Autophagy Flux Research Articles

Therapeutic effects of exendin-4 on spinal cord injury via restoring autophagy function and decreasing necroptosis in neuron.

Necroptosis is one of programmed death that may aggravate spinal cord injury (SCI). We aimed to investigate the effect and mechanism of exendin-4 (EX-4) on the recovery of motor function and necroptosis after SCI. The SD rats with left hemisection in the T10 spinal cord as SCI model were used. The behavior tests were measured within 4 weeks. The effects of EX-4 on necroptosis-associated proteins and autophagy flux were explored. In addition, the SHSY5Y cell model was introduced to explore the direct effect of EX-4 on neurons. The effect of lysosome was explored using mTOR activator and AO staining. EX-4 could improve motor function and limb strength, promote the recovery of autophagy flux, and accelerate the degradation of necroptosis-related protein at 3 d after injury in rats. EX-4 reduced lysosome membrane permeability, promoted the recovery of lysosome function and autophagy flux, and accelerated the degradation of necroptosis-related proteins by inhibiting the phosphorylation level of mTOR in the SHSY5Y cell model. Our results demonstrated that EX-4 may improve motor function after SCI via inhibiting mTOR phosphorylation level and accelerating the degradation of necroptosis-related proteins in neurons. Our findings may provide new therapeutic targets for clinical treatment after SCI.

Bilirubin, once a toxin but now an antioxidant alleviating non-alcoholic fatty liver disease in an autophagy-dependent manner in high-fat diet-induced rats: a molecular and histopathological analysis.

As an endogenous antioxidant, bilirubin has surprisingly been inversely correlated with the risk of non-alcoholic fatty liver disease (NAFLD). Thereupon, the current evaluation was designed to assess the positive effects of bilirubin on the autophagy flux, as well as the other pathogenic processes and parameters involved in the expansion of NAFLD. Thirty adult male rats weighing 150-200 g with free access to sucrose solution (18%) were randomly subdivided into 5 groups (n = 6). Subsequently, the animals were euthanized, and their blood specimens and liver tissue samples were collected to measure serum biochemical indices, liver histopathological changes, intrahepatic triglycerides content, and tissue stereological alterations. Furthermore, the expression levels of autophagy-related genes (Atgs) were measured to assess the state of the autophagy flux. Fasting blood glucose, body weight, as well as liver weight, liver-specific enzyme activity, and serum lipid profile indices markedly decreased in rats that underwent a six-week bilirubin treatment compared to the control group. In addition, histopathological studies showed that hepatic steatosis, fibrosis, inflammation, and necrosis significantly decreased in the groups that received bilirubin compared to the control animals. Bilirubin also caused significant alterations in the expression levels of the Atgs, as well as the Beclin- 1 protein. Bilirubin may have potential ameliorative effects on NAFLD-associated liver damage. Moreover, the beneficial effects of bilirubin on intrahepatic lipid accumulation and steatosis were comparable with the group that did not ever receive bilirubin.

Unlocking a New Path: An Autophagometer that Measures Flux Using a Non-Fluorescent Immunohistochemistry Method.

Macroautophagy/autophagy, a crucial cellular process, is typically measured using fluorescence-based techniques, which can be costly, complex, and impractical for clinical settings. In this paper, we introduce a novel, cost-effective, non-fluorescent immunohistochemistry (IHC) method for evaluating autophagy flux. This technique, based on antigen-antibody reactions and chromogenic detection, provides clear, quantifiable results under standard light microscopy, eliminating the need for expensive equipment and specialized reagents. Our method simplifies technical requirements, making it accessible to routine clinical laboratories and research settings with limited resources. By comparing our approach with traditional fluorescence methods, we demonstrate its superior effectiveness, cost-efficiency, and applicability to patient samples. This innovative technique has the potential to significantly advance autophagy research and improve clinical diagnostics, offering a practical and robust tool for studying autophagy mechanisms in diseases such as cancer and neurodegenerative disorders. Our non-fluorescent IHC method represents a significant step forward in evaluating autophagy flux, making it more accessible and reliable, with the promise of enhancing our understanding and treatment of autophagy-related diseases.

Lysosomal dysfunction-derived autophagy impairment of gingival epithelial cells in diabetes-associated periodontitis with altered protein acetylation

Diabetes-associated periodontitis (DP) presents severe inflammation and resistance to periodontal conventional treatment, presenting a significant challenge in clinical management. In this study, we investigated the underlying mechanism driving the hyperinflammatory response in gingival epithelial cells (GECs) of DP patients. Our findings indicate that lysosomal dysfunction under high glucose conditions leads to the blockage of autophagy flux, exacerbating inflammatory response in GECs. Single-cell RNA sequencing and immunohistochemistry analyses of clinical gingival epithelia revealed dysregulation in the lysosome pathway characterized by reduced levels of lysosome-associated membrane glycoprotein 2 (LAMP2) and V-type proton ATPase 16 kDa proteolipid subunit c (ATP6V0C) in subjects with DP. In vitro stimulation of human gingival epithelial cells (HGECs) with a hyperglycemic microenvironment showed elevated release of proinflammatory cytokines, compromised lysosomal acidity and blocked autophagy. Moreover, HGECs with deficiency in ATP6V0C demonstrated impaired autophagy and heightened inflammatory response, mirroring the effects of high glucose stimulation. Proteomic analysis of acetylation modifications identified altered acetylation levels in 28 autophagy-lysosome pathway-related proteins and 37 sites in HGECs subjected to high glucose stimulation or siATP6V0C. Overall, our finding highlights the pivotal role of lysosome impairment in autophagy obstruction in DP and suggests a potential impact of altered acetylation of relevant proteins on the interplay between lysosome dysfunction and autophagy blockage. These insights may pave the way for the development of effective therapeutic strategies against DP.

Bunyavirus SFTSV NSs utilizes autophagy to escape the antiviral innate immune response

ABSTRACT Severe fever with thrombocytopenia syndrome virus (SFTSV) nonstructural protein (NSs) is an important viral virulence factor that sequesters multiple antiviral proteins into inclusion bodies to escape the antiviral innate immune response. However, the mechanism of the NSs restricting host innate immunity remains largely elusive. Here, we found that the NSs induced complete macroautophagy/autophagy by interacting with the CCD domain of BECN1, thereby promoting the formation of a BECN1-dependent autophagy initiation complex. Importantly, our data showed that the NSs sequestered antiviral proteins such as TBK1 into autophagic vesicles, and therefore promoted the degradation of TBK1 and other antiviral proteins. In addition, the 8A mutant of NSs reduced the induction of BECN1-dependent autophagy flux and degradation of antiviral immune proteins. In conclusion, our results indicated that SFTSV NSs sequesters antiviral proteins into autophagic vesicles for degradation and to escape antiviral immune responses.

ADT-OH exhibits anti-metastatic activity on triple-negative breast cancer by combinatorial targeting of autophagy and mitochondrial fission

High basal autophagy and enhanced mitochondrial fission in triple-negative breast cancer (TNBC) cells support cell migration and promote plasticity of cancer cell metabolism. Here, we suggest a novel combination therapy approach for the treatment of TNBC that targets Drp1-mediated mitochondrial fission and autophagy pathways. Hydrogen sulfide (H2S) mediates a myriad of biological processes, including autophagy and mitochondrial function. In this study, we demonstrated that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most widely utilized sustained-release H2S donors, effectively suppresses metastasis of TNBC cells in the absence of proliferation inhibition in vitro and in vivo. ADT-OH treatment ameliorated autophagy flux by suppressing autophagosome formation and induced mitochondrial elongation through decreasing expression of dynamin-related protein 1 (Drp1) and increasing expression of mitochondrial fusion protein (Mfn2). At the same time, ADT-OH downregulated mitophagy flux and inhibited mitochondrial function, eventually leading to the inhibition of migration and invasion in TNBC cells. In vivo, intraperitoneal administration of ADT-OH revealed a potent anti-metastatic activity in three different animal models, the MDA-MB-231 orthotopic xenograft model, the 4T1-Luci orthotopic model and the 4T1-Luci tail vein metastasis model. However, ADT-OH has an extremely low water solubility, which is a significant barrier to its effectiveness. Thus, we demonstrated that the solubility of ADT-OH in water can be improved significantly by absorption with hydroxypropyl-β-cyclodextrin (CD). Remarkably, the obtained CD-ADT-OH demonstrated superior anti-cancer effect to ADT-OH in vivo. Altogether, this study describes a novel regulator of mammalian mitochondrial fission and autophagy, with potential utility as an experimental therapeutic agent for metastatic TNBC.

Protective effects of pioglitazone in renal ischemia-reperfusion injury (RIRI): focus on oxidative stress and inflammation.

Renal ischemia-reperfusion injury (RIRI) is a critical phenomenon that compromises renal function and is the most serious health concern related to acute kidney injury (AKI). Pioglitazone (Pio) is a known agonist of peroxisome proliferator-activated receptor-gamma (PPAR-γ). PPAR-γ is a nuclear receptor that regulates genes involved in inflammation, metabolism, and cellular differentiation. Activation of PPAR-γ is associated with antiinflammatory and antioxidant effects, which are relevant to the pathophysiology of RIRI. This study aimed to investigate the protective effects of Pio in RIRI, focusing on oxidative stress and inflammation. We conducted a comprehensive literature search using electronic databases, including PubMed, ScienceDirect, Web of Science, Scopus, and Google Scholar. The results of this study demonstrated that Pio has antioxidant, anti-inflammatory, and anti-apoptotic activities that counteract the consequences of RIRI. The study also discussed the underlying mechanisms, including the modulation of various pathways such as TNF-α, NF-κB signaling systems, STAT3 pathway, KIM-1 and NGAL pathways, AMPK phosphorylation, and autophagy flux. Additionally, the study presented a summary of various animal studies that support the potential protective effects of Pio in RIRI. Our findings suggest that Pio could protect the kidneys from RIRI by improving antioxidant capacity and decreasing inflammation. Therefore, these findings support the potential of Pio as a therapeutic strategy for preventing RIRI in different clinical conditions.

ROS-mediated lysosomal membrane permeabilization and autophagy inhibition regulate bleomycin-induced cellular senescence

ABSTRACT Bleomycin exhibits effective chemotherapeutic activity against multiple types of tumors, and also induces various side effects, such as pulmonary fibrosis and neuronal defects, which limit the clinical application of this drug. Macroautophagy/autophagy has been recently reported to be involved in the functions of bleomycin, and yet the mechanisms of their crosstalk remain insufficiently understood. Here, we demonstrated that reactive oxygen species (ROS) produced during bleomycin activation hampered autophagy flux by inducing lysosomal membrane permeabilization (LMP) and obstructing lysosomal degradation. Exhaustion of ROS with N-acetylcysteine relieved LMP and autophagy defects. Notably, we observed that LMP and autophagy blockage preceded the emergence of cellular senescence during bleomycin treatment. In addition, promoting or inhibiting autophagy-lysosome degradation alleviated or exacerbated the phenotypes of senescence, respectively. This suggests the alternation of autophagy activity is more a regulatory mechanism than a consequence of bleomycin-induced cellular senescence. Taken together, we reveal a specific role of bleomycin-induced ROS in mediating defects of autophagic degradation and further regulating cellular senescence in vitro and in vivo. Our findings, conversely, indicate the autophagy-lysosome degradation pathway as a target for modulating the functions of bleomycin. These provide a new perspective for optimizing bleomycin as a clinically applicable chemotherapeutics devoid of severe side-effects. Abbreviations: AT2 cells: type II alveolar epithelial cells; ATG7: autophagy related 7; bEnd.3: mouse brain microvascular endothelial cells; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCL2: C-C motif chemokine ligand 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; FTH1: ferritin heavy polypeptide 1; γ-H2AX: phosphorylated H2A.X variant histone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cells; HT22: hippocampal neuronal cell lines; Il: interleukin; LAMP: lysosomal-associated membrane protein; LMP: lysosome membrane permeabilization; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NCOA4: nuclear receptor coactivator 4; PI3K: phosphoinositide 3-kinase; ROS: reactive oxygen species; RPS6KB/S6K: ribosomal protein S6 kinase; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SAHF: senescence-associated heterochromatic foci; SASP: senescence-associated secretory phenotype; SEC62: SEC62 homolog, preprotein translocation; SEP: superecliptic pHluorin; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB

Inhibition of CSPG-PTPσ Activates Autophagy Flux and Lysosome Fusion, Aids Axon and Synaptic Reorganization in Spinal Cord Injury.

Chondroitin sulfate proteoglycans (CSPGs) and proteoglycan receptor protein tyrosine phosphatase σ (PTPσ) play a critical role in the pathology of spinal cord injury (SCI). CSPGs can be induced by autophagy inhibition in astrocyte. However, CSPG's impact on autophagy and its role in SCI is still unknown. We investigate intracellular sigma peptide (ISP) targeting PTPσ, its effects on autophagy, and synaptic reorganization in SCI. We found that ISP increased the level of autophagosome marker LC3B-II/I and decreased autophagosome degradation marker p62 in SCI, suggesting activated autophagy flux. ISP restored autophagosome-lysosome fusion-related protein syntaxin 17 (STX17) and lysosome-associated membrane protein 2 (LAMP2), indicating activated autophagosome-lysosome fusion. ISP increased pre-synaptic marker synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) expression and improved excitatory synapse marker vesicular glutamate transporter 1 (VGLUT1) and SYN in SCI, suggesting improved synaptic reorganization. ISP promoted axon marker neurofilament and growth-related GAP-43 expression in SCI. ISP rescued a preserved number of motor neurons and improved neurobehavioral recovery after SCI. Our study extended the CSPG-PTPσ inhibition role in activating autophagy flux, axon and synaptic reorganization, and functional recovery in SCI.

CTRP9 alleviates diet induced obesity through increasing lipolysis mediated by enhancing autophagy-initiation complex formation.

Recently, emerging evidence has suggested that obesity become a prevalent health threat worldwide. Reportedly, CTRP9 can ameliorate HFD induced obesity. However, the molecular mechanism underlying the role of CTRP9 in obesity remains elusive. In this study, we reported its major function in the regulation of lipolysis. First, we found that the expression of CTRP9 was decreased in mature adipocytes and white adipose tissue of obese mice. Then, we showed that overexpression adipose tissue CTRP9 alleviated diet-induced obesity and adipocytes hypertrophy, improved glucose intolerance and raised energy expenditure. Moreover, CTRP9 increased the lipolysis in vitro and vivo. Additionally, we determined that CTRP9 enhanced autophagy flux in adipocytes. Intriguingly, knock down Beclin1 by SiRNA abolished the effect of CTRP9 on lipolysis. Mechanically, CTRP9 enhanced the expression of SNX26. We demonstrated that SNX26 was a component of the ATG14L-Beclin1-VPS34 complex and enhanced the assembly of the autophagy-initiation complex. Collectively, our results suggested that CTRP9 alleviated diet induced obesity through enhancing lipolysis mediated by autophagy-initiation complex formation.

Cardiac glycosides restore autophagy flux in an iPSC-derived neuronal model of WDR45 deficiency.

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

Induction of DEPP1 by HIF Mediates Multiple Hallmarks of Ischemic Cardiomyopathy.

HIF (hypoxia inducible factor) regulates many aspects of cardiac function. We and others previously showed that chronic HIF activation in the heart in mouse models phenocopies multiple features of ischemic cardiomyopathy in humans, including mitochondrial loss, lipid accumulation, and systolic cardiac dysfunction. In some settings, HIF also causes the loss of peroxisomes. How, mechanistically, HIF promotes cardiac dysfunction is an open question. We used mice lacking cardiac pVHL (von Hippel-Lindau protein) to investigate how chronic HIF activation causes multiple features of ischemic cardiomyopathy, such as autophagy induction and lipid accumulation. We performed immunoblot assays, RNA sequencing, mitochondrial and peroxisomal autophagy flux measurements, and live cell imaging on isolated cardiomyocytes. We used CRISPR-Cas9 gene editing in mice to validate a novel mediator of cardiac dysfunction in the setting of chronic HIF activation. We identify a previously unknown pathway by which cardiac HIF activation promotes the loss of mitochondria and peroxisomes. We found that DEPP1 (decidual protein induced by progesterone 1) is induced under hypoxia in a HIF-dependent manner and localizes inside mitochondria. DEPP1 is both necessary and sufficient for hypoxia-induced autophagy and triglyceride accumulation in cardiomyocytes ex vivo. DEPP1 loss increases cardiomyocyte survival in the setting of chronic HIF activation ex vivo, and whole-body Depp1 loss decreases cardiac dysfunction in hearts with chronic HIF activation caused by VHL loss in vivo. Our findings identify DEPP1 as a key component in the cardiac remodeling that occurs with chronic ischemia.

Hesperidin Alleviates Hepatic Injury Caused by Deoxynivalenol Exposure through Activation of mTOR and AKT/GSK3β/TFEB Pathways.

Deoxynivalenol (DON) is a common agricultural mycotoxin that is chemically stable and not easily removed from cereal foods. When organisms consume food made from contaminated crops, it can be hazardous to their health. Numerous studies in recent years have found that hesperidin (HDN) has hepatoprotective effects on a wide range of toxins. However, few scholars have explored the potential of HDN in attenuating DON-induced liver injury. In this study, we established a low-dose DON exposure model and intervened with three doses of HDN, acting on male C57 BL/6 mice and AML12 cells, which served as in vivo and in vitro models, respectively, to investigate the protective mechanism of HDN against DON exposure-induced liver injury. The results suggested that DON disrupted hepatic autophagic fluxes, thereby impairing liver structure and function, and HDN significantly attenuated these changes. Further studies revealed that HDN alleviated DON-induced excessive autophagy through the mTOR pathway and DON-induced lysosomal dysfunction through the AKT/GSK3β/TFEB pathway. Overall, our study suggested that HDN could ameliorate DON-induced autophagy flux disorders via the mTOR pathway and the AKT/GSK3β/TFEB pathway, thereby reducing liver injury.

REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)—a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein – a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.

ATG10S promotes IFNL1 expression and autophagic degradation of multiple viral proteins mediated by IFNL1

ABSTRACT ATG10S is a newly discovered subtype of the autophagy protein ATG10. It promotes complete macroautophagy/autophagy, degrades multiple viral proteins, and increases the expression of type III interferons. Here, we aimed to investigate the mechanism of ATG10S cooperation with IFNL1 to degrade viral proteins from different viruses. Using western blot, immunoprecipitation (IP), tandem sensor RFP-GFP-LC3B and in situ proximity ligation assays, we showed that exogenous recombinant ATG10S protein (rHsATG10S) could enter into cells through clathrin, and ATG10S combined with ATG7 with IFNL1 assistance to facilitate ATG12–ATG5 conjugation, thereby contributing to the autophagosome formation in multiple cell lines containing different virions or viral proteins. The results of DNA IP and luciferase assays also showed that ATG10S was able to directly bind to a core motif (CAAGGG) within a binding site of transcription factor ZNF460 on the IFNL1 promoter, by which IFNL1 transcription was activated. These results clarified that ATG10S promoted autophagosome formation with the assistance of IFNL1 to ensure autophagy flux and autophagic degradation of multiple viral proteins and that ATG10S could also act as a novel transcription factor to promote IFNL1 gene expression. Importantly, this study further explored the antiviral mechanism of ATG10S interaction with type III interferon and provided a theoretical basis for the development of ATG10S into a new broad-spectrum antiviral protein drug. Abbreviation: ATG: autophagy related; ATG10S: the shorter isoform of autophagy-related 10; CC50: half cytotoxicity concentration; CCV: clathrin-coated transport vesicle; CLTC: clathrin heavy chain; CM: core motif; co-IP: co-immunoprecipitation; CPZ: chlorpromazine; ER: endoplasmic reticulum; HCV: hepatitis C virus; HBV: hepatitis B virus; HsCoV-OC43: Human coronavirus OC43; IFN: interferon; PLA: proximity ligation assay; rHsATG10S: recombinant human ATG10S protein; RLU: relative light unit; SQSTM1: sequestosome 1; ZNF: zinc finger protein

The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.

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.

Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms.

Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target. Extracellular vesicles (EVs) may act as potent anti-inflammatory agents and also modulators of autophagy in target cells. However, the molecular mechanisms by which EVs modulate autophagy flux in human microglia remain largely unexplored. In the present study, we investigated the effects of EVs derived from human oral mucosa stem cells on the autophagy in human microglia. We demonstrate that EVs promoted autophagy and autophagic flux in human microglia and that this process was dependent on the integrity of lipid rafts. Lipopolysaccharide (LPS) also activated autophagy, but combined treatment with EVs and LPS suppressed autophagy response, indicating interference between these signaling pathways. Blockage of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody suppressed EV-induced autophagy. Furthermore, inhibition of the EV-associated heat shock protein (HSP70) chaperone which is one of the endogenous ligands of the TLR4 also suppressed EV-induced lipid raft formation and autophagy. Pre-treatment of microglia with a selective inhibitor of αvβ3/αvβ5 integrins cilengitide inhibited EV-induced autophagy. Finally, blockage of purinergic P2X4 receptor (P2X4R) with selective inhibitor 5-BDBD also suppressed EV-induced autophagy. In conclusion, we demonstrate that EVs activate autophagy in human microglia through interaction with HSP70/TLR4, αVβ3/αVβ5, and P2X4R signaling pathways and that these effects depend on the integrity of lipid rafts. Our findings could be used to develop new therapeutic strategies targeting disease-associated microglia.

ATP6V1A is required for synaptic rearrangements and plasticity in murine hippocampal neurons.

Understanding the physiological role of ATP6V1A, a component of the cytosolic V1 domain of the proton pump vacuolar ATPase, in regulating neuronal development and function. Modeling loss of function of Atp6v1a in primary murine hippocampal neurons and studying neuronal morphology and function by immunoimaging, electrophysiological recordings and electron microscopy. Atp6v1a depletion affects neurite elongation, stabilization, and function of excitatory synapses and prevents synaptic rearrangement upon induction of plasticity. These phenotypes are due to an overall decreased expression of the V1 subunits, that leads to impairment of lysosomal pH-regulation and autophagy progression with accumulation of aberrant lysosomes at neuronal soma and of enlarged vacuoles at synaptic boutons. These data suggest a physiological role of ATP6V1A in the surveillance of synaptic integrity and plasticity and highlight the pathophysiological significance of ATP6V1A loss in the alteration of synaptic function that is associated with neurodevelopmental and neurodegenerative diseases. The data further support the pivotal involvement of lysosomal function and autophagy flux in maintaining proper synaptic connectivity and adaptive neuronal properties.

GPCR Function in Autophagy Control: A Systematic Approach of Chemical Intervention

Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.

The autophagic regulation of rosiglitazone-promoted adipocyte browning.

Objective: Browning of white adipocytes is considered an efficient approach to combat obesity. Rosiglitazone induces the thermogenetic program of white adipocytes, but the underlying mechanisms remain elusive. Methods: Expression levels of browning and autophagy flux markers were detected by real-time PCR and immunoblotting. H&E and Oil Red O staining were performed to evaluate the lipid droplets area. Nuclear protein extraction and immunoprecipitation were used to detect the proteins interaction. Results: In this study, we reported that rosiglitazone promoted adipocyte browning and inhibited autophagy. Rapamycin, an autophagy inducer, reversed adipocyte browning induced by rosiglitazone. Autophagy inhibition by rosiglitazone does not prevent mitochondrial clearance, which was considered to promote adipose whitening. Instead, autophagy inhibition increased p62 nuclear translocation and stabilized the PPARγ-RXRα heterodimer, which is an essential transcription factor for adipocyte browning. We found that rosiglitazone activated NRF2 in mature adipocytes. Inhibition of NRF2 by ML385 reversed autophagy inhibition and the pro-browning effect of rosiglitazone. Conclusion: Our study linked autophagy inhibition with rosiglitazone-promoted browning of adipocytes and provided a mechanistic insight into the pharmacological effects of rosiglitazone.