LC3 Protein Research Articles

Saroglitazar mitigated NASH-associated hepatic injury in dexamethasone-treated rats via modulating autophagy, apoptosis, and necroptosis

This study aimed to evaluate the possible ameliorative effects of saroglitazar (SAR) on aspects of hepatic injury in dexamethasone (DEX)-induced nonalcoholic steatohepatitis (NASH) in rats.Wistar rats received SAR (2 or 4 mg/kg/day, orally) or metformin (MET, 500 mg/kg/day, orally) for one week before and concurrently with DEX administration (8 mg/kg/day, i.p., for 6 days. Control and drug control groups received vehicle or the higher dose of SAR, respectively. At the end of the experiment, an oral glucose tolerance test (OGTT) was conducted, serum hepatic function parameters and lipid profile were assessed, and hepatic histological changes were evaluated. Moreover, hepatic p-Akt/Akt ratios, malondialdehyde (MDA) content, SREBP-1, FOXO1, LC3, cleaved caspase-3, and p-MLKL protein levels were determined. Furthermore, hepatic immunohistochemical expressions of FOXO1, caspase-3, Bcl-2, LC3, and P62 were examined.SAR (mainly at 4 mg/kg/day) significantly improved Area under the OGTT curve (P < 0.0001), hepatic function parameters, lipid profile, and hepatic histopathological features in DEX-administered rats. Moreover, SAR significantly attenuated DEX-induced increases in hepatic MDA content (P < 0.05), SREBP-1 levels (P < 0.0001), and nuclear FOXO1, caspase-3, LC3, P62, and p-MLKL protein expressions (P < 0.0001). Furthermore, SAR significantly enhanced hepatic p-Akt/Akt ratio and Bcl-2 protein expression in DEX-administered rats (P < 0.0001). The higher dose of SAR showed greater hepatoprotective effects compared to its corresponding lower dose and MET in most assessments, approaching levels similar to the control group.SAR mitigated hepatic injury associated with DEX-induced NASH in rats, suggesting it might be a potential hepatoprotective drug for patients with or at high risk of NASH.

Simvastatin Induces Autophagy and Inhibits Proliferation in Prostate Cancer Cells.

Statin has recently been studied for its effects on inducing cell death and inhibiting metastasis. Nevertheless, the precise mechanism of its anti-tumor effect is not yet fully understood. We conducted research on statin as a novel treatment for castration-resistant prostate cancer (CRPC). This study focused on autophagy in prostate cancer cells and assessed the effects of simvastatin. After administering simvastatin to PC-3 cells, we conducted a microarray analysis. Simvastatin was administered to prostate cancer cell lines (PC-3, LNCaP-LA; cultured under androgen-depleted conditions, DU145, 22RV1), and the tumor proliferation inhibition was evaluated using the MTS assay and cell count. Autophagy was measured by observing autophagosome staining under a fluorescence microscope and quantifying LC-3 protein using western blot. We also investigated the effects of rapamycin, an autophagy inducer, and chloroquine as an inhibitor. Simvastatin demonstrated a significant concentration-dependent growth inhibition effect on prostate cell lines. Moreover, a significant increase in autophagy was observed in all cell lines following simvastatin administration. When we administered simvastatin with rapamycin at a concentration that did not show a tumor growth inhibitory effect, it significantly enhanced autophagy induction compared to simvastatin alone, and also significantly enhanced the growth inhibition effect on PC-3 cells. Simvastatin induced autophagy and inhibited the proliferation of prostate cancer cell lines. The combination of simvastatin and rapamycin significantly induced autophagy and enhanced the inhibitory effect of simvastatin on proliferation. This mechanism may serve as a novel therapeutic target.

Involvement of Autophagy and Oxidative Stress-Mediated DNA Hypomethylation in Transgenerational Nephrotoxicity Induced in Rats by the Mycotoxin Fumonisin B1.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, is one of the most common pollutants in natural foods and agricultural crops. It can cause chronic and severe health issues in humans and animals. The aim of this study was to evaluate the transgenerational effects of FB1 exposure on the structure and function of the kidneys in offspring. Virgin female Wistar rats were randomly divided into three groups: group one (control) received sterile water, and groups two and three were intragastrically administered low (20 mg/kg) and high (50 mg/kg) doses of FB1, respectively, from day 6 of pregnancy until delivery. Our results showed that exposure to either dose of FB1 caused histopathological changes, such as atrophy, hypercellularity, hemorrhage, calcification, and a decrease in the glomerular diameter, in both the first and second generations. The levels of the antioxidant markers glutathione, glutathione S-transferase, and catalase significantly decreased, while malondialdehyde levels increased. Moreover, autophagy was induced, as immunofluorescence analysis revealed that LC-3 protein expression was significantly increased in both generations after exposure to either dose of FB1. However, a significant decrease in methyltransferase (DNMT3) protein expression was observed in the first generation in both treatment groups (20 mg/kg and 50 mg/kg), indicating a decrease in DNA methylation as a result of early-life exposure to FB1. Interestingly, global hypomethylation was also observed in the second generation in both treatment groups despite the fact that the mothers of these rats were not exposed to FB1. Thus, early-life exposure to FB1 induced nephrotoxicity in offspring of the first and second generations. The mechanisms of action underlying this transgenerational effect may include oxidative stress, autophagy, and DNA hypomethylation.

Metformin attenuates inflammation and boosts autophagy in the liver and intestine of chronologically aged rats

BackgroundOur previous studies found that autophagy levels in liver and intestinal segments of naturally aging rats were downregulated, and the expression of pro-inflammatory factors was increased. The increased expression of pro-inflammatory factors might be related to the downregulation of autophagy. AMPK is the most critical upstream targeting and regulating molecule of autophagy, and Metformin, as an agonist of AMPK, has the effects of anti-inflammation and anti-aging. We pretreated 29-month-old naturally aging rats with Metformin for a short period and observed the changes in autophagy levels and pro-inflammatory factors in the liver, ileum, and colon after 31 days of intervention and preliminarily investigated the mechanism of its action. Methods29-month-old SPF male Wistar rats were divided into three groups: The control group, the Metformin 100 mg/kg intervention group, and the Metformin 250 mg/kg intervention group, with eight rats in each group. At 29 months, different concentrations of Metformin (100 mg/kg, 250 mg/kg) were given by gavage once a day until 30 months, and the control group was kept generally until 30 months. Western Blot was used to assess the expression levels of AMPK, P-AMPK, LC3, and P62 proteins in the liver and intestinal tissues. Intestinal and liver tissues were immunofluorescence labeled for LC3 and P62 proteins. Moreover, RT-qPCR was conducted to detect the expression levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, and MMP-9 mRNA in liver and intestinal tissues. ResultsShort-term Metformin pretreatment (31 days) in naturally aging rats (29 months old) increased autophagy levels and down-regulated the expression of various pro-inflammatory cytokines (IL-1β, TNF-α, MMP-9, and IL-6) in various intestinal segments and the liver—the expression of LC3II protein enriched with the increase of Metformin concentration. The level of P62 protein decreased with the accumulation of Metformin concentration. And a higher concentration of Metformin was associated with increased expression of P-AMPK protein. ConclusionsMetformin intervention can boost the autophagy level in the liver and intestine and reduce the expression of aging-related inflammatory factors in aged rats, and these effects may be related to the increase of the AMPK phosphorylation level.

Autophagy and Apoptosis Cross-Talk in Response to Epigallocatechin Gallate in NALM-6 Cell Line

Background: Chemotherapy, the primary treatment for acute lymphoblastic leukemia (ALL), often yields inadequate responses. Epigallocatechin gallate (EGCG) has been shown to significantly affect tumor cells through various mechanisms, including cell cycle arrest, apoptosis, and autophagy. Objectives: The objective of this study is to explore the impact of EGCG on autophagy, apoptosis, and the interplay between them in NALM-6, a pre-B-ALL cell line. Methods: NALM-6 cells were subjected to various concentrations of EGCG for 24 and 48 hours. Additionally, NH4Cl 10 mM was used as an autophagy inhibitor to examine this mechanism. The EGCG effect on cell viability and apoptosis was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), trypan blue exclusion assay, and flow cytometry. Moreover, western blot analysis and real-time PCR were performed to investigate autophagy. Results: Our findings demonstrated that EGCG significantly affected cell proliferation and viability. It reduced cell viability by 55.24 ± 8.43% (P &lt; 0.0001) while inducing apoptosis by 51.04 ± 1.88% (P = 0.006). Furthermore, in the presence of NH4Cl, EGCG led to a 3.92 ± 1.76-fold increase in LC3 protein level (P = 0.001). It also resulted in an approximately 1.34 ± 0.34-fold enhancement in DRAM1 mRNA expression levels (P = 0.013), while reducing of LC3B by 33.3 ± 30.5% (P = 0.008), P62/SQSTM1 by 46.5 ± 28.26% (P &lt; 0.001), and Atg2B by 45.5 ± 16.25% (P &lt; 0.001). However, the inhibition of autophagy did not alter the apoptosis rate in either untreated or EGCG-treated cells. Conclusions: Overall, our findings suggest that EGCG can trigger apoptosis and autophagy in the NALM-6 cell line. However, blockage of autophagy does not appear to impact apoptosis in this cell line.

Inducin Triggers LC3-Lipidation and ESCRT-Mediated Lysosomal Membrane Repair.

Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.

The Sonic hedgehog pathway inhibitor GDC0449 induces autophagic death in human Medulloblastoma Daoy cells

ABSTRACT Medulloblastoma (MB) is a frequently occurring malignant brain tumor in children, and many of these tumors are identified by the abnormal activation of the Sonic Hedgehog (SHH) pathway. Although the Shh inhibitor GDC0449 initially shows some effectiveness in certain tumors, they eventually recur due to drug resistance mechanisms, highlighting the need for new treatment options. In this study, we explore whether GDC0449 induces autophagy in the human MB cell lines. To investigate the ultrastructural pathology changes of GDC0449-treated Daoy and D283 cells, we employed Transmission Electron Microscopy (TEM) technology to identify the expression of autophagic vacuoles. Our results indicate that GDC0449 only increases autophagy in Daoy cells by increasing the LC3-II/LC3-I ratio and autophagosome formation.We also analyzed Beclin1, LC3, Bax, and Cleaved-caspase3 protein and mRNA expression levels of autophagic and apoptotic markers using fluorescence confocal microscopy, RT-PCR, and Western blot. We found that cell autophagy and apoptosis increased in a dose-dependent manner with GDC0449 treatment. Additionally, we observed increased mammalian target of rapamycin (mTOR) phosphorylation and decreased protein kinase B (AKT/PKB), Ribosomal Protein S6, eIF4E-binding protein (4EBP1) phosphorylation in GDC0449-treated Daoy cells. It was observed that inhibiting autophagy using Beclin1 siRNA significantly blocked the apoptosis-inducing effects of GDC0449, suggesting that GDC0449 mediates its apoptotic effects by inducing autophagy.Our data suggests that GDC0449 inhibits the growth of human MB Daoy cells by autophagy-mediated apoptosis. The mechanism of GDC0449-induced autophagy in Daoy cells may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway.

The Small GTPase Rab7 Regulates Antigen Processing in B Cells in a Possible Interplay with Autophagy Machinery.

In B cells, antigen processing and peptide-antigen (pAg) presentation is essential to ignite high-affinity antibody responses with the help of cognate T cells. B cells efficiently internalize and direct specific antigens for processing and loading onto MHCII. This critical step, which enables pAg presentation, occurs in MHCII compartments (MIICs) which possess the enzymatic machinery for pAg loading on MHCII. The intracellular transport systems that guide antigen and maintain this unique compartment remain enigmatic. Here, we probed the possible functional role of two known endosomal proteins, the Rab family small GTPases Rab7 and Rab9, that are both reported to colocalize with internalized antigen. As compared to Rab9, we found Rab7 to exhibit a higher overlap with antigen and MIIC components. Rab7 also showed a higher association with antigen degradation. The inhibition of Rab7 drastically decreased pAg presentation. Additionally, we detected the strong colocalization of perinuclearly clustered and presumably MIIC-associated antigen with autophagy protein LC3. When we pharmacologically inhibited autophagy, pAg presentation was inhibited. Together, our data promote Rab7 as an important regulator of antigen processing and, considering the previously reported functions of Rab7 in autophagy, this also raises the possibility of the involvement of autophagy-related machinery in this process.

Cadmium exposure exacerbates kidney damage by inhibiting autophagy in diabetic rats

The incidence of diabetes mellitus (DM) is gradually increasing, making it a widespread global health concern. Cadmium (Cd) is a common toxic heavy metal in the environment, and cadmium exposure may be associated with diabetic nephropathy (DN). However, the mechanism of Cd-induced DN remains unclear. In this study, we aimed to determine the effect of cadmium on diabetic kidney injury and the underlying mechanism in diabetic rats and a renal tubular epithelial cell line (NRK-52E cells). Our results could provide novel insights on the nephrotoxic mechanism of cadmium. HE, PAS, and Masson staining were used to observe pathological renal injury. COL-I, COL-IV, CTSB, and CTSD protein levels were detected by immunohistochemistry and western blotting. Immunofluorescence was used to detect the fluorescence intensity of p62 and LC3 proteins in kidney tissue. TEM was used to observe the ultrastructure of mitochondria and number of autophagosomes. After cadmium exposure, DM rats showed a dramatic decrease in body weight compared to the unexposed DM group. Relative kidney weight showed a contrasting trend after cadmium exposure. Urinary microalbumin/creatinine significantly increased in normal and DM rats after cadmium exposure. However, the trend was clearer in the DM groups than in the control groups. Endogenous creatinine clearance exhibited a contrasting trend. After cadmium exposure in DM rats, MDA content significantly increased and GSH, CAT, SOD, and GSH-PX activation reduced compared to normal controls. Pathological damage was more pronounced, and the expression of autophagy related proteins and apoptosis and fibrosis proteins was significantly higher in vivo and vitro in the cadmium-exposed groups than in unexposed controls. Further, lysosomal protein levels were lower, and ROS content and autophagosome count significantly higher in the cadmium exposed groups compared to the unexposed controls. Therefore, Cadmium exposure aggravates diabetic kidney injury via autophagy inhibition.

Vital role of autophagy flux inhibition of placental trophoblast cells in pregnancy disorders induced by HEV infection

ABSTRACT Hepatitis E virus (HEV) has become one of the important pathogens that threaten the global public health. Type 3 and 4 HEV are zoonotic, which can spread vertically and cause placental damage. At the same time, autophagy plays an important role in the process of embryo development and pregnancy maintenance. However, the relationship between HEV and autophagy, especially in the placenta tissue, has not been clarified. We found lower litter rates in HEV-infected female mice, with significant intrauterine abortion of the embryo (24.19%). To explore the effects of HEV infection on placenta autophagy, chorionic cells (JEG-3) and mice placenta have been employed as research objects, while the expression of autophagy-related proteins (ATGs) has been detected in JEG-3 cells with different times of HEV inoculation. The results demonstrated that the expression of protein LC3 decreased and p62 accumulated, meanwhile ATGs such as ATG4B, ATG5, and ATG9A in JEG-3 cells have decreased significantly. In addition, the maturation of autophagosomes, which referred to the process of the combination of autophagosomes and lysosomes was prevented by HEV infection as well. All processes of autophagic flux, which include the initiation, development, and maturation three stages, were suppressed in JEG-3 cells after HEV infection. Similarly, the protein and gene expression of LC3 were significantly decreased in the placenta of pregnant mice with HEV infection. In summary, our results suggested that HEV inhibited autophagy in JEG-3 cells and placenta of pregnant mice, which might be the important pathogenic mechanisms of HEV infection leading to embryo abortion.

ATG7-mediated autophagy protects human gingival myofibroblasts from irradiation-induced apoptosis.

Apoptosis resistance of myofibroblasts is critical in pathology of irradiation-induced fibrosis and osteoradionecrosis of the jaw (ORNJ). However, molecular mechanism of apoptosis resistance induced by irradiation in oral myofibroblasts remains largely obscure. Matched ORNJ fibroblasts and normal fibroblasts pairs from gingival were primarily cultured, and myofibroblast markers of α-SMA and FAP were evaluated by qRT-PCR and western blot. CCK8 assay and flow cytometric analysis were performed to investigate the cell viability and apoptosis under irradiation treatment. Autophagy-related protein LC3 and ATG7, and punctate distribution of LC3 localization were further detected. After inhibition of autophagy with inhibitor CQ and 3-MA, as well as transfected ATG7-siRNA, cell viability and apoptosis of ORNJ and normal fibroblasts were further assessed. Compared with normal fibroblasts, ORNJ fibroblasts exhibited significantly higher α-SMA and FAP expression, increased cell, viability and decreased apoptosis under irradiation treatment. LC3-II and ATG7 were up-regulated in ORNJ fibroblasts with irradiation stimulation. After inhibition of irradiation-induced autophagic flux with lysosome inhibitor CQ, LC3-II protein was accumulated and punctate distribution of LC3 localization was increased in ORNJ fibroblasts. Moreover, autophagy inhibitor CQ and 3-MA enhanced the irradiation-induced apoptosis but inhibited viability of ORNJ fibroblasts. Silencing ATG7 with siRNA could obviously weaken irradiation-induced LC3-II expression, and promoted irradiation-induced apoptosis of ORNJ fibroblasts. After knockdown of ATG7, finally, p-AKT(Ser473) and p-mTOR(Ser2448) levels of ORNJ fibroblasts were significantly increased under irradiation. Compared with normal fibroblasts, human gingival myofibroblasts are resistant to irradiation-induced apoptosis via autophagy activation. Silencing ATG7 may evidently inhibit activation of autophagy, and promote apoptosis of gingival myofibroblasts via Akt/mTOR pathway.

LncRNA SNHG15 regulates autophagy and prevents cerebral ischaemia-reperfusion injury through mediating miR-153-3p/ATG5 axis.

Ischaemic stroke is a common cerebrovascular disease. Long non-coding RNA (lncRNA) of small nucleolar RNA host gene (SNHG15) has been supposedly performed a regulatory role in many diseases. Nonetheless, the function of SNHG15 in cerebral ischaemia-reperfusion injury has not been clarified. The OGD/R of Neuro2A cells simulated the ischaemic and reperfused states of the brain. Neuro2a cell line with stable transfection of plasmid with silent expression of SNHG15 was constructed. Neuro2a cell lines transfected with miR-153-3p mimic (miR-153-3p-mimics) and miR-153-3p inhibitor (miR-153-3p-inhibition) were constructed. Expression of SNHG15, mi R-200a, FOXO3 and ATG7 in mouse brain tissue and N2a cells was identified by qRT-PCR. Western blot (WB) analysis of mouse brain tissue and Neuro2a cells revealed the presence of the proteins ATG5, Cle-caspase-3, Bax, Bcl-2, LC3 II/I and P62 (WB). The representation and distribution of LC3B were observed by immunofluorescence. The death of cells was measured using a technique called flow cytometry (FACS). SNHG15 was highly expressed in cerebral ischaemia-reperfusion injury model. Down-regulation of SNHG15 lead to lower apoptosis rate and decreased autophagy. Dual luciferase assay and co-immunoprecipitation (CoIP) found lncRNA SNHG15/miR-153-3p/ATG5. Compared to cells transfected with NC suppression, cells transfected with miR-153-3p-inhibition had substantially greater overexpression of LC 3 II/I, ATG5, cle-Caspase-3, and Bax, as determined by a recovery experiment, the apoptosis rate was elevated, yet both P62 and Bcl-2 were significantly lower and LC3+ puncta per cells were significantly increased. Co-transfection of miR-153-3p-inhibition and sh-SNHG15 could reverse these results. LncRNA SNHG15 regulated autophagy and prevented cerebral ischaemia-reperfusion injury through mediating the miR-153-3p/ATG5 axis.

Effect of the Rubicon protein on LC3-associated phagocytosis by monocytes in the patients with severe atopic bronchial asthma

Atopic bronchial asthma is the most common and severe allergic disease among a wide range of similar diseases. The main pathogenesis of this disease is characterized by a disturbance of T lymphocyte homeostasis, which significantly worsens the general state of health. In atopic bronchial asthma, there is impaired process of T cell apoptosis. This entails dysregulation and maintenance of peripheral lymphocyte homeostasis. Normally, T cells must undergo apoptosis, and its products should be utilized by neighboring cells, or professional phagocytes: monocytes, macrophages, or dendritic cells. This process is altered in atopic bronchial asthma. The immune system disorders, such as autoimmunity, often result from dysregulation of lymphocyte apoptosis. This is especially true in cases of insufficient or missed clearance of apoptotic bodies. Recently, the research and medical communities pay much attention to efferocytosis, a form of phagocytosi which proceeds by removal of apoptotic cells by phagocytes by means of LC3-associated phagocytosis (LAP). This process initiates uptake of the particles due to interactions between the phagocyte plasma membrane receptors and apoptotic cell. Further on, a single-membrane phagosome is formed in the cell with the participation of certain autophagy proteins (Beclin-1, VPS34, UVRAG, ATG5, ATG12, ATG7, ATG4, ATG4, LC3). The phagosome is enriched with LC3 protein molecules and fused with lysosomes, in which the captured “cargo” is then lysed. As a part of our work, a detailed analysis of some key protein contents at the LAP pathway was carried out for peripheral blood monocytes of patients with severe bronchial asthma. It was found that the expression of Rubicon protein is increased, thus allowing to conclude that the LAP pathway is activated in monocytes of healthy donors, thus allowing phagocytosis of dying T cells. At the same time, the components characteristic of both autophagy and LC3-associated phagocytosis are activated in the monocytes of patients with severe atopic asthma. However, one should note that decreased expression of the Rubicon protein, a putative marker of LC3-associated phagocytosis, has been clearly confirmed.

Machine learning approaches for the identification of ligands of the autophagy marker LC3

The LC3 proteins play a crucial role in autophagy by participating to the formation of the autophagosome. Modulation of autophagy by molecular interference with LC3 proteins could help to understand this complex fundamental biological process and how it is involved in several pathologies. Identifying new LC3 ligands is a useful contribution to this aim. In the present study, we created a PubChem library of 749 compounds having a structure based on the central scaffold of novobiocin, a reported LC3A ligand. A robust, rapid and exhaustive algorithm was used for docking each compound of this database as ligands within the dihydronovobiocin binding site, providing a docking score. Remarkable reliability and consistency between docking scores and the reported binding efficiencies of known ligands was observed, validating the machine leaning protocol used in this study. Investigation of the binding mode of the ligands having the best docking score provides additional insights in possible mode of actions of the LC3 identified ligands.

Autophagy Receptor-Inspired Antibody-Fusion Proteins for Targeted Intracellular Degradation.

Autophagy is responsible for the degradation of large intracellular contents, such as unwanted protein aggregates and organelles. Impaired autophagy can therefore lead to the accumulation of pathological aggregates, correlating with aging and neurodegenerative diseases. However, a broadly applicable methodology is not available for the targeted degradation of protein aggregates or organelles in mammalian cells. Herein, we developed a series of autophagy receptor-inspired targeting chimeras (AceTACs) that can induce the targeted degradation of aggregation-prone proteins and protein aggregates (e.g., huntingtin, TDP-43, and FUS mutants), as well as organelles (e.g., mitochondria, peroxisomes, and endoplasmic reticulum). These antibody-fusion-based AceTAC degraders were designed to mimic the function of autophagy receptors, simultaneously binding with the cellular targets and the LC3 proteins on the autophagosomal membrane, eventually transporting the target to the autophagy-lysosomal process for degradation. The AceTAC degradation system provides design principles for antibody-based degradation through autophagy, largely expanding the scope of intracellular targeted degradation technologies.

Effects of autophagy inhibitor 3-methyladenine on a diabetic mice model.

To investigate the role of autophagy inhibitor 3-methyladenine (3-MA) on a diabetic mice model (DM) and the potential mechanism. Male C57BL/6J mice were randomly divided into a normal control group (NC group) and an DM group. DM were induced by multiple low-dose intraperitoneal injection of streptozotocin (STZ) 60 mg/kg·d for 5 consecutive days. DM mice were randomly subdivided into untreated group (DM group), 3-MA (10 mg/kg·d by gavage) treated group (DM+3-MA group) and chloroquine (CQ; 50 mg/kg by intraperitoneal injection) treated group (DM+CQ group). The fasting blood glucose (FBG) levels were recorded every week. At the end of experiment, retinal samples were collected. The expression levels of pro-apoptotic proteins cleaved caspase-3, cleaved poly ADP-ribose polymerase 1 (PARP1) and Bax, anti-apoptotic protein Bcl-2, fibrosis-associated proteins Fibronectin and type 1 collagen α1 chain (COL1A1), vascular endothelial growth factor (VEGF), inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α, as well as autophagy related proteins LC3, Beclin-1 and P62 were determined by Western blotting. The oxidative stress indicators 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA) were detected by commercial kits. Both 3-MA and CQ had short-term hypoglycemic effect on FBG and reduced the expression of VEGF and inflammatory factors IL-1β and TNF-α in DM mice. 3-MA also significantly alleviated oxidative stress indicators 8-OHdG and MDA, decreased the expression of fibrosis-related proteins Fibronectin and COL1A1, pro-apoptotic proteins cleaved caspase-3, cleaved PARP1, as well as the ratio of Bax/Bcl-2. CQ had no significant impact on the oxidative stress indicators, fibrosis, and apoptosis related proteins. The results of Western blotting for autophagy related proteins showed that the ratio of LC3 II/LC3 I and the expression of Beclin-1 in the retina of DM mice were decreased by 3-MA treatment, and the expression of P62 was further increased by CQ treatment. 3-MA has anti-apoptotic and anti-fibrotic effects on the retina of DM mice, and can attenuate retinal oxidative stress, VEGF expression and the production of inflammatory factors in the retina of DM mice. The underlying mechanism of the above effects of 3-MA may be related to its inhibition of early autophagy and hypoglycemic effect.

Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy

Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. The functional relevance of these interactions was confirmed by in vitro conjugation and in vivo cellular assays. Therefore, highly curved phagophoric rims not only serve as a geometric cue for hAtg3 recruitment, but also their interaction with hAtg3 promotes LC3-PE conjugation by targeting its catalytic center to the membrane surface and bringing substrates into proximity. Our studies advance the notion that autophagosome biogenesis is directly guided by the spatial interactions of Atg3 with highly curved phagophoric rims.

Co-chaperone BAG3 enters autophagic pathway via its interaction with microtubule associated protein 1 light chain 3 beta.

The co-chaperone BAG3 is a hub for a variety of cellular pathways via its multiple domains and its interaction with chaperones of the HSP70 family or small HSPs. During aging and under cellular stress conditions in particular, BAG3, together with molecular chaperones, ensures the sequestration of aggregated or aggregation-prone ubiquitinated proteins to the autophagic-lysosomal system via ubiquitin receptors. Accumulating evidence for BAG3-mediated selective autophagy independent of cargo ubiquitination led to analyses predicting a direct interaction of BAG3 with LC3 proteins. Phylogenetically, BAG3 comprises several highly conserved potential LIRs, LC3-interacting regions, which might allow for the direct targeting of BAG3 including its cargo to autophagosomes and drive their autophagic degradation. Based on pull-down experiments, peptide arrays and proximity ligation assays, our results provide evidence of an interaction of BAG3 with LC3B. In addition, we could demonstrate that disabling all predicted LIRs abolished the inducibility of a colocalization of BAG3 with LC3B-positive structures and resulted in a substantial decrease of BAG3 levels within purified native autophagic vesicles compared with wild-type BAG3. These results suggest an autophagic targeting of BAG3 via interaction with LC3B. Therefore, we conclude that, in addition to being a key co-chaperone to HSP70, BAG3 may also act as a cargo receptor for client proteins, which would significantly extend the role of BAG3 in selective macroautophagy and protein quality control.

DPP-4 Inhibitors Suppress Tau Phosphorylation and Promote Neuron Autophagy through the AMPK/mTOR Pathway to Ameliorate Cognitive Dysfunction in Diabetic Mellitus.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have been considered as incretin-based agents that signal through GLP-1R. Our high-throughput RNA sequencing (RNA-seq) and bioinformatics methods indicated that GLP-1R, downregulated in diabetes mellitus (DM), was a potential target of DPP-4 inhibitors, which was further confirmed in DM rats. Thus, this study illuminated the alleviatory mechanism of DPP-4 on cognitive dysfunction in diabetes mellitus (DM), which may be associated with GLP-1R signaling. DM rats were administered with DPP-4 inhibitors, Chloroquine (an autophagy inhibitor), Exendin 9-39 (a GLP-1R antagonist), or Compound C (a specific inhibitor of AMPK). An in vitro model of DM was induced in rat hippocampal neuronal cell line H19-7 by exposure to high glucose (HG) and high fat (HF), followed by treatment with the above inhibitors and antagonists. It was found that cognitive dysfunction was promoted, and LC3 expression was lowered in DM rats by an autophagy inhibitor. The DPP-4 inhibitors decreased cognitive dysfunction, repressed Tau phosphorylation, and enhanced GLP-1R protein level, LC3 expression, and AMPK and mTOR phosphorylation in DM rats, while GLP-1R antagonist, an autophagy inhibitor, or AMPK inhibitor counteracted these effects. Such effects were also observed in HG/HF-induced neurons. In conclusion, our data elucidated the alleviatory mechanism of DPP-4 inhibitors in the cognitive dysfunction of DM rats via the AMPK/mTOR pathway.

Blockade of CXCR4 promotes macrophage autophagy through the PI3K/AKT/mTOR pathway to alleviate coronary heart disease

ObjectiveAutophagy is important in regulating inflammation and cholesterol efflux, suggesting that targeting autophagy may slow down atherosclerosis (AS). Since the pathological basis of coronary artery disease (CAD) is atherosclerosis, it is crucial to investigate the role of autophagy in atherosclerosis. This study aimed to investigate the role of the chemokine CXC chemokine receptor 4 (CXCR4) in promoting macrophage autophagy through the phosphoinositide-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway to alleviate coronary artery disease. MethodsThe human left coronary artery and myocardium were collected to detect CXCR4, MAP1LC3(LC3) and SQSTM1(p62) expression. ApoE−/− mice were used to establish an atherosclerosis mice model, while human monocytes (THP-1) were used to establish a foam cell model and co-cultured with foam cells using siRNACXCR4. Western blotting was conducted to quantify CXCR4, PI3K/AKT/mTOR pathway protein, LC3, Beclin1 and p62 protein levels. The left coronary artery from humans and mouse aorta and myocardium were stained with Hematoxylin and Eosin (H&E), macrophages with Oil Red O staining and foam cells were assessed by Movat's staining. CXCR4 levels, PI3K/AKT/mTOR pathway protein, LC3 and p62 were detected by immunohistochemistry (IHC) and immunofluorescence assays. Detection of autophagosomes in macrophages using transmission electron microscopy. We further assessed whether the effect of CXCR4-mediated macrophage autophagy on the formation of atherosclerosis and structural changes in the myocardium was mediated via the PI3K/AKT/mTOR signaling pathway. ResultsCXCR4 and p62 proteins were upregulated in human coronary lesions, mouse aorta, myocardial tissue, and foam cells, while LC3II/LC3I was downregulated. p85 (P-PI3K), Ser473 (P-AKT), and Ser2448 (P-mTOR) phosphorylated proteins associated with the PI3K/AKT/mTOR pathway were detected in AS and foam cell models. Upregulated CXCR4 inhibited autophagy of macrophages and increased the severity of atherosclerotic lesions. After specific knockdown of CXCR4 by adeno-associated virus (AAV9-CXCR4-RNAi) and siRNACXCR4, the above indicators were reversed, macrophage autophagy was promoted, the severity of atherosclerotic lesions was reduced, and the disorganized arrangement of myocardial architecture was improved. ConclusionKnockdown of CXCR4 reduces the extent of coronary artery disease by promoting macrophage autophagy through the PI3K/AKT/mTOR pathway to attenuate atherosclerosis.