of the cytoplasmic components. This critically coordinated pathway involves sequential processing events that begin with initiation, nucleation, and elongation of phagophores, followed by the formation of double‐membrane vesicles known as autophagosomes. Finally, autophagosomes migrate towards and fuse with lysosomes in mammals and vacuoles in yeast and plants, for the eventual degradation of the intravesicular cargo. Here, we review the recent advances in our understanding of the molecular events that define the process of autophagy. [ABSTRACT FROM AUTHOR]

Plant diseases cause substantial loss to crops all over the world, reducing the quality and quantity of agricultural goods significantly. One of the world's most damaging plant diseases, rice blast poses a substantial threat to global food security. Magnaporthe oryzae causes rice blast disease, which challenges world food security by causing substantial damage in rice production annually. Autophagy is an evolutionarily conserved breakdown and recycling system in eukaryotes that regulate homeostasis, stress adaption, and programmed cell death. Recently, new studies found that the autophagy process plays a vital role in the pathogenicity of M. oryzae and the regulation mechanisms are gradually clarified. Here we present a brief summary of the recent advances, concentrating on the new findings of autophagy regulation mechanisms and summarize some autophagy-related techniques in rice blast fungus. This review will help readers to better understand the relationship between autophagy and the virulence of plant pathogenic fungi. [ABSTRACT FROM AUTHOR]

Key message: Hydrogen sulfide positively regulates autophagy and the expression of hypoxia response-related genes under submergence to enhance the submergence tolerance of Arabidopsis. Flooding seriously endangers agricultural production, and it is quite necessary to explore the mechanism of plant response to submergence for improving crop yield. Both hydrogen sulfide (H2S) and autophagy are involved in the plant response to submergence. However, the mechanisms by which H2S and autophagy interact and influence submergence tolerance have not been thoroughly elucidated. Here, we reported that exogenous H2S pretreatment increased the level of endogenous H2S and alleviated plant cell death under submergence. And transgenic lines decreased in the level of endogenous H2S, L-cysteine desulfurase 1 (des1) mutant and 35S::GFP-O-acetyl-L-serine(thiol)lyase A1 (OASA1)/des1-#56/#61, were sensitive to submergence, along with the lower transcript levels of hypoxia response genes, LOB DOMAIN 41 (LBD41) and HYPOXIA RESPONSIVE UNKNOWN PROTEIN 43 (HUP43). Submergence induced the formation of autophagosomes, and the autophagy-related (ATG) mutants (atg4a/4b, atg5, atg7) displayed sensitive phenotypes to submergence. Simultaneously, H2S pretreatment repressed the autophagosome producing under normal conditions, but enhanced this process under submergence by regulating the expression of ATG genes. Moreover, the mutation of DES1 aggravated the sensitivity of des1/atg5 to submergence by reducing the formation of autophagosomes under submergence. Taken together, our results demonstrated that H2S alleviated cell death through regulating autophagy and the expression of hypoxia response genes during submergence in Arabidopsis. [ABSTRACT FROM AUTHOR]

We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP‐4T1 syngeneic murine cancer model to study EVs in‐transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV‐associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission‐induced mitophagy preferentially populated the CD81+/PD‐L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin‐independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC‐1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti‐CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology. [ABSTRACT FROM AUTHOR]

The formation of autophagosomes and their fusion with lysosomes are key events that underpin autophagic degradation of cargoes. The core ATG8 system, which consists of the ATG8 family of ubiquitin-like proteins and the machineries that conjugate them onto autophagosomal membranes, are among the most-studied autophagy components. Despite the research focus on the core ATG8 system, there are conflicting reports regarding its essential roles in autophagy. Here, we reconcile prior observations of the core ATG8 system into a unifying model of their function that aims to consider apparently conflicting discoveries. Bypass pathways of autophagy that function independently of the core ATG8 system are also discussed. [ABSTRACT FROM AUTHOR]

Keywords: Ca 2+ signaling; autophagosome; endoplasmic reticulum; FIP200 puncta; EI24 EN Ca 2+ signaling autophagosome endoplasmic reticulum FIP200 puncta EI24 1 3 3 04/03/23 20230301 NES 230301 Autophagy is an evolutionarily conserved and tightly regulated lysosome-mediated intracellular bulk degradation pathway by which intracellular macromolecules are sequestered in autophagosomes and delivered to lysosomes for degradation and recycling. In order to assess local Ca SP 2+ sp perturbations in a submicrometer domain on the ER, they constructed a new biosensor, CYB5-GCaMP6f, to detect autophagy-induced Ca SP 2+ sp changes in a narrow spatial domain on the outer surface of the ER. They also demonstrated that Ca SP 2+ sp transients on the ER surface trigger liquid-liquid phase separation of the FIP200 complex, and that subsequently the formed FIP200 puncta aggregate to bind to the ER membrane proteins VAPA/B (VAPs) and Atlastin 2/3 (ATLs). [Extracted from the article]