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Patrice Codogno - One of the best experts on this subject based on the ideXlab platform.
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Macroautophagy signaling and regulation
Current Topics in Microbiology and Immunology, 2009Co-Authors: Audrey Esclatine, Magali Chaumorcel, Patrice CodognoAbstract:Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment. Macroautophagy is a multistep process involving: (1) signaling events that occur upstream of the molecular machinery of autophagy; (2) molecular machinery involved in the formation of the autophagosome, the initial multimembrane-bound compartment formed in the autophagic pathway; and (3) maturation of autophagosomes, which acquire acidic and degradative capacities. In this chapter we summarize what is known about the regulation of the different steps involved in autophagy, and we also discuss how Macroautophagy can be manipulated using drugs or genetic approaches that affect Macroautophagy signaling, and the subsequent formation and maturation of the autophagosomes. Modulating autophagy offers a promising new therapeutic approach to human diseases that involve Macroautophagy.
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ceramide mediated Macroautophagy involves inhibition of protein kinase b and up regulation of beclin 1
Journal of Biological Chemistry, 2004Co-Authors: Francesca Scarlatti, Riccardo Ghidoni, Chantal Auvy, Annamaria Ventruti, Giusy Sala, Francoise Cluzeaud, Alai Vandewalle, Patrice CodognoAbstract:Abstract The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls Macroautophagy, a major lysosomal catabolic pathway. Exogenous C2-ceramide stimulates Macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B1. Ceramide reverted the interleukin 13-dependent inhibition of Macroautophagy by interfering with the activation of protein kinase B. In addition, C2-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C2-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C2-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of Macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.
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amino acids interfere with the erk1 2 dependent control of Macroautophagy by controlling the activation of raf 1 in human colon cancer ht 29 cells
Journal of Biological Chemistry, 2003Co-Authors: Sophie Pattingre, Chantal Bauvy, Patrice CodognoAbstract:Activation of ERK1/2 stimulates Macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the Gα-interacting protein (GAIP) in an amino acid-dependent manner (Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P. (2000) J. Biol. Chem. 275, 39090–39095). Here we show that ERK1/2 activation by aurintricarboxylic acid (ATA) treatment induces the phosphorylation of GAIP in an amino acid-dependent manner. Accordingly, ATA challenge increased the rate of Macroautophagy, whereas epidermal growth factor did not significantly affect Macroautophagy and GAIP phosphorylation status. In fact, ATA activated the ERK1/2 signaling pathway, whereas epidermal growth factor stimulated both the ERK1/2 pathway and the class I phosphoinositide 3-kinase pathway, known to decrease the rate of Macroautophagy. Amino acids interfered with the ATA-induced Macroautophagy by inhibiting the activation of the kinase Raf-1. The role of the Ras/Raf-1/ERK1/2 signaling pathway in the GAIP- and amino acid-dependent control of Macroautophagy was confirmed in HT-29 cells expressing the Ras(G12V,T35S) mutant. Similar to the protein phosphatase 2A inhibitor okadaic acid, amino acids sustained the phosphorylation of Ser259, which is involved in the negative regulation of Raf-1. In conclusion, these results add a novel target to the amino acid signaling-dependent control of Macroautophagy in intestinal cells.
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the tumor suppressor pten positively regulates Macroautophagy by inhibiting the phosphatidylinositol 3 kinase protein kinase b pathway
Journal of Biological Chemistry, 2001Co-Authors: Sebastien Arico, Patrice Codogno, Anne Petiot, Chantal Bauvy, Peter F Dubbelhuis, Alfred J Meijer, Eric OgierdenisAbstract:The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of Macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent Macroautophagy was investigated by expressing a constitutively active form of the kinase (MyrPKB). Under these conditions a dramatic inhibition of Macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of Macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.
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distinct classes of phosphatidylinositol 3 kinases are involved in signaling pathways that control Macroautophagy in ht 29 cells
Journal of Biological Chemistry, 2000Co-Authors: Anne Petiot, Alfred J Meijer, Eric Ogierdenis, E F C Blommaart, Patrice CodognoAbstract:3-Methyladenine which stops Macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelarova, H., and Meijer, A. J. (1997) Eur. J. Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3, 4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4, 5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced Macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates Macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of Macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC(50) 5-15 nM). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Galpha(i3) protein suggesting that both class III PI3K and trimeric G(i3) protein signaling are required in the control Macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in Macroautophagy are discussed in the context of membrane recycling.
Ana Maria Cuervo - One of the best experts on this subject based on the ideXlab platform.
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huntingtin functions as a scaffold for selective Macroautophagy
Nature Cell Biology, 2015Co-Authors: Yanning Rui, Bindi Patel, Zhihua Chen, Dongsheng Chen, Antonio Tito, Gabriela David, Yamin Sun, Erin Furr Stimming, Hugo J Bellen, Ana Maria CuervoAbstract:Selective Macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. Here, we demonstrate that Huntingtin, the Huntington disease gene product, functions as a scaffold protein for selective Macroautophagy but it is dispensable for non-selective Macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with Lys-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress is attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which releases ULK1 from negative regulation by mTOR. Our data uncover an important physiological function of Huntingtin and provide a missing link in the activation of selective Macroautophagy in metazoans.
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autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin dependent manner
Molecular Biology of the Cell, 2012Co-Authors: Eloy Bejarano, David C Spray, Andrea Yuste, Bindi Patel, Henrique Girao, Carla Marques, Paulo Pereira, Ana Maria CuervoAbstract:Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of Macroautophagy to connexin degradation. Using pharmacological and genetic blockage of Macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' Macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of Macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for Macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for Macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.
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microautophagy of cytosolic proteins by late endosomes
Developmental Cell, 2011Co-Authors: Ranjit Sahu, Ana Maria Cuervo, Cristina C Clement, Susmita Kaushik, Brian Scharf, Antonia Follenzi, Elvira S Cannizzo, Ilaria Potolicchio, Edward Nieves, Laura SantambrogioAbstract:Autophagy delivers cytosolic components to lysosomes for their degradation. The delivery of autophagic cargo to late endosomes for complete or partial degradation has also been described. In this report we present evidence that distinct autophagic mechanisms control cytosolic protein delivery to late endosomes and identify a microautophagy-like process that delivers soluble cytosolic proteins to the vesicles of late endosomes/multivesicular bodies (MVBs). This microautophagy-like process has selectivity and is distinct from chaperone-mediated autophagy that occurs in lysosomes. Endosomal microautophagy occurs during MVB formation, relying on the ESCRT I and III systems for formation of the vesicles in which the cytosolic cargo is internalized. Protein cargo selection is mediated by the chaperone hsc70 and requires the cationic domain of hsc70 for electrostatic interactions with the endosomal membrane. Therefore, we propose that endosomal microautophagy shares molecular components with both the endocytic and autophagic pathways.
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Macroautophagy regulates energy metabolism during effector t cell activation
Journal of Immunology, 2010Co-Authors: Vanessa M Hubbard, Ana Maria Cuervo, Bindi Patel, Rajat Singh, Rut Valdor, Fernando MacianAbstract:Macroautophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that Macroautophagy is induced after effector T cell activation. Engagement of the TCR and CD28 results in enhanced microtubule-associated protein 1 light chain 3 (LC3) processing, increased numbers of LC3-containing vesicles, and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional KO mouse model where Atg7, a critical gene for Macroautophagy, is specifically deleted in T cells, we have found that Macroautophagy-deficient effector Th cells have defective IL-2 and IFN-γ production and reduced proliferation after stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to Macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T cells, where mitochondria and other organelles are selectively excluded. These results suggest that Macroautophagy is an actively regulated process in T cells that can be induced in response to TCR engagement to accommodate the bioenergetic requirements of activated T cells.
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constitutive activation of chaperone mediated autophagy in cells with impaired Macroautophagy
Molecular Biology of the Cell, 2008Co-Authors: Susmita Kaushik, Ashish C Massey, Noboru Mizushima, Ana Maria CuervoAbstract:Three different types of autophagy-Macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA)-contribute to degradation of intracellular components in lysosomes in mammalian cells. Although some level of basal Macroautophagy and CMA activities has been described in different cell types and tissues, these two pathways are maximally activated under stress conditions. Activation of these two pathways is often sequential, suggesting the existence of some level of cross-talk between both stress-related autophagic pathways. In this work, we analyze the consequences of blockage of Macroautophagy on CMA activity. Using mouse embryonic fibroblasts deficient in Atg5, an autophagy-related protein required for autophagosome formation, we have found that blockage of Macroautophagy leads to up-regulation of CMA, even under basal conditions. Interestingly, different mechanisms contribute to the observed changes in CMA-related proteins and the consequent activation of CMA during basal and stress conditions in these Macroautophagy-deficient cells. This work supports a direct cross-talk between these two forms of autophagy, and it identifies changes in the lysosomal compartment that underlie the basis for the communication between both autophagic pathways.
Jan D Lünemann - One of the best experts on this subject based on the ideXlab platform.
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tnf α upregulates macroautophagic processing of app β amyloid in a human rhabdomyosarcoma cell line
Journal of the Neurological Sciences, 2013Co-Authors: Christian Munz, Christian W Keller, Matthias Schmitz, Jan D Lünemann, Jens SchmidtAbstract:Sporadic inclusion body myositis is a chronic progressive, inflammatory disorder of the skeletal muscle. No effective treatment is available for this debilitating condition and the complex disease pathology is far from being understood. The major hallmark of the pathomechanisms is the co-occurrence of inflammatory as well as degenerative cascades including aggregates consisting of β-amyloid within skeletal muscle fibers. Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to be upregulated via the proinflammatory cytokine TNF-α in human skeletal muscle cells. In a human cell line from rhabdomyosarcoma as a model to study muscle cells, we here show that TNF-α-mediated upregulation of Macroautophagy modulates APP and β-amyloid load and can be blocked by inhibition of Macroautophagy. Thus, Macroautophagy may be a crucial mediator between inflammation and β-amyloid-associated degeneration in skeletal muscle.
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tnf alpha induces Macroautophagy and regulates mhc class ii expression in human skeletal muscle cells
Journal of Biological Chemistry, 2011Co-Authors: Christian W Keller, Christian Munz, Jan D Lünemann, Jens Schmidt, Claudia Fokken, Stuart Turville, Anna LunemannAbstract:Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive Macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of Macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by Macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via Macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4+ and CD8+ immune cells. These findings establish a mechanism through which TNF-α regulates both Macroautophagy and MHC class II expression and suggest that Macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle.
Christian Munz - One of the best experts on this subject based on the ideXlab platform.
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MHC Class I Internalization via Autophagy Proteins.
Methods of Molecular Biology, 2019Co-Authors: Laure-anne Ligeon, Christian MunzAbstract:Macroautophagy is a ubiquitous degradative pathway involved in innate and adaptive immunity. Its molecular machinery has been described to deliver intracellular and extracellular antigens to MHC class II loading compartment by regulating autophagosome and phagosome maturation. We recently found that the respective Atg proteins can contribute to MHC class I-restricted antigen presentation to CD8$^{+}$ T cells by regulating MHC class I surface levels in mouse dendritic cell. Indeed, we determined that MHC class I molecules are stabilized on the cell surface of murine antigen presenting cells deficient for core components of the Macroautophagy machinery such as Atg5 and Atg7. This stabilization seems to result from defective internalization of MHC class I molecules dependent on adaptor protein kinase 1 (AAK1), involved in clathrin-mediated endocytosis. Moreover, Macroautophagy-dependent stabilization of MHC class I molecules leads to enhanced CD8$^{+}$ T cell priming during influenza A virus infection in vivo, resulting in decreased pathology. In this chapter, we describe four experiments to monitor, characterize, and quantify the effect of Macroautophagy deficiency on MHC class I molecule trafficking and the subsequent CD8$^{+}$ T cell priming. First, we will show how to monitor MHC class I internalization in lung CD11c$^{+}$ cells from mice lacking key components of the Macroautophagy machinery. Then, we will propose a method to characterize the interaction between either MHC class I or Atg8/LC3 with AAK1. Finally, we will describe how to evaluate the influenza A-specific CD8$^{+}$ T cell response in mice conditionally depleted for Atg5 in their DC compartment. This set of experiments allows to characterize MHC class I internalization with the help of the molecular machinery of Macroautophagy.
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Macroautophagy proteins assist epstein barr virus production and get incorporated into the virus particles
EBioMedicine, 2014Co-Authors: Heike Nowag, Bruno Guhl, Kerstin Thriene, Susana Romao, Urs Ziegler, Joern Dengjel, Christian MunzAbstract:Epstein Barr virus (EBV) persists as a latent herpes virus infection in the majority of the adult human population. The virus can reactivate from this latent infection into lytic replication for virus particle production. Here, we report that autophagic membranes, which engulf cytoplasmic constituents during Macroautophagy and transport them to lysosomal degradation, are stabilized by lytic EBV replication in infected epithelial and B cells. Inhibition of autophagic membrane formation compromises infectious particle production and leads to the accumulation of viral DNA in the cytosol. Vice versa, pharmacological stimulation of autophagic membrane formation enhances infectious virus production. Atg8/LC3, an essential Macroautophagy protein and substrate anchor on autophagic membranes, was found in virus preparations, suggesting that EBV recruits Atg8/LC3 coupled membranes to its envelope in the cytosol. Our data indicate that EBV subverts Macroautophagy and uses autophagic membranes for efficient envelope acquisition during lytic infection.
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tnf α upregulates macroautophagic processing of app β amyloid in a human rhabdomyosarcoma cell line
Journal of the Neurological Sciences, 2013Co-Authors: Christian Munz, Christian W Keller, Matthias Schmitz, Jan D Lünemann, Jens SchmidtAbstract:Sporadic inclusion body myositis is a chronic progressive, inflammatory disorder of the skeletal muscle. No effective treatment is available for this debilitating condition and the complex disease pathology is far from being understood. The major hallmark of the pathomechanisms is the co-occurrence of inflammatory as well as degenerative cascades including aggregates consisting of β-amyloid within skeletal muscle fibers. Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to be upregulated via the proinflammatory cytokine TNF-α in human skeletal muscle cells. In a human cell line from rhabdomyosarcoma as a model to study muscle cells, we here show that TNF-α-mediated upregulation of Macroautophagy modulates APP and β-amyloid load and can be blocked by inhibition of Macroautophagy. Thus, Macroautophagy may be a crucial mediator between inflammation and β-amyloid-associated degeneration in skeletal muscle.
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tnf alpha induces Macroautophagy and regulates mhc class ii expression in human skeletal muscle cells
Journal of Biological Chemistry, 2011Co-Authors: Christian W Keller, Christian Munz, Jan D Lünemann, Jens Schmidt, Claudia Fokken, Stuart Turville, Anna LunemannAbstract:Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive Macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of Macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by Macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via Macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4+ and CD8+ immune cells. These findings establish a mechanism through which TNF-α regulates both Macroautophagy and MHC class II expression and suggest that Macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle.
Ashish C Massey - One of the best experts on this subject based on the ideXlab platform.
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lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by alzheimer related ps1 mutations
Cell, 2010Co-Authors: Haung W Yu, Asok Kumar, Panaiyur S Mohan, Corrinne M Peterhoff, Devin M Wolfe, Marta Martinezvicente, Ashish C Massey, Guy Sovak, Yasuo Uchiyama, David WestawayAbstract:Summary Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that Macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during Macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets. PaperClip
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lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by alzheimer related ps1 mutations
Cell, 2010Co-Authors: Juhyun Lee, Asok Kumar, Panaiyur S Mohan, Corrinne M Peterhoff, Devin M Wolfe, Marta Martinezvicente, Ashish C Massey, Guy Sovak, Sooyeon Lee, Yasuo UchiyamaAbstract:Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that Macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during Macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.
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constitutive activation of chaperone mediated autophagy in cells with impaired Macroautophagy
Molecular Biology of the Cell, 2008Co-Authors: Susmita Kaushik, Ashish C Massey, Noboru Mizushima, Ana Maria CuervoAbstract:Three different types of autophagy-Macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA)-contribute to degradation of intracellular components in lysosomes in mammalian cells. Although some level of basal Macroautophagy and CMA activities has been described in different cell types and tissues, these two pathways are maximally activated under stress conditions. Activation of these two pathways is often sequential, suggesting the existence of some level of cross-talk between both stress-related autophagic pathways. In this work, we analyze the consequences of blockage of Macroautophagy on CMA activity. Using mouse embryonic fibroblasts deficient in Atg5, an autophagy-related protein required for autophagosome formation, we have found that blockage of Macroautophagy leads to up-regulation of CMA, even under basal conditions. Interestingly, different mechanisms contribute to the observed changes in CMA-related proteins and the consequent activation of CMA during basal and stress conditions in these Macroautophagy-deficient cells. This work supports a direct cross-talk between these two forms of autophagy, and it identifies changes in the lysosomal compartment that underlie the basis for the communication between both autophagic pathways.
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loss of Macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus
Journal of Biological Chemistry, 2008Co-Authors: Yongjun Wang, Ana Maria Cuervo, Ashish C Massey, Susmita Kaushik, Rajat Singh, Youqing Xiang, Saul S Kane, Taneisha Grant, Mark J CzajaAbstract:Macroautophagy has been implicated as a mechanism of cell death. However, the relationship between this degradative pathway and cell death is unclear as Macroautophagy has been shown recently to protect against apoptosis. To better define the interplay between these two critical cellular processes, we determined whether inhibition of Macroautophagy could have both pro-apoptotic and anti-apoptotic effects in the same cell. Embryonic fibroblasts from mice with a knock-out of the essential Macroautophagy gene atg5 were treated with activators of the extrinsic and intrinsic death pathways. Loss of Macroautophagy sensitized these cells to caspase-dependent apoptosis from the death receptor ligands Fas and tumor necrosis factor-α (TNF-α). Atg5-/- mouse embryonic fibroblasts had increased activation of the mitochondrial death pathway in response to Fas/TNF-α in concert with decreased ATP levels. Fas/TNF-α treatment failed to up-regulate Macroautophagy, and in fact, decreased activity at late time points. In contrast to their sensitization to Fas/TNF-α, Atg5-/- cells were resistant to death from menadione and UV light. In the absence of Macroautophagy, an up-regulation of chaperone-mediated autophagy induced resistance to these stressors. These results demonstrate that inhibition of Macroautophagy can promote or prevent apoptosis in the same cell and that the response is governed by the nature of the death stimulus and compensatory changes in other forms of autophagy. Experimental findings that an inhibition of Macroautophagy blocks apoptosis do not prove that autophagy mediates cell death as this effect may result from the protective up-regulation of other autophagic pathways such as chaperone-mediated autophagy.
