bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒03‒24
seventeen papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2019 Mar 20.
      It has been indicated that the Golgi apparatus contributes to autophagy, but how it is involved in autophagosome formation and maturation is not well understood. Here we show that amino acid starvation causes trans-Golgi derived membrane fragments to colocalize with autophagosomes. Depletion of the Golgi stacking protein GORASP2/GRASP55, but not GORASP1/GRASP65, increases both MAP1LC3 (LC3)-II and SQSTM1/p62 levels. We demonstrate that GORASP2 facilitates autophagosome-lysosome fusion by physically linking autophagosomes and lysosomes through the interactions with LC3 on autophagosomes and LAMP2 on late endosomes/lysosomes. Furthermore, we provide evidence that GORASP2 interacts with BECN1 to facilitate the assembly and membrane association of the phosphatidylinositol 3-kinase (PtdIns3K) UVRAG complex. These findings indicate that GORASP2 plays an important role in autophagosome maturation during amino acid starvation.
    Keywords:  BECN1; GORASP2/GRASP55; Golgi; LAMP2; LC3; PtdIns3K complex; UVRAG; amino acid starvation; autophagosome-lysosome fusion
  2. Autophagy. 2019 Mar 22.
      Macroautophagy/autophagy is a cellular process in which cytosolic contents are degraded by lysosome in response to various stress conditions. Apart from its role in the maintenance of cellular homeostasis, autophagy also involves in regulation of cell cycle progression under nutrient-deprivation conditions. However, whether and how autophagy is regulated by the cell cycle especially during mitosis remains largely undefined. Here we show that WIPI2/ATG18B (WD repeat domain, phosphoinositide interacting 2), an autophagy-related (ATG) protein that plays a critical role in autophagosome biogenesis, is a direct substrate of CUL4-RING ubiquitin ligases (CRL4s). Upon mitosis induction, CRL4s are activated via neddylation, and recruit WIPI2 via DDB1 (damage specific DNA binding protein 1), leading to polyubiquitination and proteasomal degradation of WIPI2 and suppression of autophagy. The WIPI2 protein level and autophagy during mitosis could be rescued by knockdown of CRL4s or treatment with MLN4924/Pevonedistat, a selective inhibitor of CRLs, via suppression of NAE1 (NEDD8 activating enzyme E1 subunit 1). Moreover, restoration of WIPI2 rescues autophagy during mitosis and leads to mitotic slippage and cell senescence. Our study thus discovers a novel function of CRL4s in autophagy by targeting WIPI2 for polyubiquitination and proteasomal degradation during mitosis.
    Keywords:  CRL4s; MLN4924; WIPI2; autophagy; mitosis
  3. J Gerontol A Biol Sci Med Sci. 2019 Mar 01. pii: glz059. [Epub ahead of print]
      The mechanistic target of rapamycin (mTOR) is an essential nutrient-sensing kinase that integrates and regulates a number of fundamental cellular processes required for cell growth, cell motility, translation, metabolism and autophagy. mTOR signaling has been implicated in the progression of many human diseases and its dysregulation has been reported in several pathological processes, especially in age-related human diseases and mouse models of accelerated aging. In addition, many studies have demonstrated that regulation of mTOR activity has a beneficial effect on longevity in several mouse models of aging. However, not all mouse models of accelerated aging show positive effects on aging-associated phenotypes in response to targeting mTOR signaling. Here, we review the effects of interventions that modulate mTOR signaling on aging-related phenotypes in different mouse models of accelerated aging and discuss their implications with respect to aging and aging-related disorders.
    Keywords:  age-associated disease; dietary restriction; mTORC1; mice; rapamycin
  4. Autophagy. 2019 Mar 20.
      PSEN2 (presenilin 2) is one of the 3 proteins that, when mutated, causes early onset familial Alzheimer disease (FAD) cases. In addition to its well-known role within the γ-secretase complex (the enzyme ultimately responsible for Aβ peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca2+ homeostasis. Here, by using different FAD-PSEN2 cell models, we demonstrate that mutated PSEN2 impairs autophagy by causing a block in the degradative flux at the level of the autophagosome-lysosome fusion step. The defect does not depend on an altered lysosomal functionality but rather on a decreased recruitment of the small GTPase RAB7 to autophagosomes, a key event for normal autophagy progression. Importantly, FAD-PSEN2 action on autophagy is unrelated to its γ-secretase activity but depends on its previously reported ability to partially deplete ER Ca2+ content, thus reducing cytosolic Ca2+ response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca2+ signaling in autophagy and reveal a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for a dysfunctional quality control pathway in AD neurodegeneration.
    Keywords:  ATP2A/SERCA; Alzheimer disease; ER-mitochondria tethering; RAB7; autophagosome-lysosome fusion; calcium; presenilin
  5. Autophagy. 2019 Mar 20.
      Mitochondrial dynamics is highly implicated in a plethora of cellular processes including apoptosis and mitophagy. However, little is known about the scope and precise functions of mitochondrial dynamics proteins for mitochondrial quality control and cellular homeostasis. Whether mitochondrial dynamics proteins serve in cellular processes reliant on mitochondrial fission-fusion is still not fully explored. MIEF1/MiD51 (mitochondrial elongation factor 1) is known to promote mitochondrial fission via the recruitment of GTPase protein DNM1L/DRP1 (dynamin 1 like), but the fundamental understandings of MIEF1 for mitochondrial-dependent cellular processes are largely elusive. Here, we report novel roles of MIEF1 in responding to apoptotic stimuli and mitochondrial damage. Given our result that staurosporine (STS) treatment induced the degradation of MIEF1 via the ubiquitin-proteasome system (UPS), we are motivated to explore the role of MIEF1 in apoptosis. MIEF1 loss triggered the imbalance of BCL2 family members on the mitochondria, consequently initiating the translocation of BAX onto the mitochondria, catalyzing the decrease of mitochondrial membrane potential and promoting the release of DIABLO/SMAC (diablo IAP-binding mitochondrial protein) and CYCS (cytochrome c, somatic). We further demonstrate that MIEF1 deficiency impaired mitochondrial respiration and induced mitochondrial oxidative stress, sensitizing cells to PINK1-PRKN-mediated mitophagy. The recruitment of PRKN to depolarized mitochondria modulated the UPS-dependent degradation of MFN2 (mitofusin 2) and FIS1 (fission, mitochondrial 1) specifically, to further promote mitophagy. Our findings uncover a bridging role of MIEF1 integrating cell death and mitophagy, unlikely dependent on mitochondrial dynamics, implying new insights to mechanisms determining cellular fate.
    Keywords:  BAX; MIEF1; PRKN; ROS; apoptosis; mitochondria; mitophagy
  6. Autophagy. 2019 Mar 18. 1-2
      Deregulation of macroautophagy/autophagy, a conserved catabolic recycling pathway, has been implicated in the onset and development of several diseases. While post-translational regulation of auto-phagy-related (Atg) proteins has been an important research focus leading to significant breakthroughs in understanding autophagy regulation, less is known about the post-transcriptional regulation of ATG transcripts. In a recent study we showed that, during nitrogen starvation, the RNA-binding complex Pat1-Lsm is involved in binding and preventing the 3' to 5' exosome-mediated degradation of a specific subset of ATG mRNAs. Dephosphorylation of Pat1 at residues S456 and S457 facilitates ATG mRNA binding, resulting in ATG mRNA accumulation, Atg protein synthesis and robust autophagy induction. In addition, we present evidence that these processes are conserved in human cells. These results further elucidate our understanding of the post-transcriptional mechanism necessary for efficient induction of autophagy during stress conditions.
    Keywords:  3ʹ-5ʹ degradation; 5ʹ-3ʹ degradation; Lsm1; Xrn1; exosome; mRNA decay
  7. Dev Cell. 2019 Mar 08. pii: S1534-5807(19)30104-2. [Epub ahead of print]
      In neurons, defects in autophagosome clearance have been associated with neurodegenerative disease. Yet, the mechanisms that coordinate trafficking and clearance of synaptic autophagosomes are poorly understood. Here, we use genetic screens and in vivo imaging in single neurons of C. elegans to identify mechanisms necessary for clearance of synaptic autophagosomes. We observed that autophagy at the synapse can be modulated in vivo by the state of neuronal activity, that autophagosomes undergo UNC-16/JIP3-mediated retrograde transport, and that autophagosomes containing synaptic material mature in the cell body. Through forward genetic screens, we then determined that autophagosome maturation in the cell body depends on the protease ATG-4.2, but not the related ATG-4.1, and that ATG-4.2 can cleave LGG-1/Atg8/GABARAP from membranes. Our studies revealed that ATG-4.2 is specifically necessary for the maturation and clearance of autophagosomes and that defects in transport and ATG-4.2-mediated maturation genetically interact to enhance abnormal accumulation of autophagosomes in neurons.
    Keywords:  ATG-4.2; ATG4; UNC-16/JIP3; activity; autophagy; clearance; degradation; maturation; synapse; transport
  8. Autophagy. 2019 Mar 20.
      Impaired macroautophagy/autophagy has been implicated in experimental and human pancreatitis. However, the transcriptional control governing the autophagy-lysosomal process in pancreatitis is largely unknown. We investigated the role and mechanisms of TFEB (transcription factor EB), a master regulator of lysosomal biogenesis, in the pathogenesis of experimental pancreatitis. We analyzed autophagic flux, TFEB nuclear translocation, lysosomal biogenesis, inflammation and fibrosis in GFP-LC3 transgenic mice, acinar cell-specific tfeb knockout (KO) and tfeb and tfe3 double-knockout (DKO) mice as well as human pancreatitis samples. We found that cerulein activated MTOR (mechanistic target of rapamycin kinase) and increased the levels of phosphorylated TFEB as well as pancreatic proteasome activities that led to rapid TFEB degradation. As a result, cerulein decreased the number of lysosomes resulting in insufficient autophagy in mouse pancreas. Pharmacological inhibition of MTOR or proteasome partially rescued cerulein-induced TFEB degradation and pancreatic damage. Furthermore, genetic deletion of tfeb specifically in mouse pancreatic acinar cells increased pancreatic edema, necrotic cell death, infiltration of inflammatory cells and fibrosis in pancreas after cerulein treatment. tfeb and tfe3 DKO mice also developed spontaneous pancreatitis with increased pancreatic trypsin activities, edema and infiltration of inflammatory cells. Finally, decreased TFEB nuclear staining was associated with human pancreatitis. In conclusion, our results indicate a critical role of impaired TFEB-mediated lysosomal biogenesis in promoting the pathogenesis of pancreatitis.
  9. Autophagy. 2019 Mar 22.
      There is overwhelming evidence for an association between impaired mitochondrial function and metabolic syndrome. Mitophagy, a process that selectively removes damaged mitochondria via a specialized form of autophagy, is essential for mitochondrial quality control (mitochondrial QC) and metabolic homeostasis. We thus addressed the potential role of defective mitophagy in the pathogenesis of metabolic disorders. Mice lacking Fundc1, a newly characterized mitophagy receptor, develop more severe obesity and insulin resistance when fed a high-fat diet (HFD). Ablation of Fundc1 results in defective mitophagy and impaired mitochondrial QC in vitro and in white adipose tissue (WAT). In addition, there is more pronounced WAT remodeling with more adipose tissue-associated macrophages infiltration, more M1 macrophage polarization and thus an elevated inflammatory response. Mechanistically, hyperactivation of MAPK/JNK leads to insulin insensitivity, which can be inhibited by knocking out Mapk8/Jnk1 in fundc1 KO mice. Our results demonstrate that dysregulated mitochondrial QC due to defective mitophagy receptor FUNDC1 links with metabolic disorders via MAPK signaling and inflammatory responses.
    Keywords:  FUNDC1; MAPK; insulin resistance; mitochondrial QC; mitophagy; obesity
  10. Mol Biol Cell. 2019 Mar 20. mbcE19010046
      Studies in yeast showed that mitochondrial stressors not directly targeting the protein import machinery can cause mitochondrial Precursor Overaccumulation Stress (mPOS) in the cytosol independent of bioenergetics. Here, we demonstrate mPOS and stress responses in human cells. We show that overloading of mitochondrial membrane carriers, but not matrix proteins, is sufficient to induce cytosolic aggresomes and apoptosis. The aggresomes appear to triage unimported mitochondrial proteins. Interestingly, expression of highly unstable mutant variants of the mitochondrial carrier protein, Ant1, also induces aggresomes despite a >20-fold reduction in protein level compared to wild type. Thus, protein overloading, rather than accumulation, is critical for aggresome induction. The data suggests that the import of mitochondrial proteins is saturable and that the cytosol is limited in degrading unimported mitochondrial proteins. In addition, we found that EGR1, eEF1A1 and ubiquitin C are upregulated by Ant1 overloading. These proteins are known to promote autophagy, protein targeting to aggresomes and the processing of protein aggregates respectively. Finally, we found that overexpression of the misfolded variants of Ant1 induces additional cytosolic responses including proteasomal activation. In summary, our work captured a profound effect of unimported mitochondrial carrier proteins on cytosolic proteostasis and revealed multiple anti-mPOS mechanisms in human cells.
  11. Aging Cell. 2019 Mar 21. e12927
      RATIONALE: Age-related changes in the intervertebral discs are the predominant contributors to back pain, a common physical and functional impairment experienced by older persons. Cellular senescence, a process wherein cells undergo growth arrest and chronically secrete numerous inflammatory molecules and proteases, has been reported to cause decline in the health and function of multiple tissues with age. Although senescent cells have been reported to increase in intervertebral degeneration (IDD), it is not known whether they are causative in age-related IDD.OBJECTIVE: The study aimed to elucidate whether a causal relationship exists between cellular senescence and age-related IDD.
    METHODS AND RESULTS: To examine the impact of senescent cells on age-associated IDD, we used p16-3MR transgenic mice, which enables the selective removal of p16Ink4a -positive senescent cells by the drug ganciclovir. Disc cellularity, aggrecan content and fragmentation alongside expression of inflammatory cytokine (IL-6) and matrix proteases (ADAMTS4 and MMP13) in discs of p16-3MR mice treated with GCV and untreated controls were assessed. In aged mice, reducing the per cent of senescent cells decreased disc aggrecan proteolytic degradation and increased overall proteoglycan matrix content along with improved histological disc features. Additionally, reduction of senescent cells lowered the levels of MMP13, which is purported to promote disc degenerative changes during aging.
    CONCLUSIONS: The findings of this study suggest that systemic reduction in the number of senescent cells ameliorates multiple age-associated changes within the disc tissue. Cellular senescence could therefore serve as a therapeutic target to restore the health of disc tissue that deteriorates with age.
    Keywords:  aggrecanolysis; aging; cellular senescence; intervertebral disc; p16Ink4a; proteoglycan
  12. Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Mar 16. pii: S1388-1981(19)30041-1. [Epub ahead of print]
      The origin of the autophagosomal membrane started to be debated by scientists working in the field within one year of the modern definition of autophagy in 1963. There is now converging evidence from older and newer studies that the endoplasmic reticulum is involved in formation of autophagosomes. Thus, it is possible to trace from early morphological work - done without the benefit of molecular descriptions - to recent studies - dissecting how specific proteins nucleate autophagosome biogenesis - a long series of experimental findings that are beginning to answer the 55-year old question with some confidence. The view that has emerged is that specialised regions of the endoplasmic reticulum, in dynamic cross talk with most intracellular organelles via membrane contact sites, provide a platform for autophagosome biogenesis.
    Keywords:  Autophagy; Contact sites; Endoplasmic reticulum; Organelles
  13. Semin Cancer Biol. 2019 Mar 14. pii: S1044-579X(19)30006-9. [Epub ahead of print]
      Macroautophagy (hereafter referred to as autophagy) involves an intracellular degradation and recycling system that, in a context-dependent manner, can either promote cell survival or accelerate cellular demise. Ferroptosis was originally defined in 2012 as an iron-dependent form of cancer cell death different from apoptosis, necrosis, and autophagy. However, this latter assumption came into question because, in response to ferroptosis activators (e.g., erastin and RSL3), autophagosomes accumulate, and because components of the autophagy machinery (e.g., ATG3, ATG5, ATG4B, ATG7, ATG13, and BECN1) contribute to ferroptotic cell death. In particular, NCOA4-facilitated ferritinophagy, RAB7A-dependent lipophagy, BECN1-mediated system xc- inhibition, STAT3-induced lysosomal membrane permeabilization, and HSP90-associated chaperone-mediated autophagy can promote ferroptosis. In this review, we summarize current knowledge on the signaling pathways involved in ferroptosis, while focusing on the regulation of autophagy-dependent ferroptotic cell death. The molecular comprehension of these phenomena may lead to the development of novel anticancer therapies.
    Keywords:  Autophagy; Cell death; Ferroptosis; Iron; Lipid peroxidation
  14. Am J Physiol Lung Cell Mol Physiol. 2019 Mar 20.
      Cellular senescence is a biological process by which cells lose their capacity to proliferate yet remain metabolically active. Although originally considered a protective mechanism to limit the formation of cancer, it is now appreciated that cellular senescence also contributes to the development of disease, including common respiratory ailments, such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. While many factors have been linked to the development of cellular senescence, mitochondrial dysfunction has emerged as an important causative factor. In this study, we uncovered that the mitochondrial biogenesis pathway driven by the mTOR/PGC1α/β axis is markedly upregulated in senescent lung epithelial cells. Using two different models, we show that activation of this pathway associates with other features characteristic of enhanced mitochondrial biogenesis, including elevated number of mitochondrion per cell, increased oxidative phosphorylation and augmented mitochondrial ROS production. Furthermore, we found that pharmacological inhibition of the mTORC1 complex with rapamycin not only restored mitochondrial homeostasis but also reduced cellular senescence to bleomycin in lung epithelial cells. Likewise, mitochondrial-specific antioxidant therapy also effectively inhibited mTORC1 activation in these cells, while concomitantly reducing mitochondrial biogenesis and cellular senescence. In summary, this study provides a mechanistic link between mitochondrial biogenesis and cellular senescence in lung epithelium and suggests that strategies aimed at blocking the mTORC1/PGC1α/β axis or reducing ROS-induced molecular damage could be effective in the treatment of senescence-associated lung diseases.
    Keywords:  , idiopathic pulmonary fibrosis; cellular senescence; lung epithelium; mammalian target of rapamycin; mitochondria
  15. Mol Cell. 2019 Mar 09. pii: S1097-2765(19)30102-9. [Epub ahead of print]
      Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.
    Keywords:  BCL-2 family; MDM2; NDUFS1; apoptosis; complex I; mitochondria
  16. Autophagy. 2019 Mar 20.
      Although best understood as a degradative pathway, recent evidence demonstrates pronounced involvement of the macroautophagic/autophagic molecular machinery in cellular secretion. With either overexpression or inhibition of autophagy mediators, dramatic alterations in the cellular secretory profile occur. This affects secretion of a plethora of factors ranging from cytokines, to granule contents, and even viral particles. Encompassing a wide range of secreted factors, autophagy-dependent secretion is implicated in diseases ranging from cancer to neurodegeneration. With a growing body of evidence shedding light onto the molecular mediators, this review delineates the molecular machinery involved in selective targeting of the autophagosome for either degradation or secretion. In addition, we summarize the current understanding of factors and cargo secreted through this unconventional route, and describe the implications of this pathway in both health and disease.
    Keywords:  Autophagy-dependent secretion; IL1B; cancer; infection; neurodegeneration; secretory autophagy
  17. J Clin Invest. 2019 Mar 18. pii: 124194. [Epub ahead of print]130
      Mitofusin-2 (MFN2) is a mitochondrial outer-membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2, which cause Charcot-Marie-Tooth disease type 2A (CMT2A), primarily affect the nervous system. We generated a transgenic mouse model of CMT2A that developed severe early onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria. While mitochondrial aggregates were labeled for mitophagy, mutant MFN2 did not inhibit Parkin-mediated degradation, but instead had a dominant negative effect on mitochondrial fusion only when MFN1 was at low levels, as occurs in neurons. Finally, using a transgenic approach, we found that augmenting the level of MFN1 in the nervous system in vivo rescued all phenotypes in mutant MFN2R94Q-expressing mice. These data demonstrate that the MFN1/MFN2 ratio is a key determinant of tissue specificity in CMT2A and indicate that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease.
    Keywords:  Mouse models; Neurodegeneration; Neuromuscular disease; Neuroscience