In JoVE (1)

Other Publications (30)

Articles by Congcong He in JoVE

Other articles by Congcong He on PubMed

Autophagy and Neurodegeneration

ACS Chemical Biology. May, 2006  |  Pubmed ID: 17163674

Autophagy mediates the bulk degradation of cytosolic proteins and organelles. Recent studies using neuron-specific knockout mouse models demonstrate that autophagy deficiency leads to protein aggregation and neurodegeneration, even in the absence of disease-related aggregate-prone proteins.

Recruitment of Atg9 to the Preautophagosomal Structure by Atg11 is Essential for Selective Autophagy in Budding Yeast

The Journal of Cell Biology. Dec, 2006  |  Pubmed ID: 17178909

Autophagy is a conserved degradative pathway that is induced in response to various stress and developmental conditions in eukaryotic cells. It allows the elimination of cytosolic proteins and organelles in the lysosome/vacuole. In the yeast Saccharomyces cerevisiae, the integral membrane protein Atg9 (autophagy-related protein 9) cycles between mitochondria and the preautophagosomal structure (PAS), the nucleating site for formation of the sequestering vesicle, suggesting a role in supplying membrane for vesicle formation and/or expansion during autophagy. To better understand the mechanisms involved in Atg9 cycling, we performed a yeast two-hybrid-based screen and identified a peripheral membrane protein, Atg11, that interacts with Atg9. We show that Atg11 governs Atg9 cycling through the PAS during specific autophagy. We also demonstrate that the integrity of the actin cytoskeleton is essential for correct targeting of Atg11 to the PAS. We propose that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS that is dependent on the actin cytoskeleton during yeast vegetative growth.

Atg9 Trafficking in Autophagy-related Pathways

Autophagy. May-Jun, 2007  |  Pubmed ID: 17329962

The origin of the autophagosomal membrane and the lipid delivery mechanism during autophagy remain unsolved mysteries. Some important hints to these questions come from Atg9, which is the only integral membrane protein required for autophagosome formation and considered a membrane carrier in autophagy-related pathways. In S. cerevisiae, Atg9 cycles between peripheral sites and the pre-autophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle. We recently identified a peripheral membrane protein, Atg11, as a binding partner of Atg9, in a yeast two-hybrid screen. Based on our analysis we propose a model for Atg9 cycling. Our model suggests that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS along the actin cytoskeleton, and that this delivery process may serve as a membrane shuttle for vesicle assembly during yeast selective autophagy. Here, we discuss the implications of the model and present additional evidence that extends it with regard to membrane trafficking modes during pexophagy.

2007 Keystone Symposium on Autophagy in Health and Disease

Autophagy. Sep-Oct, 2007  |  Pubmed ID: 17617735

The first Keystone Symposium on Autophagy in Health and Disease was held in Monterey, a scenic city on the Pacific coast in central California, April 15-20, 2007. The symposium brought together approximately 280 participants, from basic researchers to physicians and journalists. The meeting was composed of a joint keynote session with the meeting "Apoptotic and Non-Apoptotic Cell Death Pathways" and eight plenary sessions, covering the molecular mechanisms of autophagy and many emerging concepts and functions of autophagy in organelle degradation, physiological regulation, cell death and survival, and disease. Three afternoon workshops focused on short talks selected from the posters, and a special discussion session led by experts dealt with techniques and concerns regarding experimental detection of autophagy. The symposium highlighted autophagy as a potential therapeutic target in a wide range of diseases, including cancer, microbial infection, myopathies and neurodegenerative disorders.

Arp2 Links Autophagic Machinery with the Actin Cytoskeleton

Molecular Biology of the Cell. May, 2008  |  Pubmed ID: 18287533

Macroautophagy involves lysosomal/vacuolar elimination of long-lived proteins and entire organelles from the cytosol. The process begins with formation of a double-membrane vesicle that sequesters bulk cytoplasm, or a specific cargo destined for lysosomal/vacuolar delivery. The completed vesicle fuses with the lysosome/vacuole limiting membrane, releasing its content into the organelle lumen for subsequent degradation and recycling of the resulting macromolecules. A majority of the autophagy-related (Atg) proteins are required at the step of vesicle formation. The integral membrane protein Atg9 cycles between certain intracellular compartments and the vesicle nucleation site, presumably to supply membranes necessary for macroautophagic vesicle formation. In this study we have tracked the movement of Atg9 over time in living cells by using real-time fluorescence microscopy. Our results reveal that an actin-related protein, Arp2, briefly colocalizes with Atg9 and directly regulates the dynamics of Atg9 movement. We propose that proteins of the Arp2/3 complex regulate Atg9 transport for specific types of autophagy.

Self-interaction is Critical for Atg9 Transport and Function at the Phagophore Assembly Site During Autophagy

Molecular Biology of the Cell. Dec, 2008  |  Pubmed ID: 18829864

Autophagy is the degradation of a cell's own components within lysosomes (or the analogous yeast vacuole), and its malfunction contributes to a variety of human diseases. Atg9 is the sole integral membrane protein required in formation of the initial sequestering compartment, the phagophore, and is proposed to play a key role in membrane transport; the phagophore presumably expands by vesicular addition to form a complete autophagosome. It is not clear through what mechanism Atg9 functions at the phagophore assembly site (PAS). Here we report that Atg9 molecules self-associate independently of other known autophagy proteins in both nutrient-rich and starvation conditions. Mutational analyses reveal that self-interaction is critical for anterograde transport of Atg9 to the PAS. The ability of Atg9 to self-interact is required for both selective and nonselective autophagy at the step of phagophore expansion at the PAS. Our results support a model in which Atg9 multimerization facilitates membrane flow to the PAS for phagophore formation.

Double Duty of Atg9 Self-association in Autophagosome Biogenesis

Autophagy. Apr, 2009  |  Pubmed ID: 19182520

The understanding of the membrane flow process during autophagosome formation is essential to illuminate the role of autophagy under various disease-causing conditions. Atg9 is the only identified integral membrane protein required for autophagosome formation, and it is thought to cycle between the membrane sources and the phagophore assembly site (PAS). Thus, Atg9 may play an important role as a membrane carrier. We report the self-interaction of Atg9 and generate an Atg9 mutant that is defective in this interaction. This mutation results in abnormal autophagy, due to altered phagophore formation as well as inefficient membrane delivery to the PAS. Based on our analyses, we discuss a model suggesting dual functions for the Atg9 complex: by reversibly binding to another Atg9 molecule, Atg9 can both promote lipid transport from the membrane origins to the PAS, and also help assemble an intact phagophore membrane.

Assaying Autophagic Activity in Transgenic GFP-Lc3 and GFP-Gabarap Zebrafish Embryos

Autophagy. May, 2009  |  Pubmed ID: 19221467

Autophagy mediates the bulk turnover of cytoplasmic constituents in lysosomes. During embryonic development in animals, a dramatic degradation of yolk proteins and synthesis of zygotic proteins takes place, leading to intracellular remodeling and cellular differentiation. Zebrafish represents a unique system to study autophagy due in part to its rapid embryonic development relative to other vertebrates. The technical advantages of this organism make it uniquely suited to various studies including high-throughput drug screens. To study autophagy in zebrafish, we identified two zebrafish Atg8 homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. Similar to yeast Atg8 and mammalian LC3, zebrafish Lc3 undergoes post-translational modification starting at the pharyngula stage during embryonic development. We observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. In addition, zebrafish Gabarap accumulates within lysosomes upon autophagy induction. Thus, we established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.

Tap42-associated Protein Phosphatase Type 2A Negatively Regulates Induction of Autophagy

Autophagy. Jul, 2009  |  Pubmed ID: 19223769

Autophagy is a highly conserved degradative process in eukaryotic cells. This process plays an integral role in cellular physiology, and the levels of autophagy must be precisely controlled to prevent cellular dysfunction. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating the induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that Tap42 and protein phosphatase type 2A (PP2A) are involved in the regulation of autophagy in yeast. Temperature-sensitive mutant alleles of TAP42 revealed that autophagy was induced without inactivation of TORC1. Absence of the Tap42-interacting protein Tip41 abolished autophagy induction in the tap42 mutants, whereas overexpression of Tip41 activated autophagy. Furthermore, inactivation of PP2A stimulated autophagy and overexpression of a catalytic subunit of PP2A blocked rapamycin-induced autophagy. Our data support a model in which autophagy is negatively regulated by the Tap42-PP2A pathway.

Regulation Mechanisms and Signaling Pathways of Autophagy

Annual Review of Genetics. 2009  |  Pubmed ID: 19653858

Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases. Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Defective autophagy plays a significant role in human pathologies, including cancer, neurodegeneration, and infectious diseases. We present our current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagy for cell survival and homeostasis. We also review the recent advances on the molecular mechanisms that regulate the autophagy machinery at various levels, from transcriptional activation to post-translational protein modification.

The Beclin 1 Interactome

Current Opinion in Cell Biology. Apr, 2010  |  Pubmed ID: 20097051

The mammalian ortholog of yeast Atg6/Vps30, Beclin 1, is an essential autophagy protein that has been linked to diverse biological processes, including immunity, development, tumor suppression, lifespan extension, and protection against certain cardiac and neurodegenerative diseases. In recent years, major advances have been made in identifying components of functionally distinct Beclin 1/class III phosphatidylinositol 3-kinase complexes, in characterizing the molecular regulation of interactions between Beclin 1 and the autophagy inhibitors, Bcl-2/BcL-X(L), and in uncovering a role for viral antagonists of Beclin 1 in viral pathogenesis. The rapidly growing list of components of the 'Beclin 1 interactome' supports a model in which autophagy, and potentially other membrane trafficking functions of Beclin 1, are governed by differential interactions with different binding partners in different physiological or pathophysiological contexts.

Analyzing Autophagy in Zebrafish

Autophagy. Jul, 2010  |  Pubmed ID: 20495344

The transparency, external development and simple drug administration of zebrafish embryos makes them a useful model for studying autophagy during embryonic development in vivo. Cloning of zebrafish lc3 and generation of a transgenic GFP-Lc3 fish line provide excellent tools to monitor autophagy in this organism. ( 1) This protocol discusses several convenient autophagy assays in zebrafish, including immunoblotting of Lc3 lipidation, microscopy imaging of GFP-Lc3 and lysosomal staining.

Exercise-induced BCL2-regulated Autophagy is Required for Muscle Glucose Homeostasis

Nature. Jan, 2012  |  Pubmed ID: 22258505

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.

Exercise Induces Autophagy in Peripheral Tissues and in the Brain

Autophagy. Oct, 2012  |  Pubmed ID: 22892563

We recently identified physical exercise as a newly defined inducer of autophagy in vivo. Exercise induced autophagy in multiple organs involved in metabolic regulation, such as muscle, liver, pancreas and adipose tissue. To study the physiological role of exercise-induced autophagy, we generated mice with a knock-in nonphosphorylatable mutation in BCL2 (Thr69Ala, Ser70Ala and Ser84Ala) (BCL2 AAA) that are defective in exercise- and starvation-induced autophagy but not in basal autophagy. We found that BCL2 AAA mice could not run on a treadmill as long as wild-type mice, and did not undergo exercise-mediated increases in skeletal glucose muscle uptake. Unlike wild-type mice, the BCL2 AAA mice failed to reverse high-fat diet-induced glucose intolerance after 8 weeks of exercise training, possibly due to defects in signaling pathways that regulate muscle glucose uptake and metabolism during exercise. Together, these findings suggested a hitherto unknown important role of autophagy in mediating exercise-induced metabolic benefits. In the present addendum, we show that treadmill exercise also induces autophagy in the cerebral cortex of adult mice. This observation raises the intriguing question of whether autophagy may in part mediate the beneficial effects of exercise in neurodegeneration, adult neurogenesis and improved cognitive function.

Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy

Autophagy. Apr, 2012  |  Pubmed ID: 22966490

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

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

Cell. Aug, 2013  |  Pubmed ID: 23954414

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 additional lysosomal 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 members in autophagy, endolysosomal trafficking, and metabolism.

WITHDRAWN: Regulation of Plasma Membrane Receptors by a New Autophagy-related BECN/Beclin Family Member

Autophagy. May, 2014  |  Pubmed ID: 24569622

Regulation of Plasma Membrane Receptors by a New Autophagy-related BECN/Beclin Family Member

Autophagy. Aug, 2014  |  Pubmed ID: 24991830

We have recently shown the roles of an autophagy gene in the regulation of metabolism and metabolic diseases. We identified Becn2/Beclin 2, a novel mammalian specific homolog of Becn1/Beclin 1, characterized the functions of the gene product in autophagy and agonist-induced lysosome-mediated downregulation of a subset of G protein-coupled receptors (GPCRs), and proposed a model of dual functions of BECN2 in these 2 lysosomal degradation pathways. Further analyses revealed that knockout of Becn2 dramatically decreases embryonic survival in homozygotes, and leads to metabolic dysregulation in heterozygotes, which is likely caused by disruption of GPCR signaling. This finding suggests that besides autophagy, BECN/Beclin family members may play a role in the regulation of a broader spectrum of intracellular signaling pathways.

A Comparative Study of Aroma-active Compounds Between Dark and Milk Chocolate: Relationship to Sensory Perception

Journal of the Science of Food and Agriculture. Apr, 2015  |  Pubmed ID: 25043370

The most important aroma-active compounds of two types of chocolate and cocoa liquor used for their production were analysed by gas chromatography-olfactometry-mass spectrometry (GC-O-MS) and aroma extract dilution analysis (AEDA). Furthermore, the relationship between odorants and sensory perception of chocolate was measured by quantitative analysis, sensory evaluation and correlation analysis. In addition, some chemicals were added to the original dark or milk chocolate to validate their roles in the aroma property of chocolate.

Salinity Regulation of the Interaction of Halovirus SNJ1 with Its Host and Alteration of the Halovirus Replication Strategy to Adapt to the Variable Ecosystem

PloS One. 2015  |  Pubmed ID: 25853566

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.

Postharvest Exogenous Application of Abscisic Acid Reduces Internal Browning in Pineapple

Journal of Agricultural and Food Chemistry. Jun, 2015  |  Pubmed ID: 26007196

Internal browning (IB) is a postharvest physiological disorder causing economic losses in pineapple, but there is no effective control measure. In this study, postharvest application of 380 μM abscisic acid (ABA) reduced IB incidence by 23.4-86.3% and maintained quality in pineapple fruit. ABA reduced phenolic contents and polyphenol oxidase and phenylalanine ammonia lyase activities; increased catalase and peroxidase activities; and decreased O2(·-), H2O2, and malondialdehyde levels. This suggests ABA could control IB through inhibiting phenolics biosynthesis and oxidation and enhancing antioxidant capability. Furthermore, the efficacy of IB control by ABA was not obviously affected by tungstate, ABA biosynthesis inhibitor, nor by diphenylene iodonium, NADPH oxidase inhibitor, nor by lanthanum chloride, calcium channel blocker, suggesting that ABA is sufficient for controlling IB. This process might not involve H2O2 generation, but could involve the Ca(2+) channels activation. These results provide potential for developing effective measures for controlling IB in pineapple.

Emerging Roles of Autophagy in Metabolism and Metabolic Disorders

Frontiers in Biology. Apr, 2015  |  Pubmed ID: 26989402

The global prevalence of metabolic disorders is an immediate threat to human health. Genetic features, environmental aspects and lifestyle changes are the major risk factors determining metabolic dysfunction in the body. Autophagy is a housekeeping stress-induced lysosomal degradation pathway, which recycles macromolecules and metabolites for new protein synthesis and energy production and regulates cellular homeostasis by clearance of damaged protein or organelles. Recently, a dramatically increasing number of literatures has shown that defects of the autophagic machinery is associated with dysfunction of multiple metabolic tissues including pancreatic β cells, liver, adipose tissue and muscle, and is implicated in metabolic disorders such as obesity and insulin resistance. Here in this review, we summarize the representative works on these topics and discuss the versatile roles of autophagy in the regulation of cellular metabolism and its possible implication in metabolic diseases.

Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (3rd Edition)

Autophagy. Jan, 2016  |  Pubmed ID: 26799652

A Real-Time PCR Method to Detect the Population Level of Halovirus SNJ1

PloS One. 2016  |  Pubmed ID: 27192212

Although viruses of haloarchaea are the predominant predator in hypersaline ecosystem, the culture studies about halovirus-host systems are infancy. The main reason is the tradition methodology (plaque assay) for virus-host interaction depends on culturable and susceptible host. Actually, more than 90% of haloarchaea are unculturable. Therefore, it is necessary to establish an approach for detecting the dynamics of virus in hypersaline environment without culture. In this study, we report a convenient method to determine the dynamics of halovirus SNJ1 based on quantitative real-time PCR (qPCR). All findings showed that the qPCR method was specific (single peak in melt curves), accurate (a good linear relationship between the log of the PFU and the Ct values, R2 = 0.99), reproducible (low coefficient of variations, below 1%). Additionally, the physicochemical characteristics of the samples tested did not influence the stability of qPCR. Therefore, the qPCR method has the potential value in quantifying and surveying haloviruses in halophilic ecological system.

Autophagy Activation by Novel Inducers Prevents BECN2-mediated Drug Tolerance to Cannabinoids

Autophagy. Sep, 2016  |  Pubmed ID: 27305347

Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.

MicroRNAs-103/107 Coordinately Regulate Macropinocytosis and Autophagy

The Journal of Cell Biology. Dec, 2016  |  Pubmed ID: 27872138

Macropinocytosis, by which cells ingest large amounts of fluid, and autophagy, the lysosome-based catabolic process, involve vesicular biogenesis (early stage) and turnover (end stage). Much is known about early-stage events; however, our understanding of how the end stages of these processes are governed is incomplete. Here we demonstrate that the microRNA-103/107(miR-103/107) family, which is preferentially expressed in the stem cell-enriched limbal epithelium, coordinately regulates aspects of both these activities. Loss of miR-103/107 causes dysregulation of macropinocytosis with the formation of large vacuoles, primarily through up-regulation of Src, Ras, and Ankfy1. Vacuole accumulation is not a malfunction of early-stage autophagy; rather, miR-103/107 ensure proper end-stage autophagy by regulating diacylglycerol/protein kinase C and cyclin-dependent kinase 5 signaling, which enables dynamin to function in vacuole clearance. Our findings unveil a key biological function for miR-103/107 in coordinately suppressing macropinocytosis and preserving end-stage autophagy, thereby contributing to maintenance of a stem cell-enriched epithelium.

SNAPIN is Critical for Lysosomal Acidification and Autophagosome Maturation in Macrophages

Autophagy. Dec, 2016  |  Pubmed ID: 27929705

We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H(+) pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.

Mechanism of Internal Browning of Pineapple: The Role of Gibberellins Catabolism Gene (AcGA2ox) and GAs

Scientific Reports. Dec, 2016  |  Pubmed ID: 27982026

Internal browning (IB), a physiological disorder (PD) that causes severe losses in harvested pineapple, can be induced by exogenous gibberellins (GAs). Over the years, studies have focused on roles of Gibberellin 2-oxidase (GA2oxs), the major GAs catabolic enzyme in plants, in the regulation of changes in morphology or biomass. However, whether GA2oxs could regulate PD has not been reported. Here, a full-length AcGA2ox cDNA was isolated from pineapple, with the putative protein sharing 23.59% to 72.92% identity with GA2oxs from five other plants. Pineapples stored at 5 °C stayed intact, while those stored at 20 °C showed severe IB. Storage at 5 °C enhanced AcGA2ox expression and decreased levels of a GAs (GA4) 'compared with storage at 20 °C. However, at 20 °C, exogenous application of abscisic acid (ABA) significantly suppressed IB. ABA simultaneously upregulated AcGA2ox and reduced GA4. Ectopic expression of AcGA2ox in Arabidopsis resulted in reduced GA4, lower seed germination, and shorter hypocotyls and roots, all of which were restored by exogenous GA4/7. Moreover, in pineapple, GA4/7 upregulated polyphenol oxidase, while storage at 5 °C and ABA downregulated it. These results strongly suggest the involvement of AcGA2ox in regulation of GAs levels and a role of AcGA2ox in regulating IB.

Identification of Natural Products with Neuronal and Metabolic Benefits Through Autophagy Induction

Autophagy. Jan, 2017  |  Pubmed ID: 27791467

Autophagy is a housekeeping lysosomal degradation pathway important for cellular survival, homeostasis and function. Various disease models have shown that upregulation of autophagy may be beneficial to combat disease pathogenesis. However, despite several recently reported small-molecule screens for synthetic autophagy inducers, natural chemicals of diverse structures and functions have not been included in the synthetic libraries, and characterization of their roles in autophagy has been lacking. To discover novel autophagy-regulating compounds and study their therapeutic mechanisms, we used analytic chemistry approaches to isolate natural phytochemicals from a reservoir of medicinal plants used in traditional remedies. From this pilot plant metabolite library, we identified several novel autophagy-inducing phytochemicals, including Rg2. Rg2 is a steroid glycoside chemical that activates autophagy in an AMPK-ULK1-dependent and MTOR-independent manner. Induction of autophagy by Rg2 enhances the clearance of protein aggregates in a cell-based model, improves cognitive behaviors in a mouse model of Alzheimer disease, and prevents high-fat diet-induced insulin resistance. Thus, we discovered a series of autophagy-inducing phytochemicals from medicinal plants, and found that one of the compounds Rg2 mediates metabolic and neurotrophic effects dependent on activation of the autophagy pathway. These findings may help explain how medicinal plants exert the therapeutic functions against metabolic diseases.

Blueberry Anthocyanin-Enriched Extracts Attenuate Fine Particulate Matter (PM2.5)-Induced Cardiovascular Dysfunction

Journal of Agricultural and Food Chemistry. Jan, 2017  |  Pubmed ID: 27996266

Blueberry anthocyanin-enriched extracts (BAE) at three doses (0.5, 1.0, and 2.0 g/kg) were administered by oral gavage to rats exposed to 10 mg/kg fine particulate matter (PM2.5) three times a week. A positive control group was exposed to PM2.5 without BAE treatment. We analyzed heart rate (HR), electrocardiogram (ECG), and histopathology, and biomarkers of cardiovascular system injuries, systemic inflammation, oxidative stress, endothelial function, and apoptosis. Results indicated that BAE, particularly at 1.0 g/kg, improved ECG and decreased cytokine levels in PM2.5-exposed rats. These changes were accompanied by an increase in interleukin 10 levels and superoxide dismutase activity in heart tissue and Bcl-2 protein expression, as well as a decrease in interleukin 6, malondialdehyde, endothelin 1, and angiotensin II levels and a reduction in Bax protein expression. This study demonstrates that BAE at certain doses can protect the cardiovascular system from PM2.5-induced damage.

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