CircRNA circUSP36 impairs the stability of NEDD4L mRNA through recruiting PTBP1 to enhance ULK1-mediated autophagic granulosa cell death.

BACKGROUND: Hypercholesterolemia is defined as a high risk factor for causing female infertility by changing the cholesterol level in granulosa cells to impair the microenvironment of oocyte development and maturation. High blood levels of oxidized low-density lipoprotein (ox-LDL) undergoes an increase of autophagic granulosa cell death. Unfortunately, this underlying molecular mechanism remains largely elusive. We aim to uncover the role of circ-ubiquitin specific peptidase 36 (USP36) in autophagic granulosa cell death. METHODS: Exposure of ox-LDL on the ovarian granulosa cell-like human granulosa (KGN) cells line was established for simulating the situation of hypercholesterolemia in vitro. Levels of circUSP36 and ULK1 were detected using real-time polymerase chain reaction (RT-PCR). Cell viability and apoptosis were assessed using (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Immunofluorescence staining of LC3 was performed to evaluate activity of autophagy. Western blot was employed to determine expression of apoptosis and autophagy-associated markers. RNA immunoprecipitation (RIP) and RNA pull-down assays were subjected to verify the circUSP36-PTBP1-NEDD4L regulatory axis. RESULTS: Treatment of ox-LDL induced aberrantly up-regulated circUSP36. Knockdown of circUSP36 alleviated cell apoptosis and excessive autophagy of granulosa cells triggered by ox-LDL. Mechanistically, reinforced expression of circUSP36 guided and facilitated PTBP1 binding to the coding region (CDS) of NEDD4L, resulting in NEDD4L mRNA decay. ULK1 was regulated by the circUSP36-PTBP1-NEDD4L axis in granulosa cells, thereby contributing to autophagic granulosa cell death. CONCLUSIONS: In summary, ox-LDL fostered autophagic granulosa cell death through circUSP36-mediated NEDD4L mRNA decay, thus elevating ULK1 expression.

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease.

The CAG expansion of huntingtin (mHTT) associated with Huntington disease (HD) is a ubiquitously expressed gene, yet it prominently damages the striatum and cortex, followed by widespread peripheral defects as the disease progresses. However, the underlying mechanisms of neuronal vulnerability are unclear. Previous studies have shown that SUMO1 (small ubiquitin-like modifier-1) modification of mHtt promotes cellular toxicity, but the in vivo role and functions of SUMO1 in HD pathogenesis are unclear. Here, we report that SUMO1 deletion in Q175DN HD-het knockin mice (HD mice) prevented age-dependent HD-like motor and neurological impairments and suppressed the striatal atrophy and inflammatory response. SUMO1 deletion caused a drastic reduction in soluble mHtt levels and nuclear and extracellular mHtt inclusions while increasing cytoplasmic mHtt inclusions in the striatum of HD mice. SUMO1 deletion promoted autophagic activity, characterized by augmented interactions between mHtt inclusions and a lysosomal marker (LAMP1), increased LC3B- and LAMP1 interaction, and decreased interaction of sequestosome-1 (p62) and LAMP1 in DARPP-32-positive medium spiny neurons in HD mice. Depletion of SUMO1 in an HD cell model also diminished the mHtt levels and enhanced autophagy flux. In addition, the SUMOylation inhibitor ginkgolic acid strongly enhanced autophagy and diminished mHTT levels in human HD fibroblasts. These results indicate that SUMO is a critical therapeutic target in HD and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.

Vorinostat in autophagic cell death: A critical insight into autophagy-mediated, -associated and -dependent cell death for cancer prevention.

Histone deacetylases (HDACs) inhibit the acetylation of crucial autophagy genes, thereby deregulating autophagy and autophagic cell death (ACD) and facilitating cancer cell survival. Vorinostat, a broad-spectrum pan-HDAC inhibitor, inhibits the deacetylation of key autophagic markers and thus interferes with ACD. Vorinostat-regulated ACD can have an autophagy-mediated, -associated or -dependent mechanism depending on the involvement of apoptosis. Molecular insights revealed that hyperactivation of the PIK3C3/VPS34-BECN1 complex increases lysosomal disparity and enhances mitophagy. These changes are followed by reduced mitochondrial biogenesis and by secondary signals that enable superactivated, nonselective or bulk autophagy, leading to ACD. Although the evidence is limited, this review focuses on molecular insights into vorinostat-regulated ACD and describes critical concepts for clinical translation.

COPB2: a transport protein with multifaceted roles in cancer development and progression.

The Coatomer protein complex subunit beta 2 (COPB2) is involved in the formation of the COPI coatomer protein complex and is responsible for the transport of vesicles between the Golgi apparatus and the endoplasmic reticulum. It plays an important role in maintaining the integrity of these cellular organelles, as well as in maintaining cell homeostasis. More importantly, COPB2 plays key roles in embryonic development and tumor progression. COPB2 is regarded as a vital oncogene in several cancer types and has been implicated in tumor cell proliferation, survival, invasion, and metastasis. Here, we summarize the current knowledge on the roles of COPB2 in cancer development and progression in the context of the hallmarks of cancer.

The Sonic Hedgehog signaling pathway regulates autophagy and migration in ovarian cancer.

BACKGROUND: The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying mechanism in ovarian cancer are still not entirely understood. METHODS: We detected the expressions of SHH and SQSTM1 in borderline ovarian tumor tissues, epithelial ovarian cancer (EOC) tissues and benign ovarian tumor tissues. Cyclopamine (Cyp, a well-known inhibitor of SHH signaling pathway) and chloroquine (CQ, the pharmaceutical inhibitor of autophagy) were used in vivo and in vitro (autophagic flux, CCK-8 assay, wound healing assay, transwell assay, tumor xenograft model). The mechanism of action was explored through Quantitative RT-PCR and Western Blot. RESULTS: We found up-regulation of SHH and accumulation of SQSTM1/P62 in epithelial ovarian cancer. Cyp induced autophagy through the PI3K/AKT signaling pathway. Moreover, low-dose Cyp and chloroquine (CQ) significantly promoted the migratory ability of SKOV3 cells. CONCLUSIONS: Our findings suggest that inhibition of the SHH pathway and autophagy may be a potential and effective therapy for the treatment of ovarian cancer.

Cellular degradation systems in ferroptosis.

In eukaryotic cells, macromolecular homeostasis requires selective degradation of damaged units by the ubiquitin-proteasome system (UPS) and autophagy. Thus, dysfunctional degradation systems contribute to multiple pathological processes. Ferroptosis is a type of iron-dependent oxidative cell death driven by lipid peroxidation. Various antioxidant systems, especially the system xc--glutathione-GPX4 axis, play a significant role in preventing lipid peroxidation-mediated ferroptosis. The endosomal sorting complex required for transport-III (ESCRT-III)-dependent membrane fission machinery counteracts ferroptosis by repairing membrane damage. Moreover, cellular degradation systems play a dual role in regulating the ferroptotic response, depending on the cargo they degrade. The key ferroptosis repressors, such as SLC7A11 and GPX4, are degraded by the UPS. In contrast, the overactivation of selective autophagy, including ferritinophagy, lipophagy, clockophagy and chaperone-mediated autophagy, promotes ferroptotic death by degrading ferritin, lipid droplets, circadian proteins, and GPX4, respectively. Autophagy modulators (e.g., BECN1, STING1/TMEM173, CTSB, HMGB1, PEBP1, MTOR, AMPK, and DUSP1) also determine the ferroptotic response in a context-dependent manner. In this review, we provide an updated overview of the signals and mechanisms of the degradation system regulating ferroptosis, opening new horizons for disease treatment strategies.

Suppressing BCL-XL increased the high dose androgens therapeutic effect to better induce the Enzalutamide-resistant prostate cancer autophagic cell death.

Most patients with advanced prostate cancer (PCa) initially respond well to androgen deprivation therapy (ADT) with antiandrogens, but most of them eventually become resistant to ADT. Here, we found that the antiandrogen Enzalutamide-resistant (EnzR) PCa cells can be suppressed by hyper-physiological doses of the androgen DHT. Mechanism dissection indicates that while androgens/androgen receptor (AR) can decrease BCL-2 expression to induce cell death, yet they can also simultaneously increase anti-apoptosis BCL-XL protein expression via decreasing its potential E3 ubiquitin ligase, PARK2, through transcriptionally increasing the miR-493-3p expression to target PARK2. Thus, targeting the high dose DHT/AR/miR-493-3p/PARK2/BCL-XL signaling with BCL-XL-shRNA can increase high-dose-DHT effect to better suppress EnzR cell growth via increasing the autophagic cell death. A preclinical study using in vivo mouse model also validated that suppressing BCL-XL led to enhance high dose DHT effect to induce PCa cell death. The success of human clinical trials in the future may help us to develop a novel therapy using high dose androgens to better suppress CRPC progression.

Inhibition of prostaglandin-degrading enzyme 15-PGDH rejuvenates aged muscle mass and strength.

Treatments are lacking for sarcopenia, a debilitating age-related skeletal muscle wasting syndrome. We identifed increased amounts of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the prostaglandin E2 (PGE2)-degrading enzyme, as a hallmark of aged tissues, including skeletal muscle. The consequent reduction in PGE2 signaling contributed to muscle atrophy in aged mice and results from 15-PGDH-expressing myofibers and interstitial cells, such as macrophages, within muscle. Overexpression of 15-PGDH in young muscles induced atrophy. Inhibition of 15-PGDH, by targeted genetic depletion or a small-molecule inhibitor, increased aged muscle mass, strength, and exercise performance. These benefits arise from a physiological increase in PGE2 concentrations, which augmented mitochondrial function and autophagy and decreased transforming growth factor-ß signaling and activity of ubiquitin-proteasome pathways. Thus, PGE2 signaling ameliorates muscle atrophy and rejuvenates muscle function, and 15-PGDH may be a suitable therapeutic target for countering sarcopenia.

Autophagy modulating agents as chemosensitizers for cisplatin therapy in cancer.

Although cisplatin is one of the most common antineoplastic drug, its successful utilisation in cancer treatment is limited by the drug resistance. Multiple attempts have been made to find potential cisplatin chemosensitisers which would overcome cancer cells resistance thus improving antineoplastic efficacy. Autophagy modulation has become an important area of interest regarding the aforementioned topic. Autophagy is a highly conservative cellular self-digestive process implicated in response to multiple environmental stressors. The high basal level of autophagy is a common phenomenon in cisplatin-resistant cancer cells which is thought to grant survival benefit. However current evidence supports the role of autophagy in either promoting or limiting carcinogenesis depending on the context. This encourages the search of substances modulating the process to alleviate cisplatin resistance. Such a strategy encompasses not only simple autophagy inhibition but also harnessing the process to induce autophagy-dependent cell death. In this paper, we briefly describe the mechanism of cisplatin resistance with a special emphasis on autophagy and we give an extensive literature review of potential substances with cisplatin chemosensitising properties related to autophagy modulation.

Overexpression of Prolidase Induces Autophagic Death in MCF-7 Breast Cancer Cells.

BACKGROUND/AIMS: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. METHODS: The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. CONCLUSION: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death.

Ganoderic acid D induces synergistic autophagic cell death except for apoptosis in ESCC cells.

ETHNOPHAMACOLOGICAL RELEVANCE: Ganoderma lucidum has been used as a medicinal mushroom for more than 2000 years in China. Ganoderic acid D (GAD) as a representative active triterpenoid from Ganoderma lucidum is known to possess anticancer activity. However, the mechanism involved in its anticancer cell process is still largely elusive. AIM OF THE STUDY: Our study aimed to investigate the anticancer effects of GAD on the esophageal squamous cell carcinoma (ESCC) cells and the underlying mechanisms at the cell level. MATERIALS AND METHODS: EC9706 and Eca109 cells were treated with GAD (0, 10, 20, 40 µM) for 24 h. The cell viability, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis rate, caspase-3 activity, autophagic flux, lysosomal function were examined. Cell cycle, apoptotic, autophagy and mTOR signal pathway related proteins such as P53, Cyclin B1, CytoC, PARP, Beclin-1, P62, LC3, PI3K, AKT and mTOR were analyzed by Western blot approach. RESULTS: GAD inhibited cell proliferation and induced both apoptosis and autophagic cell death. In particular, we found that in the early stage of the autophagic process, GAD could initiate and enhance the autophagy signal while in the late stage it on the contrary could block the autophagic flux by impairing the autophagosome-lysosome fusion and inhibited the lysosomal degradation. Besides the autophagic cell death, GAD also induced the apoptosis mediated by caspase-related process in parallel. The mechanism involved for the synergistic apoptotic and autophagic cell death was also explored. We found that GAD down-regulated the expression of PI3K, AKT and mTOR phosphorylated proteins in the mTOR signaling pathway which thus led to the synergistic effect on apoptosis and autophagic cell death in the ESCC cells. CONCLUSIONS: In summary, this study has documented that GAD may inhibit cell proliferation through the mTOR pathway in ESCC cells, and induce synergistic apoptosis and autophagic cell death by disrupting the autophagic flux. This work therefore also suggests that GAD may be used as an efficient anticancer adjuvant for ESCC cancer therapy.

Heme Induces BECN1/ATG5-Mediated Autophagic Cell Death via ER Stress in Neurons.

Intracerebral hemorrhage (ICH) is a serious medical problem, and effective treatment is limited. Hemorrhaged blood is highly toxic to the brain, and heme, which is mainly released from hemoglobin, plays a vital role in neurotoxicity. However, the specific mechanism involved in heme-mediated neurotoxicity has not been well studied. In this study, we investigated the neurotoxicity of heme in neurons. Neurons were treated with heme, and cell death, autophagy, and endoplasmic reticulum (ER) stress were analyzed. In addition, the relationship between autophagy and apoptosis in heme-induced cell death and the downstream effects were also assessed. We showed that heme induced cell death and autophagy in neurons. The suppression of autophagy using either pharmacological inhibitors (3-methyladenine) or RNA interference of essential autophagy genes (BECN1 and ATG5) decreased heme-induced cell death in neurons. Moreover, the ER stress activator thapsigargin increased cell autophagy and the cell death ratio following heme treatment. Autophagy promoted heme-induced cell apoptosis and cell death through the BECN1/ATG5 pathway. Our findings suggest that heme potentiates neuronal autophagy via ER stress, which in turn induces cell death via the BECN1/ATG5 pathway. Targeting ER stress-mediated autophagy might be a promising therapeutic strategy for ICH.

Autophagy role in environmental pollutants exposure.

During the last decades, the potential harmfulness derived from the exposure to environmental pollutants has been largely demonstrated, with associated damages ranging from geno- and cyto-toxicity to tissue malfunction and alterations in organism physiology. Autophagy is an evolutionarily-conserved cellular mechanism essential for cellular homeostasis, which contributes to protect cells from a wide variety of intracellular and extracellular stressors. Due to its pivotal importance, its correct functioning is directly linked to cell, tissue and organismal fitness. Environmental pollutants, particularly industrial compounds, are able to impact autophagic flux, either by increasing it as a protective response, by blocking it, or by switching its protective role toward a pro-cell death mechanism. Thus, the understanding of the effects of chemicals exposure on autophagy has become highly relevant, offering new potential approaches for risk assessment, protection and preventive measures to counteract the detrimental effects of environmental pollutants on human health.

NMMHC IIA triggers neuronal autophagic cell death by promoting F-actin-dependent ATG9A trafficking in cerebral ischemia/reperfusion.

Previous findings have shown that non-muscle myosin heavy-chain IIA (NMMHC IIA) is involved in autophagy induction triggered by starvation in D. melanogaster; however, its functional contribution to neuronal autophagy remains unclear. The aim of this study is to explore the function of NMMHC IIA in cerebral ischemia-induced neuronal autophagy and the underlying mechanism related to autophagy-related gene 9A (ATG9A) trafficking. Functional assays and molecular mechanism studies were used to investigate the role of NMMHC IIA in cerebral ischemia-induced neuronal autophagy in vivo and in vitro. A middle cerebral artery occlusion (MCAO) model in mice was used to evaluate the therapeutic effect of blebbistatin, a myosin II ATPase inhibitor. Herein, either depletion or knockdown of NMMHC IIA led to increased cell viability in both primary cultured cortical neurons and pheochromocytoma (PC12) cells exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). In addition, NMMHC IIA and autophagic marker LC3B were upregulated by OGD/R, and inhibition of NMMHC IIA significantly reduced OGD-induced neuronal autophagy. Furthermore, NMMHC IIA-induced autophagy is through its interactions with F-actin and ATG9A in response to OGD/R. The NMMHC IIA-actin interaction contributes to ATG9A trafficking and autophagosome formation. Inhibition of the NMMHC IIA-actin interaction using blebbistatin and the F-actin polymerization inhibitor cytochalasin D significantly suppressed ATG9A trafficking and autophagy induction. Furthermore, blebbistatin significantly improved neurological deficits and infarct volume after ischemic attack in mice, accompanied by ATG9A trafficking and autophagy inhibition. These findings demonstrate neuroprotective effects of NMMHC IIA inhibition on regulating ATG9A trafficking-dependent autophagy activation in the context of cerebral ischemia/reperfusion.

Overexpression of FNTB and the activation of Ras induce hypertrophy and promote apoptosis and autophagic cell death in cardiomyocytes.

Farnesyltransferase (FTase) is an important enzyme that catalyses the modification of protein isoprene downstream of the mevalonate pathway. Previous studies have shown that the tissue of the heart in the suprarenal abdominal aortic coarctation (AAC) group showed overexpression of FTaseß (FNTB) and the activation of the downstream protein Ras was enhanced. FTase inhibitor (FTI) can alleviate myocardial fibrosis and partly improve cardiac remodelling in spontaneously hypertensive rats. However, the exact role and mechanism of FTase in myocardial hypertrophy and remodelling are not fully understood. Here, we used recombinant adenovirus to transfect neonatal rat ventricular cardiomyocytes to study the effect of FNTB overexpression on myocardial remodelling and explore potential mechanisms. The results showed that overexpression of FNTB induces neonatal rat ventricular myocyte hypertrophy and reduces the survival rate of cardiomyocytes. FNTB overexpression induced a decrease in mitochondrial membrane potential and increased apoptosis in cardiomyocytes. FNTB overexpression also promotes autophagosome formation and the accumulation of autophagy substrate protein, LC3II. Transmission electron microscopy (TEM) and mCherry-GFP tandem fluorescent-tagged LC3 (tfLC3) showed that FNTB overexpression can activate autophagy flux by enhancing autophagosome conversion to autophagolysosome. Overactivated autophagy flux can be blocked by bafilomycin A1. In addition, salirasib (a Ras farnesylcysteine mimetic) can alleviate the hypertrophic phenotype of cardiomyocytes and inhibit the up-regulation of apoptosis and autophagy flux induced by FNTB overexpression. These results suggest that FTase may have a potential role in future treatment strategies to limit the adverse consequences of cardiac hypertrophy, cardiac dysfunction and heart failure.

Ferroptosis is a type of autophagy-dependent cell death.

Macroautophagy (hereafter referred to as autophagy) involves an intracellular degradation and recycling system that, in a context-dependent manner, can either promote cell survival or accelerate cellular demise. Ferroptosis was originally defined in 2012 as an iron-dependent form of cancer cell death different from apoptosis, necrosis, and autophagy. However, this latter assumption came into question because, in response to ferroptosis activators (e.g., erastin and RSL3), autophagosomes accumulate, and because components of the autophagy machinery (e.g., ATG3, ATG5, ATG4B, ATG7, ATG13, and BECN1) contribute to ferroptotic cell death. In particular, NCOA4-facilitated ferritinophagy, RAB7A-dependent lipophagy, BECN1-mediated system xc- inhibition, STAT3-induced lysosomal membrane permeabilization, and HSP90-associated chaperone-mediated autophagy can promote ferroptosis. In this review, we summarize current knowledge on the signaling pathways involved in ferroptosis, while focusing on the regulation of autophagy-dependent ferroptotic cell death. The molecular comprehension of these phenomena may lead to the development of novel anticancer therapies.

Prognostic Significance of LC3B and p62/SQSTM1 Expression in Gastric Adenocarcinoma.

BACKGROUND/AIM: Autophagy is a cellular mechanism that recycles cellular components to maintain homeostasis. To investigate the clinical implication of autophagy in gastric cancer, the autophagy markers with autophagosome formation, LC3B and selective autophagy substrate p62/SQSTM1 (P62) were validated. MATERIALS AND METHODS: LC3B and p62 expression was examined using immunohistochemistry, western blot assays, and reverse-transcription polymerase chain reaction (RT-PCR). The relationship of LC3B and p62 expression in gastric adenocarcinomas with clinicopathological parameters, including patient survival, were analyzed. RESULTS: Normal gastric mucosae exhibit no LC3B and p62 expression, while tubular adenoma and gastric adenocarcinomas exhibit variable nuclear or cytoplasmic p62 expression. High LC3B, high cytoplasmic p62, and low nuclear p62 protein expression in gastric adenocarcinomas is positively correlated with poor prognostic factors including survival. CONCLUSION: Dynamic LC3B and p62 changes are suggested to be involved in gastric tumorigenesis and cancer progression. LC3B and p62 could be used as prognostic biomarkers and potential therapeutic targets for gastric adenocarcinomas.

A Double Negative Feedback Loop between mTORC1 and AMPK Kinases Guarantees Precise Autophagy Induction upon Cellular Stress.

Cellular homeostasis is controlled by an evolutionary conserved cellular digestive process called autophagy. This mechanism is tightly regulated by the two sensor elements called mTORC1 and AMPK. mTORC1 is one of the master regulators of proteostasis, while AMPK maintains cellular energy homeostasis. AMPK is able to promote autophagy by phosphorylating ULK1, the key inducer of autophagosome formation, while mTORC1 downregulates the self-eating process via ULK1 under nutrient rich conditions. We claim that the feedback loops of the AMPK-mTORC1-ULK1 regulatory triangle guarantee the appropriate response mechanism when nutrient and/or energy supply changes. In our opinion, there is an essential double negative feedback loop between mTORC1 and AMPK. Namely, not only does AMPK downregulate mTORC1, but mTORC1 also inhibits AMPK and this inhibition is required to keep AMPK inactive at physiological conditions. The aim of the present study was to explore the dynamical characteristic of AMPK regulation upon various cellular stress events. We approached our scientific analysis from a systems biology perspective by incorporating both theoretical and molecular biological techniques. In this study, we confirmed that AMPK is essential to promote autophagy, but is not sufficient to maintain it. AMPK activation is followed by ULK1 induction, where protein has a key role in keeping autophagy active. ULK1-controlled autophagy is always preceded by AMPK activation. With both ULK1 depletion and mTORC1 hyper-activation (i.e., TSC1/2 downregulation), we demonstrate that a double negative feedback loop between AMPK and mTORC1 is crucial for the proper dynamic features of the control network. Our computer simulations have further proved the dynamical characteristic of AMPK-mTORC1-ULK1 controlled cellular nutrient sensing.

ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress.

Age-related macular degeneration (AMD) is an ocular disease with retinal degeneration. Retinal pigment epithelium (RPE) degeneration is mainly caused by long-term oxidative stress. Kinase activity could be either protective or detrimental to cells during oxidative stress; however, few reports have described the role of kinases in oxidative stress. In this study, high-throughput screening of kinome siRNA library revealed that erb-b2 receptor tyrosine-protein kinase 2 (ERBB2) knockdown reduced reactive oxygen species (ROS) production in ARPE-19 cells during oxidative stress. Silencing ERBB2 caused an elevation in microtubule associated protein light chain C3-II (MAP1LC3B-II/I) conversion and sequesterone (SQSTM)1 protein level. ERBB2 deprivation largely caused an increase in autophagy-regulating protease (ATG4B) expression, a protease that negatively recycles MAP1LC3-II at the fusion step between the autophagosome and lysosome, suggesting ERBB2 might modulate ATG4B for autophagy induction in oxidative stress-stimulated ARPE-19 cells. ERBB2 knockdown also caused an accumulation of nuclear factor erythroid 2-related factor 2 (NRF2) and enhanced its transcriptional activity. In addition, ERBB2 ablation or treatment with autophagy inhibitors reduced oxidative-induced cytotoxic effects in ARPE-19 cells. Furthermore, ERBB2 silencing had little or no additive effects in ATG5/7-deficient cells. Taken together, our results suggest that ERBB2 may play an important role in modulating autophagic RPE cell death during oxidative stress, and ERBB2 may be a potential target in AMD prevention.

Hedgehog and Wingless signaling are not essential for autophagy-dependent cell death.

Autophagy-dependent cell death is a distinct mode of regulated cell death required in a context specific manner. One of the best validated genetic models of autophagy-dependent cell death is the removal of the Drosophila larval midgut during larval-pupal transition. We have previously shown that down-regulation of growth signaling is essential for autophagy induction and larval midgut degradation. Sustained growth signaling through Ras and PI3K blocks autophagy and consequently inhibits midgut degradation. In addition, the morphogen Dpp plays an important role in regulating the correct timing of midgut degradation. Here we explore the potential roles of Hh and Wg signaling in autophagy-dependent midgut cell death. We demonstrate that Hh and Wg signaling are not involved in the regulation of autophagy-dependent cell death. However, surprisingly we found that one key component of these pathways, the Drosophila Glycogen Synthase Kinase 3, Shaggy (Sgg), may regulate midgut cell size independent of Hh and Wg signaling.