bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒12‒01
fourteen papers selected by
Viktor Korolchuk, Newcastle University

  1. Nat Rev Mol Cell Biol. 2019 Nov 25.
      Exciting new discoveries have transformed the view of the lysosome from a static organelle dedicated to the disposal and recycling of cellular waste to a highly dynamic structure that mediates the adaptation of cell metabolism to environmental cues. Lysosome-mediated signalling pathways and transcription programmes are able to sense the status of cellular metabolism and control the switch between anabolism and catabolism by regulating lysosomal biogenesis and autophagy. The lysosome also extensively communicates with other cellular structures by exchanging content and information and by establishing membrane contact sites. It is now clear that lysosome positioning is a dynamically regulated process and a crucial determinant of lysosomal function. Finally, growing evidence indicates that the role of lysosomal dysfunction in human diseases goes beyond rare inherited diseases, such as lysosomal storage disorders, to include common neurodegenerative and metabolic diseases, as well as cancer. Together, these discoveries highlight the lysosome as a regulatory hub for cellular and organismal homeostasis, and an attractive therapeutic target for a broad variety of disease conditions.
  2. J Biol Chem. 2019 Nov 24. pii: jbc.RA119.010892. [Epub ahead of print]
      mTOR Complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn, activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of TSC complex subunit 2 (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.
    Keywords:  Phospholipase D; amino acid; cancer biology; cancer therapy; growth factor; lysosome; mTOR complex (mTORC); phosphatidic acid; phospholipid vesicle
  3. Adv Exp Med Biol. 2019 ;1206 85-108
      AMPK is an evolutionarily conserved serine/threonine-protein kinase that acts as an energy sensor in cells and plays a key role in the upregulation of catabolism and inactivation of anabolism. Under various physiological and pathological conditions, AMPK can be phosphorylated by an upstream kinase and bind to AMP or ADP rather than ATP, leading to its activation. Activated AMPK regulates a variety of metabolic processes, including autophagy. AMPK promotes autophagy directly by phosphorylating autophagy-related proteins in the mTORC1, ULK1, and PIK3C3/VPS34 complexes or indirectly by regulating the expression of autophagy-related genes downstream of transcription factors such as FOXO3, TFEB, and BRD4. AMPK can also upregulate the autophagic degradation of mitochondria (mitophagy), as it can induce fragmentation of damaged mitochondria in the network and promote the translocation of the autophagy machinery to damaged mitochondria. In this section, we will detail the molecular structure of AMPK, how its activity is regulated, and its pivotal role in regulating autophagy and mitophagy.
    Keywords:  AMPK; Autophagy; Mitophagy
  4. Biochim Biophys Acta Mol Cell Res. 2019 Nov 21. pii: S0167-4889(19)30221-6. [Epub ahead of print]1867(2): 118613
      Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.
    Keywords:  Autophagy; Beclin1; Lafora disease; Laforin; Malin; PI3KC3; Protein degradation; Ubiquitination
  5. Cells. 2019 Nov 27. pii: E1531. [Epub ahead of print]8(12):
      Alterations in the autophagosomal-lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal-lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.
    Keywords:  Batten disease; CLN3; autophagy; neuronal ceroid lipofuscinosis
  6. Adv Exp Med Biol. 2019 ;1206 421-434
      Autophagy plays an important role in the renewal of cellular components, which function in energy production, metabolism, and clearance of damaged organelles. Both macroautophagy and microautophagy are involved in these processes. Although it was thought that nonselective macroautophagy is responsible for the clearance of damaged or old organelles, recent studies show that the clearance of cellular organelles depends on selective processes. Mitophagy is a process for selective degradation of mitochondria, which is well documented. The selective autophagy for other organelles includes endoplasmic reticulum autophagy (reticulophagy) and peroxisome autophagy (pexophagy). Autophagy is a routine pathway for cells to degrade unused proteins and damaged organelles in cells. Autophagy selectively removes dysfunctional cellular components but not damages the normally functioning organelles, to maintain the homeostasis of cells. In addition to the maintenance of the homeostasis of cells, autophagy clears the damaged organelles in disease or injury conditions to achieve cellular quality control. In some differentiated cells, such as red blood cells, some organelles are removed during the maturation, including mitochondria. The autophagy system can selectively clear the mitochondria and other organelles, which lead to the maturation of red blood cells. Dysfunction of autophagy impairs the clearance of damaged organelles, which results in injury of cells. In the maturation of red blood cells, failure to clear the cellular organelles by autophagy will disturb the normal differentiation of red blood cells, leading to a series of diseases such as anemia.
    Keywords:  Autophagy; Fission; Fusion; Mitochondria; Mitophagy; PINK1; Parkin
  7. Nat Commun. 2019 Nov 28. 10(1): 5426
      Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 or TSC2, which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. Here we use cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. We show that loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor. Our results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC.
  8. Mol Cell Biol. 2019 Nov 25. pii: MCB.00256-19. [Epub ahead of print]
      Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mis-localize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mis-localization and aggregation are implicated in ALS pathology, though the mechanism(s) of TDP-43 and FUS cytoplasmic toxicity remains unclear. Recently, we determined that the endocytic function aids turnover (i.e. protein degradation) of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 co-factor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in S. cerevisiae Cdc48 physically interacts and co-localizes with TDP-43, as does VCP in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function, but not autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identifies a role for Cdc48/VCP and endocytosis function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise.
  9. Adv Exp Med Biol. 2019 ;1206 287-326
      Autophagy is an evolutionarily conserved lysosome-dependent intracellular degradation process that is essential for the maintenance of cellular homeostasis and adaptation to cellular stresses in eukaryotic cells. The most well-characterized type of autophagy, the macroautophagy, involves the progressive sequestration of cytoplasmic components into dedicated double-membraned vesicles called autophagosomes, which ultimately fuse with lysosomes to initiate the autophagic degradation of the sequestered cargo. In the past decade, our understanding of the molecular mechanism of macroautophagy has significantly evolved, with particular contributions from the biochemical and structural characterizations of autophagy-related proteins. In this chapter, we focus on some autophagy regulatory proteins involved in the macroautophagy pathway, summarize their currently known structures, and discuss their relevant molecular mechanisms from a perspective of structural biology.
    Keywords:  Autophagy; Autophagy regulatory proteins; Macroautophagy; Structural biology
  10. Mol Metab. 2019 Dec;pii: S2212-8778(19)30916-0. [Epub ahead of print]30 192-202
      OBJECTIVE: A decay in intracellular NAD+ levels is one of the hallmarks of physiological decline in normal tissue functions. Accordingly, dietary supplementation with NAD+ precursors can prevent, alleviate, or even reverse multiple metabolic complications and age-related disorders in diverse model organisms. Within the constellation of NAD+ precursors, nicotinamide riboside (NR) has gained attention due to its potent NAD+ biosynthetic effects in vivo while lacking adverse clinical effects. Nevertheless, NR is not stable in circulation, and its utilization is rate-limited by the expression of nicotinamide riboside kinases (NRKs). Therefore, there is a strong interest in identifying new effective NAD+ precursors that can overcome these limitations.METHODS: Through a combination of metabolomics and pharmacological approaches, we describe how NRH, a reduced form of NR, serves as a potent NAD+ precursor in mammalian cells and mice.
    RESULTS: NRH acts as a more potent and faster NAD+ precursor than NR in mammalian cells and tissues. Despite the minor structural difference, we found that NRH uses different steps and enzymes to synthesize NAD+, thus revealing a new NRK1-independent pathway for NAD+ synthesis. Finally, we provide evidence that NRH is orally bioavailable in mice and prevents cisplatin-induced acute kidney injury.
    CONCLUSIONS: Our data identify a new pathway for NAD+ synthesis and classify NRH as a promising new therapeutic strategy to enhance NAD+ levels.
    Keywords:  Metabolism; NAD(+); Nicotinamide riboside
  11. Science. 2019 Nov 28. pii: eaau2753. [Epub ahead of print]
      Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine-binding. The phosphorylated LARS1 showed decreased leucine-binding, which may inhibit protein synthesis and help save energy. Leucine, not used to anabolic process, may be available to catabolic pathway for energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival deprived of glucose. Thus, dependent on the availability of glucose, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.
  12. Mol Brain. 2019 Nov 29. 12(1): 100
      Due to their post-mitotic state, metabolic demands and often large polarised morphology, the function and survival of neurons is dependent on an efficient cellular waste clearance system both for generation of materials for metabolic processes and removal of toxic components. It is not surprising therefore that deficits in protein clearance can tip the balance between neuronal health and death. Here we discuss how autophagy and lysosome-mediated degradation pathways are disrupted in several neurological disorders. Both genetic and cell biological evidence show the diversity and complexity of vesicular clearance dysregulation in cells, and together may ultimately suggest a unified mechanism for neuronal demise in degenerative conditions. Causative and risk-associated mutations in Alzheimer's disease, Frontotemporal Dementia, Amyotrophic Lateral Sclerosis, Parkinson's disease, Huntington's disease and others have given the field a unique mechanistic insight into protein clearance processes in neurons. Through their broad implication in neurodegenerative diseases, molecules involved in these genetic pathways, in particular those involved in autophagy, are emerging as appealing therapeutic targets for intervention in neurodegeneration.
    Keywords:  Autophagy; Endo-lysosome; Mitophagy; Neurodegeneration
  13. Adv Exp Med Biol. 2019 ;1206 67-83
      Autophagy plays a crucial role in maintaining cellular homeostasis, and is closely related to the occurrence of variety of human diseases. It is known that autophagy occurs in response to various environmental stresses such as nutrient deficiency, growth factor deficiency, and hypoxia. Induced autophagy eliminates the damage caused by these stresses and returns to normal levels when the stresses are relieved. To comprehend the induction of autophagy under various stress conditions and the effects of autophagy on the life processes of cells, it is necessary to understand how autophagy is regulated. Many studies have shown that a number of signal transduction pathways are involved in the regulation of autophagy. Among these pathways, some pathways converge at the target of rapamycin (TOR), a highly conserved kinase important for autophagy regulation. This review will focus on the role of TOR signaling pathway in the regulation of autophagy.
    Keywords:  Autophagy; Regulation; TOR
  14. Oxid Med Cell Longev. 2019 ;2019 3809308
      Autophagy is a self-digestive process that degrades intracellular components, including damaged organelles, to maintain energy homeostasis and to cope with cellular stress. Autophagy plays a key role during development and adult tissue homeostasis, and growing evidence indicates that this catalytic process also has a direct role in modulating aging. Although autophagy is essentially protective, depending on the cellular context and stimuli, autophagy outcome can lead to either abnormal cell growth or cell death. The autophagic process requires a tight regulation, with cellular events following distinct stages and governed by a wide molecular machinery. Reactive oxygen species (ROS) have been involved in autophagy regulation through multiple signaling pathways, and mitochondria, the main source of endogenous ROS, have emerged as essential signal transducers that mediate autophagy. In the present review, we aim to summarize the regulatory function of mitochondria in the autophagic process, particularly regarding the mitochondrial role as the coordination node in the autophagy signaling pathway, involving mitochondrial oxidative stress, and their participation as membrane donors in the initial steps of autophagosome assembly.