Browsing by Subject "Autophagy"
Now showing 1 - 20 of 28
- Results Per Page
- Sort Options
Item Autophagy in Antiviral Immunity(2012-08-15) Orvedahl, Anthony Walter; Levine, BethAutophagy is an evolutionarily conserved pathway in which cytoplasmic material is sequestered in a double-membrane vesicle and delivered to the lysosome for degradation. During times of stress, autophagy functions to generate essential nutrients through the degradation of non-essential cytoplasmic contents. It is also the only known mechanism for removal of damaged or superfluous organelles and cytoplasmic contents that are too large to be degraded by the proteasome. Given the critical role for autophagy in stress response and in maintaining cell cytoplasmic quality control, it is not surprising that autophagy plays an essential role in the host response to infection, and that microbes have evolved mechanisms to counteract or evade autophagy. In this work, we studied the role of autophagy inhibition in a mouse model of herpes simplex virus type I (HSV-1) encephalitis, investigated the role of autophagy in protection against Sindbis virus infection of the central nervous system, and identified novel host genes involved in targeting viral proteins to the autophagy pathway. We found that the HSV-1 encoded neurovirulence protein ICP34.5 interacted with the host autophagy protein Beclin 1, and that this interaction was essential for HSV-1 neurovirulence. This was the first example of a viral virulence protein that targets host autophagy, and provided evidence that autophagy functions in innate immunity to viruses. In the second study, we found that the host autophagy gene Atg5 was required to protect against lethal Sindbis virus CNS diseases, and that autophagy targeted viral proteins for degradation in brains of infected mice and cells in vitro. We found that the autophagy adaptor protein p62 was involved in targeting viral proteins for autophagic degradation and this promoted survival of infected cells. This study demonstrated that clearance of viral proteins by autophagy was an important mechanism for cellular and organismal survival during viral infection. Lastly, we performed a genome-wide siRNA screen to identify novel host factors required for autophagic targeting of viral proteins. We identified previously unappreciated cellular networks and genes that were involved in targeting viral proteins for autophagy. One of these factors, SMURF1, is an E3 ubiquitin ligase that not only functions to target viral proteins, but is also involved in targeting damaged mitochondria for autophagic clearance.Item Autophagy in the pathogenesis of disease(2008-06-13) Levine, BethThe protocol file is a published article. Due to publisher restrictions, direct access to the protocol file in this collection is not available. The publisher's final edited version of this article is available at Cell. A free full-text version of the article is available through PubMed Central (PMC): https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/18191218/Item Autophagy in Zellweger Syndrome Spectrum Disorder and Cancer(2016-05-17) Lee, Ming Yeh; Tu, Benjamin; DeBerardinis, Ralph J.; Hill, Joseph A.; Levine, BethAutophagy is a lysosomal degradation pathway that breaks down unwanted proteins and organelles from the cytoplasm to regenerate cellular building blocks. This process is constitutively active at low basal levels, and can be upregulated by stress stimuli to promote cellular homeostasis. In this work, we investigated two aspects of autophagy regulation and relevance to human diseases. First, we examined how autophagy selectively removes viral components and damaged mitochondria from the cytoplasm through PEX13, a peroxin protein mutated in Zellweger syndrome spectrum (ZSS). Second, we examined the role of autophagy as a potential mechanism contributing to exercise-mediated protection against cancer progression. PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with ZSS disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurodevelopmental, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy), and that disease-associated PEX13 mutants I326T and W313G are defective in mitophagy. The selective mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13-mediated mitophagy may contribute to ZSS pathogenesis. In the second part of this study, we evaluated physiological functions regulated by exercise-induced autophagy, including changes to the metabolome, proteome, and breast cancer progression. A previous study from our laboratory demonstrated that exercise is a potent inducer of autophagy and that autophagy contributes to exercise-mediated metabolic benefits. Therefore, we speculate that autophagy may contribute to exercise-mediated protection against other diseases. Although many epidemiological and laboratory studies have provided strong evidence that physical exercise can decrease cancer development and mortality, the mechanisms are poorly understood. Using the E0771 injectable murine breast cancer, we show that exercise delays cancer progression in wild-type, but not in Bcl-2 AAA mice or Beclin 1 heterozygous knockout mice that are deficient in exercise-induced autophagy. We identified candidate factors and pathways regulated by exercise-induced autophagy, including plasma levels of pyrimidine, branched chain amino acids, LIF, and IL-15, as well as skeletal muscle expression of IDH2 and NDUFA13. Further studies are required to elucidate the metabolomic and proteomic alterations regulated by exercise-induced autophagy and the mechanism by which exercise-induced autophagy protects against tumor progression.Item Cardiomyocyte Autophagy Is Induced by Protein Aggregation in Heart Disease(2009-06-19) Tannous, Paul; Hill, Joseph A.Autophagy is associated with diverse forms of myocardial stress. When I initiated my studies activators of this pathway had not been identified in the heart, nor was it clear weather autophagy is an adaptive or maladaptive response in the stressed myocardium. My initial research focused on autophagy in hypertension-induced heart failure, the most common cardiovascular disease in Western nations. Early evidence demonstrated generation of reactive oxygen species, protein damage, and protein aggregation in the acute period of pressure overload. Given the simultaneous presence of autophagosomes and aggregates, and autophagy's role in bulk degradation, I postulated these events were mechanistically linked. I designed experiments to test the hypotheses that protein aggregates are activators of autophagy in the heart, and that autophagy functions in aggregate clearance. Here I report novel findings that link pressure overload-induced protein aggregation to increased cardiomyocyte autophagy. Specifically, in the pressure-stressed ventricle 1) generation of reactive oxygen species is an early pathological event, 2) there is extensive protein aggregation with higher-order processing into aggresomes, 3) protein aggregation induces cardiomyocyte autophagy, and 4) in this setting autophagy functions in its role as a mechanism of bulk protein degradation. These findings are the first to demonstrate proteinopathy of non-genetic etiology contributes to hypertension-induced heart failure and that protein aggregates are robust activators of cardiomyocyte autophagy. To directly address the role of autophagy in cardiomyocyte clearance of toxic protein species, I turned my attention to CryABR120G-induced desmin-related cardiomyopathy (DRCM), an aggregate-associated disease with autosomal dominant inheritance. My studies demonstrated that 1) autophagy is activated by CryABR120G-induced protein aggregation, 2) aggregate formation is inversely proportional to the degree of autophagic activity and 3) blunting autophagy accelerates pathological myocardial remodeling and the onset of heart failure. Extending this work to clinical medicine, we observed increased autophagy in the skeletal muscle from patients with desmin-related skeletal myopathy. Cumulatively these data are the first to demonstrate autophagy is induced in DRCM and functions as a protective cellular response. These findings suggest autophagy is a pathway amenable to therapeutic intervention in patients suffering from myofibrillar myopathy, a disease class for which there are limited therapeutic options.Item Characterization of mTOR Inhibition and Autophagy Inhibition in Non-Small Cell Lung Cancer(2014-04-14) Britt, Rebecca; DeBerardinis, Ralph J.; Minna, John D.; Levine, Beth; Mendelson, Carole R.Lung cancer continues to be the leading cause of cancer related death in both men and women. Pre-clinical studies of targeted therapies are needed in order to improve upon the chemotherapeutics that are currently in use. The ability to identify subsets of patient tumors which will respond to a particular targeted agent using biomarkers to indicate an acquired vulnerability will improve selection of effective therapeutics and minimize time and money wasted on ineffective drugs. The goal of this dissertation has been to characterize NSCLC response to mTOR inhibition and determine whether there are any molecular biomarkers that can predict response. mTOR is a central regulator of several pro-oncogenic signaling pathways and plays a role in cell growth, proliferation, metabolism, and inhibition of autophagy. Early studies examining mTOR inhibitors were limited by a lack of proper patient selection and the inability of first generation drugs to completely inhibit mTOR signaling. In the present study, we screened a panel of well-characterized NSCLC cell lines with three mTOR inhibitors, classical mTORC1 inhibitor rapamycin, and two novel dual mTORC1/2 inhibitors, Torin1 and AZD8055 in order to identify potential biomarkers that may be used to predict response to these agents. Additionally, in order to further characterize vulnerabilities to mTOR related genes within lung cancer subsets, we performed a genetic knockdown screen individually targeting 55 genes in this important pathway. Because inhibition of mTOR frequently leads to a cytostatic rather than cytotoxic effect, mTOR targeting agents may have greater utility when used in combination with other chemo- and targeted-agents. Therefore, we screened the three mTOR inhibitors in combination with the chemotherapy doublet paclitaxel/carboplatin or the targeted agent erlotinib. Finally, mTOR inhibition and other drug treatments have been shown to lead to autophagy activation. This process of cellular "self-eating" is thought to protect cancer cells from low nutrient availability and therapy induced stress. We screened NSCLC cells for their response to autophagy inhibitors alone and in combination with chemo- and targeted-therapy agents. The studies described in this thesis led us to the following conclusions. A subset of NSCLCs are more responsive to rapamycin than to mTORC1/2 inhibition by Torin1, or AZD8055, and sensitivity to mTOR inhibition is associated with RTK activation such as ERBB2 amplification or EGFR mutation or amplification, while KRAS and/or LKB1 mutations were associated with resistance. RNAi knockdown of various components related to mTOR signaling and autophagy produce a heterogeneous growth effect response in NSCLCs cells, and potentially define subset-specific vulnerabilities. mTOR inhibitors sensitize NSCLC cells to standard targeted- and chemotherapy agents erlotinib and paclitaxel/carboplatin doublet in an additive or synergistic manner, with the greatest level of synergy occurring in cell lines which are resistant to single agent therapies, including those with KRAS mutations. Finally, inhibition of autophagy using chloroquine is not likely to be a successful therapeutic approach in lung cancer as no significant growth effect was seen at physiologically relevant concentrations and no sensitization to standard chemo- or targeted-therapies were observed.Item Characterization of Vibrio VopS, an AMPylator of Rho GTPases(2009-06-19) Yarbrough, Melanie Leann; Orth, KimVibrio parahaemolyticus is a gram-negative marine bacterium that causes gastroenteritis associated with the consumption of contaminated shellfish. The emergence of pandemic strains of V. parahaemolyticus has increased the need for characterization of the virulence factors of this pathogen. Sequencing of the genome of a clinical isolate revealed the presence of two type III secretion systems (T3SSs), one on each chromosome. The T3SS on chromosome one (T3SS1) has been shown to be responsible for cytotoxicity in HeLa cells, and it shares a high degree of homology to the T3SS of the Yersinia spp. Our studies have shown that infection of HeLa cells with a strain of V. parahaemolyticus capable of secreting only from T3SS1 indicated that T3SS1 mediates several events during infection including the rapid induction of autophagy, cell rounding, and finally lysis of the cell. Defining the T3SS1-mediated events of infection gives insight into virulence mechanisms of V. parahaemolyticus that have not been well characterized and provide a basis for the elucidation of the functions associated with T3SS1 effectors. One of the T3SS effectors, VopS, contains a Filamentation induced by cAMP (Fic) domain that we have shown is critical for the function of this effector. Our studies have found that VopS inhibits Rho GTPase signaling during infection by directly modifying Rho, Rac, and Cdc42, preventing their interaction with downstream effectors. These observations reveal a unique activity for VopS, which targets a pathway that is critical in the cellular response to V. parahaemolyticus infection. In addition, they provide insight into a novel post-translational modification that may expand our knowledge of eukaryotic cell signaling. Fic domains are found in proteins from several bacterial and eukaryotic species and are recognized by their conserved motif, HPFX(D/E)GNGR. The presence of Fic domains in higher eukaryotes suggested that this modification could be utilized in cell signaling. Our preliminary studies indicated that AMPylation is utilized by eukaryotes. We have shown that a Fic protein from humans, HYPE, possesses auto-AMPylation activity, confirming our hypothesis that these domains are involved in AMPylation. Ongoing and future studies seek to identify the substrates of HYPE activity and identify other components involved in this new layer of eukaryotic cell signaling.Item Characterization of VOPQ, A Type III Secreted Effector Protein from Vibrio Parahaemolyticus(2009-06-15) Burdette, Dara Lesley; Orth, KimVibrio parahaemolyticus is a Gram-negative bacterium responsible for gastroenteritis associated with the consumption of raw or undercooked shellfish. Its most well-characterized virulence factors are hemolysins that cause b-hemolysis on a special blood agar. Mutants lacking these hemolysins are still virulent in animal and tissue culture models of infection. These phenomena can be attributed in part to one of two type III secretion systems; one on chromosome 1 and the other on chromosome 2. We demonstrate that Vibrio parahaemolyticus utilizes the type III secretion system on chromosome 1 to induce a temporally regulated series of events that initiates with the induction of autophagy, followed by cellular rounding and finally cellular lysis and death. To the best of our knowledge, no other Gram-negative extracellular bacterium has been shown to induce autophagy during infection. To understand the mechanism of Vibrio parahaemolyticus induced cell death, we focused our analysis on VopQ, a type III secreted effector encoded by the type III locus on chromosome 1. We demonstrate that VopQ contributes to cytotoxicity as DvopQ strains induce cell lysis less efficiently. In addition, VopQ is necessary and sufficient for the induction of autophagy during infection. VopQ-mediated autophagy occurs independently of phosphatidylinositol 3-kinases and prevents phagocytosis. Additional experiments using Saccharomyces cerevisiae demonstrate VopQ induces autophagy and cell death through an evolutionarily conserved mechanism. Results presented herein delineate a novel virulence mechanism used by Vibrio parahaemolyticus to cause disease. This study also highlights the effector VopQ as a novel inducer of autophagy and a key mediator of cytotoxicity during infection.Item Doxorubicin Inhibits Cardiomyocyte Autophagic Flux by Suppressing Lysosomal Acidification(2015-04-09) Li, Dan; Sadek, Hesham A.; Hill, Joseph A.; Levine, Beth; Amatruda, James F.The clinical use of doxorubicin is limited by cardiotoxicity. Dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases. However, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of acute doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, I first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, and is reminiscent of the effects seen in patients of chronic doxorubicin cardiomyopathy. Next, via multiple assays I showed that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. Moreover, I went on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, I studied animals with diminished autophagic activity due to haploinsufficiency for Beclin 1. Beclin 1+/- mice exposed to doxorubicin manifested restored cardiac autophagic flux, and were protected in terms of structural and functional changes within the myocardium. Conversely, animals over-expressing Beclin 1 manifested an amplified cardiotoxic response, correlating with their aggravated accumulation of autolysosomes in cardiomyocytes after doxorubicin treatments. In summary, I report here that doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Further, reducing autophagy initiation may protect against doxorubicin cardiotoxicity.Item Epithelial Cell Autophagy in Antibacterial Defense of the Small Intestine(2013-10-14) Benjamin, Jamaal Louis; Yarovinsky, Felix; Hooper, Lora V.; Levine, Beth; Sperandio, VanessaThe intestines of all mammals are colonized with a diverse microbiota that provide metabolic benefits to their hosts. However, this symbiotic relationship can break down when resident bacteria opportunistically invade the intestinal barrier, leading to pathologies such as inflammatory bowel disease (IBD), and bacteremia. As a result, epithelial cell innate immune responses play an essential role in preventing bacterial invasion of host tissues and maintaining a symbiotic host-bacterial relationship. Autophagy is emerging as an important component of innate immunity. Mounting evidence suggests that dysregulation of the autophagy-independent function of autophagy genes can lead to inflammatory bowel disease. However, little is known about the role of autophagy-dependent gene function in controlling interactions between intestinal bacteria and the intestinal epithelium in vivo. In this study, I have demonstrated that small intestinal epithelial cell autophagy is essential for protection against tissue invasion by intestinal pathogens and opportunistically invasive commensals. I have shown that small intestinal autophagy is an early innate immune response that functions in an epithelial cell-intrinsic MyD88-dependent, NOD2-independent manner. Utilizing mice deficient in small intestinal epithelial cell autophagy (Atg5∆IEC), I have determined that epithelial cell autophagy is required to limit pathogen dissemination to extraintestinal sites. This study thus shows that autophagy is a critical mechanism of innate immune defense that protects intestinal epithelial surfaces from bacterial invasion. My findings may lead to new insights into how autophagy protects against gastrointestinal infections and maintains homeostasis with the intestinal microbiota.Item From Feast to Famine: A Tale of Satiety and Hunger Hormones(2016-04-18) Zhang, Yuanyuan; Repa, Joyce J.; Horton, Jay D.; Chen, Zhijian J.; Goldstein, Joseph L.; Brown, Michael S.Ghrelin is a peptide hormone secreted mainly from the stomach. It has a unique octanoylation on Ser-3 by Ghrelin-O-Acyltransferase (GOAT). We have previously shown that Goat−/− mice developed severe hypoglycemia under 60% calorie restriction. Liver autophagy has been reported to play a crucial role in maintaining blood glucose during fasting. The present work was carried out to explore whether autophagy plays a role in the onset of hypoglycemia in Goat−/− mice. We observed a deficiency in autophagy in livers of calorie-restricted Goat−/− mice by showing lower expression level of LC3-II, an autophagy marker. This was further demonstrated by showing 10-fold fewer autolysosomes in livers of calorie-restricted Goat−/− mice as compared to the control mice (20 electron microscopic images analyzed for each group). We then went on to show that the deficiency in autophagy in Goat−/− mice can be restored by infusion of growth hormone. It can also be restored by injections of lactate, a gluconeogenic precursor, or octanoate, a fatty acid that spares the usage of glucose. Protein expression of p- STAT 5, a downstream target of growth hormone action, was significantly lower in livers of calorie-restricted Goat−/− mice, and was restored by infusion of growth hormone and by injections of lactate or octanoate. Protein expression levels of LC3-II and p-STAT 5 showed a strong correlation (r2=0.87, p<10-6) through the time course of calorie-restriction. Considered together, these data suggest that the onset of autophagy during calorie restriction is strongly correlated with the ghrelin-growth hormone axis, and that autophagy plays an important role in maintaining blood glucose homeostasis during chronic starvation.Item Identifying Novel Functions of the WNK Pathway(2017-11-20) Gallolu Kankanamalage, Sachith Sandaruwan Perera; Burma, Sandeep; Seemann, Joachim; Luby-Phelps, Katherine; Cobb, Melanie H.The with no lysine [K] (WNK) pathway consists of WNK kinases, their downstream target kinases, oxidative stress responsive (OSR)1 and SPS/Ste20-related proline-alanine-rich kinase (SPAK), and OSR1/SPAK substrates, cation chloride cotransporters. The pathway regulates ion transport across cell membranes, among other functions, and is implicated in human diseases including hypertension, cancer and neurological diseases. However, the functions of WNK pathway beyond cotransporter regulation have not been extensively studied. The purpose of my work has been to understand novel functions of the WNK pathway. I demonstrated that WNK1, largest and ubiquitously expressed WNK isoform, is an inhibitor of autophagy, an intracellular degradation pathway. WNK1 inhibited the class III phosphatidylinositol 3-kinase (PI3KC3) complex which acts upstream in the autophagy pathway. In addition, WNK1 inhibited the unc-51-like kinase 1 (ULK1) complex that acts upstream of PI3KC3. WNK1 also inhibited AMP-activated protein kinase (AMPK), the upstream activator of ULK1. The actions of WNK1 on the AMPK-ULK1 axis only partially mediated its effects on autophagy. WNK1 directly bound UV radiation resistance-associated gene (UVRAG) in vitro and had an overlapping localization with it in cells, and autophagy induction led to a decrease in this property. OSR1 had no significant effect on autophagy while SPAK acted as an autophagy inhibitor. Therefore, WNK pathway most likely inhibits autophagy through multiple mechanisms. I also discovered that OSR1 regulates the cellular localization of inward-rectifier potassium channel (Kir) 2.3 that contains an OSR1/SPAK recognition motif, and is activated by WNK. OSR1 promoted Kir2.3 localization to shift towards the cell membrane in the presence of sodium chloride. Similar to OSR1, WNK kinase activity also promoted the change in localization of Kir2.3 elicited by NaCl. Therefore, I suggest that activated WNK induces Kir2.3 channel activity by driving it to the cell membrane.Item The Mechanism and Function of Autophagy Induction by Cytosolic DNA(2018-07-11) Gui, Xiang; Olson, Eric N.; Hooper, Lora V.; Wang, Zhigao; Chen, Zhijian J.Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects pathogen infections or tissue damage by binding to microbial or self-DNA in the cytoplasm. Upon binding to DNA, cGAS produces cGAMP that binds and activates the adaptor protein stimulator of interferon genes (STING), which activates the kinases IKK and TBK1 to induce interferons and other inflammatory cytokines. Here, we report that STING also activates autophagy and induces cell death through a mechanism independent of TBK1 and IRF3 activation, which canonically triggers innate immunity signaling. Upon binding to cGAMP, STING translocates to the ER-Golgi intermediate compartments (ERGIC) and the Golgi in a process that depends on the COP-II complex and ARF GTPases. The STING-containing ERGIC serves as a membrane source for LC3 lipidation, a key step in autophagosome biogenesis. Interestingly, STING lacking its C tail for interferon signaling is still capable of membrane trafficking and autophagy induction. Through endosomes or autophagosomes, STING is further degraded in the lysosome to shut down its activation. Interestingly, we determined that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Furthermore, sea anemone STING induces autophagy but not interferons in response to stimulation by cGAMP, suggesting that induction of autophagy is a primordial function of the cGAS-STING pathway.Item Mechanistic Studies of Autophagy Initiation in Mammalian Cells(2011-08-10) Shang, Libin; Wang, XiaodongMacroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells use to adapt to starvation and other stresses. During autophagy, portions of cytoplasmic materials are engulfed into specialized double-membrane structures to form autophagosomes, which then fuse with lysosomes to degrade their cargos and regenerate nutrients. Initiation of autophagy has been extensively studied in budding yeast Saccharomyces cerevisiae. However, various significant differences exist between yeast and mammals. To pinpoint how mammalian autophagy is initiated, I first adopted proteomic approaches to identify associating partners of Unc-51-like kinase 1 (Ulk1), key initiator for mammalian autophagy. Two novel proteins, mAtg13 and Atg101, were found to interact with Ulk1 stoichiometrically. Knockdown of either mAtg13 or Atg101 led to decreased autophagy, and autophagy could be rescued with exogenous expression, suggesting the two proteins were critical for mammalian autophagy initiation. I then observed Ulk1 undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. I found phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin (mTOR) kinase and AMP-activated protein kinase (AMPK). AMPK interacts with Ulk1 in a nutrient-dependent manner, and proper phosphorylations on Ulk1 are crucial for Ulk1/AMPK association, as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this interaction. Compared to its wild-type counterpart, this Ulk1-S758A mutant initiates starvation-induced autophagy faster at early time points, but does not alter the maximum capacity of autophagy when starvation prolongs. With this layer of regulation, mammalian autophagy is capable of responding to environmental changes more promptly than previously considered.Item Mitophagy in Heart Failure: A Selective Autophagic Degradation of Mitochondria(2009-06-05) Yang, Kai-Chun (Daniel); Hill, Joseph A.INTRODUCTION: Cardiovascular disease is associated with declines in mitochondrial function. Autophagy is a lysosomal-dependent process through which cytoplasmic proteins and organelles can be degraded and has recently been shown to participate in remodeling of the myocardium in a variety of cardiac pathologies. Autophagy can be either non-selective or selective for damaged protein aggregates or organelles. Reactive oxygen species (ROS) generated in mitochondria causes mitochondrial permeability transition (MPT) and induces selective degradation of mitochondria (mitophagy). We hypothesized that mitophagy contributes to remodeling of the heart under severe oxidative stress. METHODS: Mice were subjected to hemodynamic overload by severe thoracic aortic constriction (sTAC). qPCR was used to measure the abundance of mtDNA relative to nuclear DNA. Changes in proteins and cardiac function were also assessed. RESULTS: Decreases in mtDNA abundance were time dependent after sTAC (-47%±17 at day 2, p<0.1; -73%±10 at day 4, p<0.05) (n=2 each) and correlated with increased mortality (37% at day 2; 75% at day 4). The decline in mtDNA was greater in the basal septum (-88%±2, p<0.01) than in the left ventricular free wall (- 42%±15, p<0.15) (n=4) day 8 post-sTAC. The basal septum is where we have observed the largest increases in autophagic activity and protein carbonylation, a ROS-mediated protein modification. Daily injections with cyclosporine (CsA), an inhibitor of both MPT and calcineurin, blunted load-induced mtDNA loss (-28%±2 with CsA vs -83%±10 with vehicle treatment, p<0.01) (n=3) at 4 days post-sTAC. Furthermore, CsA improved survival at 4 days-post sTAC (40% mortality with CsA vs 75% with vehicle) (n=5-8). Mice with increased ROS generation due to a disruption of the cardiac isoform of the cytochrome-c oxidase subunit COXVIaH were more sensitive to pressure overload-induced loss of mtDNA and mitochondrial proteins. CONCLUSION: MtDNA abundance declines in this model of load-induced heart failure and is associated with increased autophagic activity and ROS generation. Short-term application of CsA can blunt mtDNA loss and improve survival.Item Novel Insights into the Regulation of Autophagy in Saccharomyces Cerevisiae(2011-12-15) Wu, Xi; Tu, BenjaminAutophagy is an evolutionarily conserved pathway for the degradation of intracellular contents. How autophagy is regulated, especially upon changes in metabolic and nutritional state, remains poorly understood. In Saccharomyces cerevisiae, autophagy is normally triggered by nutrient starvation. However, by using a prototrophic strain, I discovered that autophagy can be strongly induced upon switch from a rich medium (YPL) to a minimal medium (SL) without nutrient starvation. This new autophagy-inducing condition was termed SL-induced autophagy. Growth measurement confirmed that SL-induced autophagy was important for cellular homeostasis and growth following medium switch. A genetic screen uncovered IML1, NPR2, NPR3 and PBP1, which are all required for SL-induced autophagy, but not for nitrogen-starvation-induced autophagy. Iml1p, Npr2p and Npr3p function in the same complex and regulate autophagosome formation. During SL-induced autophagy, Iml1p can localize to the pre-autophagosomal structures, consistent with the role of the Iml1p complex in autophagosome formation. Moreover, a conserved domain in Iml1p was identified to be required for SL-induced autophagy as well as complex formation. I discovered that sulfur containing amino acids, but not non-sulfur containing amino acids, can specifically inhibit SL-induced autophagy. I further demonstrated that cysteine is a key metabolite that inhibits SL-induced autophagy by regulating cellular processes related to cysteine metabolism. Cysteine does not suppress SL-induced autophagy by regulating oxidative stress, protein urmylation and thiolation of cytosolic tRNAs. Future studies will be required to reveal the exact mechanism through which cysteine inhibits SL-induced autophagy. I also discovered that autophagy can be significantly induced upon depletion of a Fe-S cluster containing protein, Rli1p, and other factors that are also involved in rRNA processing and translation initiation. Interestingly, IML1, NPR2, NPR3 and PBP1 are also important for Rli1p-depletion-induced mitophagy. These results strongly suggest the mechanistic link between SL-induced autophagy and ribosome biogenesis or translation regulation. Collectively, my studies have demonstrated the existence of additional mechanisms that regulate autophagy in response to relatively more subtle changes in metabolic and nutritional state.Item Paneth Cell-Dependent Intestinal Homeostasis During T. Gondii Infection(2017-04-14) Burger, Elise Sylvie; Hooper, Lora V.; Yarovinsky, Felix; van Oers, Nicolai S. C.; Burstein, Ezra; Brown, Michael S.; Amatruda, James F.The intestinal epithelial barrier faces the arduous task of constantly monitoring and controlling the diverse bacteria which colonize the gut. Key for this balance are the Paneth cells, highly specialized secretory epithelial cells in the intestinal crypts which secrete anti-microbial peptides to regulate the microbiota. Breakdown of the symbiotic host-microbe relationship, possibly due to Paneth cell dysfunction, leads to intestinal pathologies such as inflammatory bowel disease (IBD) and infection. The protozoan parasite Toxoplasma gondii triggers severe small intestinal immunopathology that recapitulates many characteristics of IBD such as elevated IFN-g and microbiota-mediated inflammation, along with disappearance of Paneth cells. However, little is known about what happens to these Paneth cells and if their loss exacerbates inflammation. In this study, we have generated a number of fluorescent Paneth cell-specific reporter mice to demonstrate that acute T. gondii infection leads to Paneth cell death by an IFN-g-dependent pathway. Our results also revealed that constitutive elimination of Paneth cells does not enhance susceptibility to T. gondii infection, enhance inflammation, or increase damage to the intestinal epithelium, suggesting that Paneth cell retention at the peak of inflammation may be detrimental and these cells may be targeted for controlled elimination early during T. gondii infection. We also demonstrate that the microbiota triggers basal Paneth cell-specific autophagy via induction of IFN-g. Deletion of Atg5 in Paneth cells resulted in exaggerated intestinal inflammation characterized by the complete destruction of the intestinal crypts seen in pan-epithelial Atg5 deficient mice. An evaluation of Atg5 in intestinal organoids and in T. gondii-infected mice revealed that lack of functional autophagy in Paneth cells resulted in increased sensitivity to TNF, leading to exaggerated microbiota and IFN-g-dependent intestinal immunopathology. Our results reveal that Paneth cell expression of Atg5 is essential for tissue protection against cytokine-mediated immunopathology during acute gastrointestinal infection. Together, these findings have broad implications for the role of Paneth cells during both acute infection and chronic intestinal inflammation.Item RalGDS-Dependent Cardiomyocyte Autophagy Is Necessary for Load-Induced Ventricular Hypertrophy(2014-05-01) Rifki, Oktay Feridun; White, Michael A.; Hill, Joseph A.; Olson, Eric N.; Levine, BethRecent work has demonstrated that autophagy, a phylogenetically conserved, lysosome-mediated pathway of protein degradation, is a key participant in pathological cardiac remodeling. One common feature of cell growth and autophagy is membrane biogenesis and processing. The exocyst, an octomeric protein complex involved in vesicle trafficking, is implicated in numerous cellular processes, yet its role in cardiomyocyte plasticity is unknown. Here, I set out to explore the role of small G protein-dependent membrane trafficking in stress-induced cardiomyocyte remodeling and autophagy. To explore underlying mechanisms, I tested in cultured neonatal cardiomyocytes two isoforms of Ral that are downstream of RalGDS (RalA, RalB) and whose actions are mediated by the exocyst. In these experiments, mTOR inhibition was maintained in response to starvation and Torin 1 despite RalA or RalB knockdown; however, autophagy was diminished only in NRCM's with RalB knockdown, implicating RalB as required for cardiomyocyte autophagy. Hearts from mice lacking RalGDS (Ralgds-/-), a guanine exchange factor (GEF) for the Ral family of small GTPases, were similar to wild-type (WT) littermates in terms of ventricular structure, contractile performance, and gene expression. However, Ralgds-/- hearts manifested a blunted growth response (p<0.05) to TAC-mediated pressure-overload stress as determined by heart weight to body weight ratios Ventricular chamber size and contractile performance were preserved in response to TAC in Ralgds-/- mice. Interestingly, TAC-induced activation of the fetal gene program was similar in both genotypes despite the relative lack of hypertrophic growth in mutant hearts. Ralgds-/- mice also exhibited diminished load-induced cardiomyocyte autophagy. Consistent with the TAC findings, Ralgds-/- mice manifested a blunted autophagic response to 24-hour fasting, suggesting a generalized defect in autophagy. Together, these data implicate RalGDS-mediated induction of autophagy as a critical feature of load-induced cardiac hypertrophy.Item The Regulation and Function of Drosophila Acinus(2017-12-01) Tyra, Lauren Katherine; Johnson, Jane E.; Smith, Dean P.; Pan, Duojia; Krämer, HelmutAutophagy and growth control are two processes critical to organisms that mutually antagonize and regulate on each other. Several well known connections between these processes have been described, but here I describe a new link. Using Drosophila melanogaster as a model system, my thesis research has identified Acinus and Atg1, already known for their functions in autophagy, as growth suppressors. Further, my data show that this suppression is, at least in part, mediated by Atg1 phosphorylating and thereby inhibiting the pro-growth transcriptional co-activator Yorkie. Genetic gain- and loss-of function experiments indicate that this Atg1 function depends on Acinus. This work provides a new role for Atg1 in inhibition of growth and thereby adding a new regulatory pressure on Yorkie. Furthermore, my data indicate that Acinus’ function in promoting basal autophagy is based on its starvation-independent activation of the Atg1 kinase.Item The Regulation of Autophagy and Its Role in Mitotic Exit(2014-07-14) An, Zhenyi; Tu, Benjamin; Cobb, Melanie H.; White, Michael A.Autophagy is an evolutionarily conserved pathway in which cells enclose cytoplasmic contents in double membrane vesicles and deliver them to the lysosome for degradation. Autophagy plays critical regulatory roles in cancer, aging, neurodegeneration, immunity and many other physiological processes. Autophagy can be induced by multiple conditions such as starvation, viral infection, exercise and oxidative stress. In this work, we studied the role of autophagy in starvation-induced cell cycle arrest and quiescence entry, and studied the function and regulation of a major phosphorylation site of Beclin 1, serine 90. In the first study, we found that the in response to starvation, autophagy-deficient yeasts failed to arrest properly in G1/G0, but arrested in telophase with a quiescent-specific phenotype. In a second study, we found that Beclin 1 serine 90 is a major phosphorylation site of Beclin 1, which is induced by multiple stresses such as starvation and osmotic stress. The phosphorylation of Beclin 1 serine 90 leads to the activation of autophagy and inhibition of tumorigenesis. We identified MK2/3 as kinases that positively regulate autophagy by phosphorylating Beclin 1 at amino acid residue serine 90. We also found that Beclin 1 serine 90 phosphorylation is negatively regulated by Bcl-2 and positively regulated by AMPK. Beclin 1 serine 90 phosphorylation is also important for cell survival during high osmotic stress. Taken together, these results suggest that Beclin 1 serine 90 phosphorylation is a critical event in autophagy induction, which is tightly regulated by multiple kinases and regulatory proteins.Item The Role of Autophagy in Early Development and Tumor Suppression Using a Zebrafish Model System(2013-06-25) Lee, Eunmyong; Brugarolas, James B.; Abrams, John M.; Cleaver, Ondine; Levine, Beth; Amatruda, James F.Autophagy is an evolutionarily conserved lysosomal degradation pathway which involves the sequestration of cytoplasmic components into a double membraned structure called the autophagosome. By using genetically manipulated autophagy-deficient models, important roles for autophagy in development and tumorigenesis have been suggested. Genetic analyses indicate that autophagy is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryonic development. To address this question, the role of autophagy in early development was examined using zebrafish, a model system for studying vertebrate tissue and organ morphogenesis. Active autophagy was observed in multiple tissues during early embryonic development, as evidenced by the presence of autophagosomes in electron microscope images or GFP-LC3 puncta in autophagy reporter fish line Tg(cmv