Molecular Mechanisms Of Autophagy Research Articles

Early-stage autophagy protects nucleus pulposus cells from glucose deprivation-induced degeneration via the p-eIF2α/ATF4 pathway.

Autophagy is a double-edged sword in cellular survival, but its effects on nucleus pulposus (NP) cells are yet to be clarified. This study explored the role and molecular mechanisms of autophagy in the survival of NP cells under nutrient deprivation. Glucose limitation induced time-dependent morphological changes, proteoglycan degradation, and apoptosis in NP cells. Glucose deprivation triggered the activation of early-stage autophagy, evident as increases in LC3-II and ATG12-ATG5 expression and the number of GFP-LC3 puncta. Importantly, at early time points, the autophagy inhibitor 3-MA significantly enhanced the apoptosis of NP cells, suggesting that early-stage autophagy protects cells against glucose deprivation. Interestingly, the p-eIF2α/ATF4 unfolded protein response pathway was activated in NP cells deprived of glucose, and a deficiency in ATF4 attenuated the activation of early-stage autophagy and increased apoptosis. ATF4 silencing also inhibited the late-stage accumulation of reactive oxygen species (ROS) and apoptosis. Together, our results demonstrate an interplay between endoplasmic reticulum stress, ROS production, and the early-stage autophagy induced by glucose deprivation in NP cells. These findings may provide a therapeutic strategy for the treatment of degenerative intervertebral disc disease.

Up-regulation of the active form of small GTPase Rab13 promotes macroautophagy in vascular endothelial cells

The importance of macroautophagy (hereafter referred to as autophagy) in vascular endothelial cell (VEC) biology and dysfunction is increasingly recognized, but the molecular mechanisms of autophagy in VECs in the presence of serum are still poorly understood. Previously, we identified pterostilbene as a potent autophagy inducer of VECs in the presence of serum. In this study, we used pterostilbene as a tool to induce VEC autophagy and identified the differentially expressed genes using high-throughput DAN microarray. The small GTPase Ras-related protein in brain 13 (Rab13) was found to be the most significantly up-regulated gene in pterostilbene-treated human umbilical VECs (HUVECs). Knockdown of Rab13 blocked pterostilbene-induced mTOR inhibition and autophagy, whereas overexpression of the GTP-containing active form of Rab13 induced mTOR inhibition and autophagy in HUVECs. Using a combination of immunofluorescence and co-immunoprecipitation (co-IP) assays, we demonstrated that pterostilbene or up-regulation of the active form of Rab13 promoted the interaction between Rab13 and growth factor receptor-bound protein 2 (Grb2). Knockdown of Grb2 suppressed pterostilbene or up-regulation of the active form of Rab13-induced autophagy. Further mechanistic studies revealed that Rab13 activated the downstream AMP-activated protein kinase (AMPK) and blocked mammalian target of rapamycin (mTOR) signaling by its functional interaction with Grb2 to regulate autophagy in HUVECs. Our study firmly establishes Rab13 as a novel regulator of autophagy in VECs under nutrient-enriched conditions.

Molecular mechanisms of autophagy in regulating renal aging and interventional effects of Chinese herbal medicine

Aging is the gradual functional recession of the living tissues or organs caused by a variety of genetic and environmental factors together. Autophagy is a process of degrading cytoplasmic components mediated by lysosomes in eukaryotic cells. Kidney is a typical target organ of aging. Autophagy regulates renal aging. Decrease in autophagy can accelerate renal aging,whereas,increase in autophagy can delay renal aging. During the process of regulating renal aging,the mammalian target of rapamycin (mTOR) and its related signaling pathways including the adenosine monophosphate activated protein kinase (AMPK)/mTOR,the phosphatidylinositol 3-kinase (PI3K)/ serine-threonine kinase(Akt)/mTOR,the AMPK/silent information regulation 1 (Sirt1) and transforming growth factor β (TGF-β) play the important roles in renal aging. Regulating the key signaling molecules in these pathways in vivo can control renal aging. Some Chinese herbal medicine (CHM) and their extracts with the effects of nourishing kidney or activating stasis, such as Cordyceps sinensis, curcumin and resveratrol have the beneficial effects on renal aging and/or autophagy. Therefore,revealing the pharmacological effects of CHM in anti-renal aging based on the molecular mechanisms of autophagy will become one of the development trends in the future study.

Autophagy in Diabetic Retinopathy.

Autophagy is an important homeostatic cellular process encompassing a number of consecutive steps indispensable for degrading and recycling cytoplasmic materials. Basically autophagy is an adaptive response that under stressful conditions guarantees the physiological turnover of senescent and impaired organelles and, thus, controls cell fate by various cross-talk signals. Diabetic retinopathy (DR) is a serious microvascular complication of diabetes and accounts for 5% of all blindness. Although, various metabolic disorders have been linked with the onset of DR, due to the complex character of this multi-factorial disease, a connection between any particular defect and DR becomes speculative. Diabetes increases inflammation, advanced glycation end products (AGEs) and oxidative stress in the retina and its capillary cells. Particularly, a great number of evidences suggest a mutual connection between oxidative stress and other major metabolic abnormalities implicated in the development of DR. In addition, the intricate networks between autophagy and apoptosis establish the degree of cellular apoptosis and the progression of DR. Growing data underline the crucial role of reactive oxygen species (ROS) in the activation of autophagy. Depending on their delicate balance both redox signaling and autophagy, being detrimental or beneficial, retain opposing effects. The molecular mechanisms of autophagy are very complex and involve many signaling pathways cooperating at various steps. This review summarizes recent advances of the possible molecular mechanisms in autophagic process that are involved in pathophysiology of DR. In-depth analysis on the molecular mechanisms leading to autophagy in the retinal pigment epithelial (RPE) will be helpful to plan new therapies aimed at preventing or improving the progression of DR.

Molecular mechanisms of autophagy and its role in cancer development

Autophagy is an evolutionary process preserved in eukaryotes, which removes harmful components and maintains cell homeostasis in response to a variety of extracellular stimuli. It is involved in both physiological and pathological conditions, including cancer.The role of autophagy in the treatment of cancer is described as a “double-edged sword”, which reflects its involvement in tumor suppression, survival and subsequent proliferation of tumor cells. Recent advances are useful for planning appropriate adjustments to inhibit or promote autophagy in order to obtain therapeutic efficacy in cancer patients. The objectives of this review are to clarify the role of autophagy in cancer and to highlight the need for more research in the field.

Autophagy, Innate Immunity and Tissue Repair in Acute Kidney Injury.

Kidney is a vital organ with high energy demands to actively maintain plasma hemodynamics, electrolytes and water homeostasis. Among the nephron segments, the renal tubular epithelium is endowed with high mitochondria density for their function in active transport. Acute kidney injury (AKI) is an important clinical syndrome and a global public health issue with high mortality rate and socioeconomic burden due to lack of effective therapy. AKI results in acute cell death and necrosis of renal tubule epithelial cells accompanied with leakage of tubular fluid and inflammation. The inflammatory immune response triggered by the tubular cell death, mitochondrial damage, associative oxidative stress, and the release of many tissue damage factors have been identified as key elements driving the pathophysiology of AKI. Autophagy, the cellular mechanism that removes damaged organelles via lysosome-mediated degradation, had been proposed to be renoprotective. An in-depth understanding of the intricate interplay between autophagy and innate immune response, and their roles in AKI pathology could lead to novel therapies in AKI. This review addresses the current pathophysiology of AKI in aspects of mitochondrial dysfunction, innate immunity, and molecular mechanisms of autophagy. Recent advances in renal tissue regeneration and potential therapeutic interventions are also discussed.

Molecular mechanisms of autophagy in plants: Role of ATG8 proteins in formation and functioning of autophagosomes

Autophagy is an efficient way of degradation and removal of unwanted or damaged intracellular components in plant cells. It plays an important role in recycling of intracellular structures (during starvation, removal of cell components formed during plant development or damaged by various stress factors) and in programmed cell death. Morphologically, autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which are essential for the isolation and degradation of cytoplasmic components. Among autophagic (ATG) proteins, ATG8 from the ubiquitin-like protein family plays a key role in autophagosome formation. ATG8 is also involved in selective autophagy, fusion of autophagosome with the vacuole, and some other intracellular processes not associated with autophagy. In contrast to yeasts that carry a single ATG8 gene, plants have multigene ATG8 families. The reason for such great ATG8 diversity in plants remains unclear. It is also unknown whether all members of the ATG8 family are involved in the formation and functioning of autophagosomes. To answer these questions, the identification of the structure and the possible functions of plant proteins from ATG8 family is required. In this review, we analyze the structures of ATG8 proteins from plants and their homologs from yeast and animal cells, interactions of ATG8 proteins with functional ligands, and involvement of ATG8 proteins in different metabolic processes in eukaryotes.

MicroRNA-27a Promotes Inefficient Lysosomal Clearance in the Hippocampi of Rats Following Chronic Brain Hypoperfusion.

Chronic brain hypoperfusion (CBH) induces the accumulation of abnormal cellular proteins, accompanied by cognitive decline, and the autophagic-lysosomal system is abnormal in dementia. Whether CBH accounts for autophagic-lysosomal neuropathology remains unknown. Here, we show that CBH significantly increased the number of autophagic vacuoles (AVs) with high LC3-II levels, but decreased SQSTM1 and cathepsin D levels in the hippocampi of rats following bilateral common carotid artery occlusion (2VO) for 2weeks. Further studies showed that microRNA-27a (Mir27a) was upregulated at 2weeks compared with the sham group. Additionally, LAMP-2 proteins were downregulated by Mir27a overexpression, upregulated by Mir27a inhibition, and unchanged by binding-site mutations or miR-masks, indicating that lamp-2 is the target of Mir27a. Knockdown of endogenous Mir27a prevented the reduction of LAMP-2 protein expression as well as the accumulation of AVs in the hippocampi of 2VO rats. Overexpression of Mir27a induced,while the knockdown of Mir27a reduced, the accumulation of AVs and the LC3-II level in cultured neonatal rat neurons. The results revealed that CBH in rats at 2weeks could induce inefficient lysosomal clearance, which is regulated by the Mir27a-mediated downregulation of LAMP-2 protein expression. These findings provide an insight into a novel molecular mechanism of autophagy at the miRNA level.

The role of autophagy in mechanisms of tumor cell death

Autophagy is an evolutionarily established process of maintaining the cell integrity by lysosomal recycling of their own cytosolic proteins, macromolecules, organelles, and protein aggregates. Autophagy is regulated by a group of autophagy-related genes (ATG) and is detected at the basal level in almost all cells of the body. Alterations in the autophagy-associated signaling pathways often occur in various pathologies, including malignant neoplasms. Numerous experimental and clinical trials demonstrated the important role of autophagy in mechanisms of chemoresistance or chemosensitivity to a wide range of antitumor agents. This review describes the molecular mechanisms of autophagy and its interaction with apoptosis. The possibilities of modulating the autophagy level in tumor cells to increase the efficacy of anticancer therapy are estimated.

Morphological and ultrastructural examination of senescence in Morchella elata

In recent years, the artificial cultivation of Morchella mushrooms that belong to Elata Clade, including Morchella elata, has been developed rapidly in China. However, the prominent problem of spawn aging has been frustrating the morel farming. In this paper, aging in M. elata was achieved from 12 to 17 subcultures and lifespan of 1536–2256h by successive subculturing. The lifespan can be roughly divided into juvenile phase and senescent phase with respect to the mycelia linear growth rate. After a certain period of rapid growth with almost constant rate at the juvenile phase, the isolate entered the senescent phase characterized by slow down of mycelia growth, producing pigments ahead of time and final death of the apical hyphae. The period of the senescent phase was definitely 240–288h; while that of the juvenile phase was diverse relying on different isolates. Moreover, microscopic study showed that angles between the leading and primary hyphae increased constantly with aging. In senesced hyphal cells of M. elata, the typical characteristics of autophagy (enlargement of vacuoles and existence of organelles sequestrated lysosomes) and apoptosis (condensation of the cytoplasm and nuclear and plasmolysis) were observed. In addition, in the final stage of senescence, the apical hyphae collapsed with the plasma membrane and all the cellular organelles disrupted, indicated a necrotic mode of cell death. Taken together, these data revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The characterization and molecular mechanism of autophagy, apoptosis and necrosis need further study and the systematic study of morel aging will be beneficial for the healthy development of morel farming.

Tu1841 Investigation of Indomethacin-Induced Small Intestinal Mucosal Damage in Atg5-Knock-out Mice, and the Molecular Mechanisms of Autophagy and Apoptosis

Background: In recent years, it has been shown that nonsteroidal anti-inflammatory drugs (NSAIDs) damage the mucosa of both the stomach and the small intestine. Various points are still unclear with respect to the pathology of NSAID-induced small intestinal mucosal damage, but attention has been given to the type of pathology that involves autophagy and cell death. For the present study, mice were prepared in which the Atg5 gene, essential for autophagy induction, was knocked out, in the small intestine only[w1] . Small intestinal mucosal damage induced by oral administration of indomethacin, an NSAID, was then compared between knock-out and normal mice. In addition, in order to elucidate the molecular mechanism, a study was carried out with IEC-6 cells, which are normal rat epithelial cells. Methods: Indomethacin was administered to normal mice and knock-out mice in the non-fasted state, and after 24 hours the entire damaged area of the small intestine was measured. In addition, Atg5-knock-out IEC-6 cells were administered indomethacin, and cell viability was determined. Furthermore, LC3, PARP1, Erk1/2, Nrf2, and HO-1 expression was investigated by the Western blot method. Results: The mean surface areas of small intestinal damage in normal mice and Atg5-knock-out mice were 16.26 ± 3.24 mm2 and 7.09 ± 2.24 mm2, respectively. Indomethacin-induced damage was also lower in Atg5-knock-out IEC-6 cells than in normal IEC-6 cells, and ERK, Nrf2, and HO-1 expression was higher in the former. Conclusions: It was confirmed that loss of autophagy results in reduction of indomethacin-induced small intestinal mucosal damage both in vivo and in vitro. It is known that HO-1 is induced by Nrf2, and Nrf2 is induced via the Erk pathway, suggesting the possibility that increased HO-1 expression mediated by the Erk-Nrf2 pathway, due to inhibition of autophagy, is the mechanism by which cytotoxicity is inhibited.

Recent progress in research on molecular mechanisms of autophagy in the heart.

Dysregulation of autophagy, an evolutionarily conserved process for degradation of long-lived proteins and organelles, has been implicated in the pathogenesis of human disease. Recent research has uncovered pathways that control autophagy in the heart and molecular mechanisms by which alterations in this process affect cardiac structure and function. Although initially thought to be a nonselective degradation process, autophagy, as it has become increasingly clear, can exhibit specificity in the degradation of molecules and organelles, such as mitochondria. Furthermore, it has been shown that autophagy is involved in a wide variety of previously unrecognized cellular functions, such as cell death and metabolism. A growing body of evidence suggests that deviation from appropriate levels of autophagy causes cellular dysfunction and death, which in turn leads to heart disease. Here, we review recent advances in understanding the role of autophagy in heart disease, highlight unsolved issues, and discuss the therapeutic potential of modulating autophagy in heart disease.

Autophagy and vascularization

The molecular mechanism of autophagy is complicated and it always affects the occurrence and development of tumor in many different degrees,so it is the new hot spot in pathological study area.The angiogenesis includes physiological vascularization and pathological vascularization,and the pathological new vessels which provide nutrition for the continuous growth of tumors have a close relationship to the development and metastasis of tumor.The process of angiogenesis can be affected by regulating the autophagy of vascular endothelial cells,which provides a new method for tumor therapy. Key words: Autophagy; Neoplasms; Vascularization, pathologic

Autophagy and Its Implication in Antiviral Immunity

Autophagy is a catabolic mechanism developed in eukaryotes in order to maintain cellular homeostasis and development. It is also an essential constituent of the immune system since it is involved in the MHC class I and II antigen generation and exerts an antiviral effect. However many viruses are either able to inhibit or induce autophagy to avoid their own elimination or to favor their replication by interacting with different complexes of the autophagy pathway. This review summarizes the molecular mechanisms of autophagy and its importance in immunity. It describes the different strategies that viruses have adopted to hijack this process.

The Role of Autophagy in Early Mammalian Embryonic Development

During oogenesis, the oocyte stores a large amount of maternally provided products, including mRNAs and proteins. However, after fertilization, these products are rapidly degraded and new materials are synthesized from the zygotic genome. This oocyte-to-embryo transition, also known as the maternal-to-zygotic transition, is conserved in many species and plays a pivotal role in development, because non-degraded products can hamper further embryonic development. Given that the time of early embryonic development is so rapid, ubiquitin/proteasome-mediated turnover of individual proteins is probably not sufficient to remove maternal products during the oocyte-to-embryo transition. Autophagy is an evolutionally conserved degradation system in which portions of the cytoplasm sequestered by double membrane structures called autophagosomes are delivered to lysosomes for degradation. The basic roles of autophagy are the generation of amino acids for energy and the maintenance of cellular quality. In addition to the fundamental functions of autophagy, the unique role of autophagy in removing random cytoplasmic contents including mitochondria, peroxisomes, and even lipids, may contribute to extensive cellular remodeling during the oocyte-to-embryo transition. Here we briefly review the history and molecular mechanisms of autophagy, and discuss the function of autophagy in early mammalian embryogenesis.

Beclin 2 Functions in Autophagy, Degradation of G Protein-Coupled Receptors, and Metabolism

The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additionallysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family membersin autophagy, endolysosomal trafficking, and metabolism.

ChemInform Abstract: Small‐Molecule Regulators of Autophagy and Their Potential Therapeutic Applications

AbstractReview: 123 refs.

Small‐Molecule Regulators of Autophagy and Their Potential Therapeutic Applications

Autophagy is a highly conserved process in which damaged proteins and organelles are sequestered in double-membrane autophagosomes and delivered to lysosomes for degradation and recycling. As an efficient response to cellular stress, autophagy is essential for the maintenance of cellular homeostasis. Defective autophagy is associated with a variety of diseases, including cancer. This article summarizes current knowledge about the molecular mechanism of autophagy and its role in tumorigenesis. Particular focus is placed on the development of small-molecule regulators of autophagy and their potential application as anticancer therapeutic agents.

Autophagy inhibition enhances etoposide-induced cell death in human hepatoma G2 cells

Induction of autophagy usually acts as a survival mechanism of cancer cells in response to chemotherapy. However, the function and molecular mechanism of autophagy in human hepatoma cells under drug treatment is still not clear. To address this issue, we established an experimental model in which HepG2 cells were treated with etoposide, a widely used anticancer agent. We demonstrate the etoposide-induced accumulation of GFP-LC3 dots by fluorescent microscopy, the up-regulation of LC3-II protein expression by Western blotting and the increased number of autophagic vacuoles by electron microscopy, confirming the activation of autophagy by etoposide in HepG2 cells. Inhibition of autophagy by either 3-methyladenine (3MA) or beclin-1 small interfering RNA enhanced etoposide-induced cell death. Furthermore, activation of p53 and AMPK was detected in etoposide-treated cells and inhibition of AMPK triggered apoptosis through suppression of autophagy. On the other hand, inactivation of p53 promoted cell survival through augmentation of autophagy. Collectively, these findings indicate that etoposide-induced autophagy promotes hepatoma cell adaptation and survival, and that autophagy inhibition improves the chemotherapeutic effect of etoposide. Moreover, AMPK activation is clearly associated with etoposide-induced autophagy. We conclude that manipulation of AMPK may be a promising approach of adjuvant chemotherapy for hepatocellular carcinoma.

The Role of Macroautophagy in Development of Filamentous Fungi

Autophagy (macroautophagy) is a bulk degradative pathway by which cytoplasmic components are delivered to the vacuole for recycling. This process is conserved from yeast to human, where it is implicated in cancer and neurodegenerative diseases. During the last decade, many ATG genes involved in autophagy have been identified, initially in Saccharomyces cerevisiae. This review summarizes the knowledge on the molecular mechanisms of autophagy using yeast as model system. Although many of the core components involved in autophagy are conserved from yeast to human, there are, nevertheless, significant differences between these organisms, for example, during autophagy initiation. Autophagy also plays an essential role in filamentous fungi especially during differentiation. Remarkably, in these species autophagy may reflect features of both yeast and mammals. This is exemplified by the finding that filamentous fungi lack the S. cerevisiae clade-specific Atg31 protein, but contain Atg101, which is absent in this clade. A reappraisal of genome data further suggests that, similar to yeast and mammals, filamentous fungi probably also contain two distinct phosphatidylinositol 3-kinase complexes. This review also summarizes the state of knowledge on the role of autophagy in filamentous fungi during differentiation, such as pathogenic development, programmed cell death during heteroincompatibility, and spore formation.