bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2021‒02‒28
33 papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2021 Feb 26. 1-17
      Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two clinically distinct classes of neurodegenerative disorders. Yet, they share a range of genetic, cellular, and molecular features. Hexanucleotide repeat expansions (HREs) in the C9orf72 gene and the accumulation of toxic protein aggregates in the nervous systems of the affected individuals are among such common features. Though the mechanisms by which HREs cause toxicity is not clear, the toxic gain of function due to transcribed HRE RNA or dipeptide repeat proteins (DPRs) produced by repeat-associated non-AUG translation together with a reduction in C9orf72 expression are proposed as the contributing factors for disease pathogenesis in ALS and FTD. In addition, several recent studies point toward alterations in protein homeostasis as one of the root causes of the disease pathogenesis. In this review, we discuss the effects of the C9orf72 HRE in the autophagy-lysosome pathway based on various recent findings. We suggest that dysfunction of the autophagy-lysosome pathway synergizes with toxicity from C9orf72 repeat RNA and DPRs to drive disease pathogenesis.Abbreviation: ALP: autophagy-lysosome pathway; ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ASO: antisense oligonucleotide; C9orf72: C9orf72-SMCR8 complex subunit; DENN: differentially expressed in normal and neoplastic cells; DPR: dipeptide repeat protein; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; ER: endoplasmic reticulum; FTD: frontotemporal dementia; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; HRE: hexanucleotide repeat expansion; iPSC: induced pluripotent stem cell; ISR: integrated stress response; M6PR: mannose-6-phosphate receptor, cation dependent; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: neurodegenerative disorder; RAN: repeat-associated non-ATG; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SLC66A1/PQLC2: solute carrier family 66 member 1; SMCR8: SMCR8-C9orf72 complex subunit; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; WDR41: WD repeat domain 41.
    Keywords:  Amyotrophic lateral sclerosis (ALS); autophagy; axonal transport; c9orf72; dipeptide repeat protein (DPR); frontotemporal dementia (FTD); lysosome; smcr8; wdr41
  2. J Cell Biol. 2021 May 03. pii: e202004010. [Epub ahead of print]220(5):
      The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.
  3. Autophagy. 2021 Feb 25.
      The mechanisms orchestrating recycling of lysosomes through autophagic lysosome reformation (ALR) is incompletely understood. Previous data show that genetic depletion of BLOC1S1/GCN5L1/BORCS1 increases autolysosome (AL) accumulation. We postulated that this phenotype may manifest due to perturbed ALR. We explored this in control and bloc1s1 liver-specific knockout (LKO) mouse hepatocytes, showing that in response to nutrient-deprivation LKO's fail to initiate ALR due to blunted lysosomal tubulation. As kinesin motor proteins and the intracellular cytoskeleton are requirements for tubular formation from ALs, we explored the interaction of BLOC1S1 with motor proteins and cytoskeletal factors. BLOC1S1 interacts with the ARL8B-KIF5B (GTPase and kinesin motor protein) complex to recruit KIF5B to ALs. Furthermore, BLOC1S1 interacts with the actin nucleation promoting factor WHAMM, which is an essential structural protein in the initiation of lysosomal tubulation (LT). Interestingly, the genetic reintroduction of BLOC1S1 rescues LT in LKO hepatocytes, but not when KIF5B is concurrently depleted. Finally, given the central role of MTORC1 signaling in ALR initiation, it was interesting that MTORC1 activity was increased despite the absence of LT in LKO hepatocytes. Concurrently, inhibition of MTORC1 abolished BLOC1S1 reconstitution-mediated rescue of LT in LKO hepatocytes. Taken together these data demonstrate that the functional interaction of BLOC1S1 with the kinesin binding complex and the actin cytoskeleton are a requirement for LT which, in parallel with MTORC1 signaling, initiate lysosome recycling via ALR.
    Keywords:  Autophagic lysosome reformation; GCN5L1; MTORC1; autophagy; hepatocyte; lysosomal tubulation; lysosome
  4. Autophagy. 2021 Feb 25. 1-3
      Phase-separated droplets with liquid-like properties can be degraded by macroautophagy/autophagy, but the mechanism underlying this degradation is poorly understood. We have recently derived a physical model to investigate the interaction between autophagic membranes and such droplets, uncovering that intrinsic wetting interactions underlie droplet-membrane contacts. We found that the competition between droplet surface tension and the increasing tendency of growing membrane sheets to bend determines whether a droplet is completely engulfed or isolated in a piecemeal fashion, a process we term fluidophagy. Intriguingly, we found that another critical parameter of droplet-membrane interactions, the spontaneous curvature of the membrane, determines whether the droplet is degraded by autophagy or - counterintuitively - serves as a platform from which autophagic membranes expand into the cytosol. We also discovered that the interaction of membrane-associated LC3 with the LC3-interacting region (LIR) found in the autophagic cargo receptor protein SQSTM1/p62 and many other autophagy-related proteins influences the preferred bending directionality of forming autophagosomes in living cells. Our study provides a physical account of how droplet-membrane wetting underpins the structure and fate of forming autophagosomes.
    Keywords:  Autophagy; condensate; droplet; isolation membrane; membrane; p62; phase separation; piecemeal autophagy; wetting
  5. FEBS Lett. 2021 Feb 22.
      Mitophagy is one of the selective autophagy pathways that catabolizes dysfunctional or superfluous mitochondria. Under mitophagy-inducing conditions, mitochondria are labeled with specific molecular landmarks that recruit the autophagy machinery to the surface of mitochondria, enclosed into autophagosomes, and delivered to lysosomes (vacuoles in yeast) for degradation. As damaged mitochondria are the major sources of reactive oxygen species, mitophagy is critical for mitochondrial quality control and cellular health. Moreover, appropriate control of mitochondrial quantity via mitophagy is vital for the energy supply-demand balance in cells and whole organisms, cell differentiation, and developmental programs. Thus, it seems conceivable that defects in mitophagy could elicit pleiotropic pathologies such as excess inflammation, tissue injury, neurodegeneration, and ageing. In this review, we will focus on the molecular basis and physiological relevance of mitophagy, and potential of mitophagy as a therapeutic target to overcome such disorders.
    Keywords:  adaptor; ageing; autophagy; inflammation; mitochondria; neurodegeneration; ubiquitin
  6. Reprod Sci. 2021 Feb 22.
      The placenta is important for pregnancy maintenance, and autophagy is documented to be essential for placental development. Autophagy is responsible for degrading and recycling cellular misfolded proteins and damaged organelles. Mitophagy is a selective type of autophagy, where the autophagic machinery engulfs the damaged mitochondria for degradation, and there is reciprocal crosstalk between autophagy and mitochondria. Within these processes, 5'-AMP-activated protein kinase (AMPK) plays an important role. However, the role of AMPK regulation in both autophagy and mitochondria in primary human trophoblasts is unknown. In this study, we address this question by investigating changes in mRNA expression and the abundance of autophagy- and mitochondria-related proteins in isolated human trophoblasts after treatment with AMPK agonists and antagonists. We found that compared to the control group, autophagy was slightly suppressed in the AMPK agonist group and significantly enhanced autophagy in the AMPK antagonist group. However, the expressions of genes related to autophagosome-lysosome fusion were reduced, while genes related to lysosomal function were unchanged in both groups. Furthermore, mitophagy and mitochondrial fusion/fission were both impaired in the AMPK agonist and antagonist groups. Although mitochondrial biogenesis was enhanced in both groups, the function of mitochondrial fatty acid oxidation was increased in the AMPK agonist group but decreased in the AMPK antagonist group. Overall, our study demonstrates that AMPK regulation negatively modulates autophagy and consequently affects mitophagy, mitochondrial fusion/fission, and function in primary human trophoblasts.
    Keywords:  Autophagy; Human trophoblasts; Mitochondrial fusion/fission; Mitophagy
  7. Hum Mol Genet. 2021 Feb 25. pii: ddab057. [Epub ahead of print]
      Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurons, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be cause of LBs formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusions formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterised the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2 deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.
  8. Autophagy. 2021 Feb 25. 1-3
      Neurons are long-lived cells that communicate via release of neurotransmitter at specialized contacts termed synapses. The maintenance of neuronal health and the regulation of synaptic function requires the efficient removal of damaged or dispensable proteins and organelles from synapses. How macroautophagy/autophagy contributes to neuronal and synaptic protein turnover, and what its main physiological substrates are in healthy neurons is largely unknown. We have now shown that loss of neuronal autophagy facilitates presynaptic neurotransmission by controlling the axonal endoplasmic reticulum and, thereby, axonal and synaptic calcium homeostasis.
    Keywords:  Autophagy; SV protein; calcium; endoplasmic reticulum; er-phagy; neurotransmission; reticulophagy; ryanodine receptor; synapse
  9. J Cell Sci. 2021 Feb 23. pii: jcs.249276. [Epub ahead of print]
      A genome-wide screen recently identified SEC24A as a novel mediator of thapsigargin-induced cell death in HAP1 cells. Here, we determined the cellular mechanism and specificity of SEC24A-mediated cytotoxicity. Measurement of calcium levels using organelle-specific fluorescent indicator dyes showed that calcium efflux from endoplasmic reticulum (ER) and influx into mitochondria were significantly impaired in SEC24A knockout cells. Furthermore, SEC24A knockout cells also showed ∼44% less colocalization of mitochondria and peripheral tubular ER. Knockout of SEC24A, but not its paralogs SEC24B, SEC24C, or SEC24D, rescued HAP1 cells from cell death induced by three different inhibitors of Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase (SERCA) but not from cell death induced by a topoisomerase inhibitor. Thapsigargin-treated SEC24A knockout cells showed a ∼2.5-fold increase in autophagic flux and ∼10-fold reduction in apoptosis compared to wild-type cells. Taken together, our findings indicate that SEC24A plays a previously unrecognized role in regulating association and calcium flux between the ER and mitochondria, thereby impacting processes dependent on mitochondrial calcium levels, including autophagy and apoptosis.
    Keywords:  Apoptosis; Autophagy; Calcium; ER stress; Mitochondrial-associated membranes; SEC24A; SERCA; Thapsigargin
  10. Exp Eye Res. 2021 Feb 18. pii: S0014-4835(21)00057-9. [Epub ahead of print]205 108492
      We assess the effect of autophagy inhibition on photoreceptor (PR) survival during experimental retinal detachment (RD) and examine the and examine the relationship between autophagy and the expression of glycolytic enzymes HK2 and PKM2 in the retina. We find that inhibiting autophagy by genetic knock out of the autophagy activator Atg5 in rod PRs resulted in increased apoptotic and necroptotic cell death during RD, demonstrated by elevated terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, caspase 8 activity, transcript levels of Fas receptor and RIPK3 as compared to controls. The absence of autophagy in rods resulted in downregulation of hexokinase 2 and pyruvate kinase muscle isozyme 2 levels. More than 460 proteins were identified by mass spectroscopy in autophagosomes isolated from detached retinas compared with less than 150 proteins identified in autophagosomes from attached retinas. Among various cellular compartments, proteins from cytoskeleton, cytoplasm and intracellular organelles constituted a large portion of increased autophagosome contents. These proteins represent numerous biological processes, including phototransduction, cell-cell signaling, metabolism and inflammation. Our findings suggest that competent autophagy machinery is necessary for PR homeostasis and improving PR survival during periods of nutrient deprivation.
    Keywords:  Aerobic glycolysis; Atg5; Autophagosome; Autophagy; Retinal detachment
  11. Mol Biol Cell. 2021 Feb 24. mbcE20070500
      Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane-bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane-bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of both domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad, and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to understanding how the C-terminal lipase domain recognizes autophagic bodies to degrade them. [Media: see text] [Media: see text].
  12. Dev Cell. 2021 02 22. pii: S1534-5807(21)00113-1. [Epub ahead of print]56(4): 400-402
      Many pathogens are capable of disrupting autophagy within host cells. In this issue of Developmental Cell, Miao et al. discover that the SARS-CoV-2 protein ORF3a inhibits autophagosome-lysosome fusion by dysregulating the HOPS complex.
  13. Eur Rev Med Pharmacol Sci. 2021 Feb;pii: 24875. [Epub ahead of print]25(3): 1641-1649
      Autophagy is a main metabolic process in which eukaryotic cells use lysosomes to eliminate abnormal proteins and damaged organelles to maintain cell homeostasis. Studies have revealed that neurodegenerative diseases, tumor, hepatic diseases, etc. are related to abnormal autophagy processes in recent years. Recent studies have shown that TFEB is a major transcription regulator of autophagy-lysosomal pathway (ALP) transcriptional regulation, which positively regulates the expression of autophagy and lysosomal biogenesis-related genes, thereby promoting autophagosome formation, autophagosome-lysosome fusion, and degradation of autophagy substrates. It has also been found that TFEB promotes clearance of intracellular substrates through lysosomal exocytosis. Therefore, the study of biological functions and related regulatory mechanisms of TFEB will provide important clues and theoretical basis for further explaining its physiological pathogenesis and the treatment of related diseases.
  14. Front Oncol. 2020 ;10 619727
      Cancer progression involves a variety of pro-tumorigenic biological processes including cell proliferation, migration, invasion, and survival. A cellular pathway implicated in these pro-tumorigenic processes is autophagy, a catabolic route used for recycling of cytoplasmic components to generate macromolecular building blocks and energy, under stress conditions, to remove damaged cellular constituents to adapt to changing nutrient conditions and to maintain cellular homeostasis. During autophagy, cells form a double-membrane sequestering a compartment termed the phagophore, which matures into an autophagosome. Following fusion with the lysosome, the cargo is degraded inside the autolysosomes and the resulting macromolecules released back into the cytosol for reuse. Cancer cells use this recycling system during cancer progression, however the key autophagy players involved in this disease is unclear. Accumulative evidences show that autophagy receptors, crucial players for selective autophagy, are overexpressed during cancer progression, yet the mechanisms whereby pro-tumorigenic biological processes are modulated by these receptors remains unknown. In this review, we summarized the most important findings related with the pro-tumorigenic role of autophagy receptors p62/SQSTM1, NBR1, NDP52, and OPTN in cancer progression. In addition, we showed the most relevant cargos degraded by these receptors that have been shown to function as critical regulators of pro-tumorigenic processes. Finally, we discussed the role of autophagy receptors in the context of the cellular pathways implicated in this disease, such as growth factors signaling, oxidative stress response and apoptosis. In summary, we highlight that autophagy receptors should be considered important players of cancer progression, which could offer a niche for the development of novel diagnosis and cancer treatment strategies.
    Keywords:  aggressiveness; autophagy; autophagy receptors; cancer progression; metastasis
  15. Curr Biol. 2021 Feb 17. pii: S0960-9822(21)00146-9. [Epub ahead of print]
      Mutations in WDR45 and WDR45B cause the human neurological diseases β-propeller protein-associated neurodegeneration (BPAN) and intellectual disability (ID), respectively. WDR45 and WDR45B, along with WIPI1 and WIPI2, belong to a WD40 repeat-containing phosphatidylinositol-3-phosphate (PI(3)P)-binding protein family. Their yeast homolog Atg18 forms a complex with Atg2 and is required for autophagosome formation in part by tethering isolation membranes (IMs) (autophagosome precursor) to the endoplasmic reticulum (ER) to supply lipid for IM expansion in the autophagy pathway. The exact functions of WDR45/45B are unclear. We show here that WDR45/45B are specifically required for neural autophagy. In Wdr45/45b-depleted cells, the size of autophagosomes is decreased, and this is rescued by overexpression of ATG2A, providing in vivo evidence for the lipid transfer activity of ATG2-WIPI complexes. WDR45/45B are dispensable for the closure of autophagosomes but essential for the progression of autophagosomes into autolysosomes. WDR45/45B interact with the tether protein EPG5 and target it to late endosomes/lysosomes to promote autophagosome maturation. In the absence of Wdr45/45b, formation of the fusion machinery, consisting of SNARE proteins and EPG5, is dampened. BPAN- and ID-related mutations of WDR45/45B fail to rescue the autophagy defects in Wdr45/45b-deficient cells, possibly due to their impaired binding to EPG5. Promoting autophagosome maturation by inhibiting O-GlcNAcylation increases SNARE complex formation and facilitates the fusion of autophagosomes with late endosomes/lysosomes in Wdr45/45b double knockout (DKO) cells. Thus, our results uncover a novel function of WDR45/45B in autophagosome-lysosome fusion and provide molecular insights into the development of WDR45/WDR45B mutation-associated diseases.
    Keywords:  BPAN; ID; WDR45; WDR45B; autophagy
  16. Sci Rep. 2021 Feb 22. 11(1): 4342
      Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.
  17. Ageing Res Rev. 2021 Feb 21. pii: S1568-1637(21)00056-8. [Epub ahead of print] 101309
      Mitochondria are highly dynamic organelles capable of adapting their network, morphology, and function, playing a role in oxidative phosphorylation and many cellular processes in most cell types. Skeletal muscle is a very plastic tissue, subjected to many morphological changes following diverse stimuli, such as during myogenic differentiation and regenerative myogenesis. For some time now, mitochondria have been reported to be involved in myogenesis by promoting a bioenergetic remodeling and assisting myoblasts in surviving the process. However, not much is known about the interplay between mitochondrial quality control and myogenic differentiation. Sestrin2 (SESN2) is a well described regulator of autophagy and antioxidant responses and has been gaining attention due to its role in aging-associated pathologies and redox signaling promoted by reactive oxygen species (ROS) in many tissues. Current evidence involving SESN2-associated pathways suggest that it can act as a potential regulator of mitochondrial quality control following induction by ROS under stress conditions, such as during myogenesis. Yet, there are no studies directly assessing SESN2 involvement in myogenic differentiation. This review provides novel insights pertaining the involvement of SESN2 in myogenic differentiation by analyzing the interactions between ROS and mitochondrial remodeling.
    Keywords:  SESN2; differentiation; mitochondria; mitohormesis; myogenesis
  18. J Toxicol Pathol. 2021 Jan;34(1): 73-82
      Autophagy is a lysosomal-dependent degradation pathway in eukaryotic cells. Recent studies have reported that autophagy can facilitate the activation of hepatic stellate cells (HSCs) and fibrogenesis of the liver during long-term carbon tetrachloride (CCl4) exposure. However, little is known about the role of autophagy in CCl4-induced acute hepatic failure (AHF). This study aimed to identify whether modulation of autophagy can affect CCl4-induced AHF and evaluate the upstream signaling pathways mediated by CCl4-induced autophagy in rats. The accumulation of specific punctate distribution of endogenous LC3-II, increased expression of LC3-II, Atg5, and Atg7 genes/proteins, and decreased expression of p62 gene were observed after acute liver injury was induced by CCl4 in rats, indicating that CCl4 resulted in a high level of autophagy. Moreover, loss of autophagic function by using chloroquine (CQ, an autophagic inhibitor) aggravated liver function, leading to increased expression of p21 (a cyclin-dependent kinase inhibitor) in CCl4-treated rats. Furthermore, the AMPK-mTORC1-ULK1 axis was found to serve a function in CCl4-induced autophagy. These results reveal that AMPK-mTORC1-ULK1 signaling-induced autophagy has a protective role in CCl4-induced hepatotoxicity by inhibiting the p21 pathway. This study suggests a useful strategy aimed at ameliorating CCl4-induced acute hepatotoxicity by autophagy.
    Keywords:  AMPK-mTOR-ULK1 axis; autophagy; carbon tetrachloride (CCl4); p21; rat
  19. Free Radic Biol Med. 2021 Feb 17. pii: S0891-5849(21)00093-9. [Epub ahead of print]
      AIMS: The senescence of alveolar epithelial type 2 (AT2) cells is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Cigarette smoke (CS) is a strong risk factor for IPF and it is also a pro-senescent factor. Here we aimed to investigate whether and how CS induces AT2 cells senescence via a SIRT1/autophagy dependent pathway. Our results showed that CS extract (CSE) reduced autophagy and mitophagy and increased mitochondrial reactive oxygen species (mitoROS) in MLE-12 cells, an AT2 cell line. The autophagy inducer rapamycin (RAPA) and the mitochondria-targeted antioxidant mitoquinone (mitoQ) inhibited CSE-related senescence and decreased mitoROS. Next, we found that CSE promoted DNA damage, downregulated the nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide (NADH) ratio and suppressed SIRT1 activity. Activating SIRT1 with its activator SRT1720 attenuated senescence through an autophagy-dependent pathway. The NAD+ precursor nicotinamide mononucleotide and the poly ADP-ribose polymerase (PARP1) inhibitor olaparib also exerted anti-senescent effects by activating SIRT1. Moreover, the results showed that mitoQ and RAPA, in turn, elevated SIRT1 activity by inhibiting DNA damage. Consistent with these results, SRT1720 and mitoQ mitigated CS-induced AT2 cells senescence and lung fibrosis in vivo. Moreover, autophagy in AT2 cells was rescued by SRT1720. Taken together, our results suggested that CS-induced senescence of AT2 cells was due to decreased autophagy mediated by SIRT1 inactivation, which was attributed to competitive consumption of NAD+ caused by DNA damage-induced PARP1 activation. The reduction in autophagy, in turn, decreased SIRT1 activity by promoting mitochondrial oxidative stress-related DNA damage, thereby establishing a positive feedback loop between SIRT1 and autophagy in CS-induced AT2 cells senescence. Consequently, CS-inactivated SIRT1 promoted autophagy-dependent senescence of AT2 cells to induce pulmonary fibrosis.
    Keywords:  Pulmonary fibrosis; SIRT1; autophagy; cigarette smoke; senescence
  20. Autophagy. 2021 Feb 25. 1-15
      Glioma is the most common primary malignant brain tumor with poor survival and limited therapeutic options. The non-psychoactive phytocannabinoid cannabidiol (CBD) has been shown to be effective against glioma; however, the molecular target and mechanism of action of CBD in glioma are poorly understood. Here we investigated the molecular mechanisms underlying the antitumor effect of CBD in preclinical models of human glioma. Our results showed that CBD induced autophagic rather than apoptotic cell death in glioma cells. We also showed that CBD induced mitochondrial dysfunction and lethal mitophagy arrest, leading to autophagic cell death. Mechanistically, calcium flux induced by CBD through TRPV4 (transient receptor potential cation channel subfamily V member 4) activation played a key role in mitophagy initiation. We further confirmed TRPV4 levels correlated with both tumor grade and poor survival in glioma patients. Transcriptome analysis and other results demonstrated that ER stress and the ATF4-DDIT3-TRIB3-AKT-MTOR axis downstream of TRPV4 were involved in CBD-induced mitophagy in glioma cells. Lastly, CBD and temozolomide combination therapy in patient-derived neurosphere cultures and mouse orthotopic models showed significant synergistic effect in both controlling tumor size and improving survival. Altogether, these findings showed for the first time that the antitumor effect of CBD in glioma is caused by lethal mitophagy and identified TRPV4 as a molecular target and potential biomarker of CBD in glioma. Given the low toxicity and high tolerability of CBD, we therefore propose CBD should be tested clinically for glioma, both alone and in combination with temozolomide.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; Baf-A1: bafilomycin A1; CANX: calnexin; CASP3: caspase 3; CAT: catalase; CBD: cannabidiol; CQ: chloroquine; DDIT3: DNA damage inducible transcript 3; ER: endoplasmic reticulum; GBM: glioblastoma multiforme; GFP: green fluorescent protein; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; SLC8A1: solute carrier family 8 member A1; SQSTM1: sequestosome 1; TCGA: The cancer genome atlas; TEM: transmission electron microscopy; TMZ: temozolomide; TRIB3: tribbles pseudokinase 3; TRPC: transient receptor potential cation channel subfamily C; TRPV4: transient receptor potential cation channel subfamily V member 4.
    Keywords:  Cannabidiol; cannabinoid; glioblastoma; glioma; mitophagy; trpv4
  21. Nat Commun. 2021 Feb 26. 12(1): 1322
      The ubiquitin-proteasome system (UPS) and autophagy are two major quality control processes whose impairment is linked to a wide variety of diseases. The coordination between UPS and autophagy remains incompletely understood. Here, we show that ubiquitin ligase UBE3C and deubiquitinating enzyme TRABID reciprocally regulate K29/K48-branched ubiquitination of VPS34. We find that this ubiquitination enhances the binding of VPS34 to proteasomes for degradation, thereby suppressing autophagosome formation and maturation. Under ER and proteotoxic stresses, UBE3C recruitment to phagophores is compromised with a concomitant increase of its association with proteasomes. This switch attenuates the action of UBE3C on VPS34, thereby elevating autophagy activity to facilitate proteostasis, ER quality control and cell survival. Specifically in the liver, we show that TRABID-mediated VPS34 stabilization is critical for lipid metabolism and is downregulated during the pathogenesis of steatosis. This study identifies a ubiquitination type on VPS34 and elucidates its cellular fate and physiological functions in proteostasis and liver metabolism.
  22. Inhal Toxicol. 2021 Feb 25. 1-14
      Lysosomes offer a unique arrangement of degradative, exocytic, and signaling capabilities that make their continued function critical to cellular homeostasis. Lysosomes owe their function to the activity of lysosomal ion channels and transporters, which maintain concentration gradients of H+, K+, Ca2+, Na+, and Cl- across the lysosomal membrane. This review examines the contributions of lysosomal ion channels to lysosome function, showing how ion channel function is integral to degradation and autophagy, maintaining lysosomal membrane potential, controlling Ca2+ signaling, and facilitating exocytosis. Evidence of lysosome dysfunction in a variety of disease pathologies creates a need to understand how lysosomal ion channels contribute to lysosome dysfunction. For example, the loss of function of the TRPML1 Ca2+ lysosome channel in multiple lysosome storage diseases leads to lysosome dysfunction and disease pathogenesis while neurodegenerative diseases are marked by lysosome dysfunction caused by changes in ion channel activity through the TRPML1, TPC, and TMEM175 ion channels. Autoimmune disease is marked by dysregulated autophagy, which is dependent on the function of multiple lysosomal ion channels. Understanding the role of lysosomal ion channel activity in lysosome membrane permeability and NLRP3 inflammasome activation could provide valuable mechanistic insight into NLRP3 inflammasome-mediated diseases. Finally, this review seeks to show that understanding the role of lysosomal ion channels in lysosome dysfunction could give mechanistic insight into the efficacy of certain drug classes, specifically those that target the lysosome, such as cationic amphiphilic drugs.
    Keywords:  BK; Lysosome; NLRP3; TMEM175; TRPML1; autophagy; crystalline silica; lysosomal membrane permeability
  23. Biochim Biophys Acta Mol Basis Dis. 2021 Feb 19. pii: S0925-4439(21)00037-5. [Epub ahead of print] 166104
      Depolarized/damaged mitochondria aggregate at the perinuclear region prior to mitophagy in cells treated with mitochondrial stressors. However, the cellular mechanism(s) by which damaged mitochondria are transported and remain aggregated at the perinuclear region is unknown. Here, we demonstrate that mitofusins (Mfn1/2) are post-translationally modified by SUMO2 (Small Ubiquitin-related Modifier 2) in Human embryonic kidney 293 (Hek293) cells treated with protonophore CCCP and proteasome inhibitor MG132, both known mitochondrial stressors. SUMOylation of Mfn1/2 is not for their proteasomal degradation but facilitate mitochondrial congression at the perinuclear region in CCCP- and MG132-treated cells. Additionally, congressed mitochondria (mito-aggresomes) colocalize with LC3, ubiquitin, and SUMO2 in CCCP-treated cells. Knowing that SUMO functions as a "molecular glue" to facilitate protein-protein interactions, we propose that SUMOylation of Mfn1/2 may congress, glues, and confines damaged mitochondria to the perinuclear region thereby, protectively quarantining them from the heathy mitochondrial network until their removal via mitophagy in cells.
    Keywords:  26S Proteasome; Autophagy; Mitochondria; Mitofusin; Mitophagy; Small Ubiquitin Modifier (SUMO); ubiquitin
  24. Front Oncol. 2020 ;10 607149
      One of the most notoriously altered genes in human cancer is the tumor-suppressor TP53, which is mutated with high frequency in more cancers than any other tumor suppressor gene. Beyond the loss of wild-type p53 functions, mutations in the TP53 gene often lead to the expression of full-length proteins with new malignant properties. Among the defined oncogenic functions of mutant p53 is its effect on cell metabolism and autophagy. Due to the importance of autophagy as a stress adaptive response, it is frequently dysfunctional in human cancers. However, the role of p53 is enigmatic in autophagy regulation. While the complex action of the wild-type p53 on autophagy has extensively been described in literature, in this review, we focus on the conceivable role of distinct mutant p53 proteins in regulating different autophagic pathways and further discuss the available evidence suggesting a possible autophagy stimulatory role of mutant p53. Moreover, we describe the involvement of different autophagic pathways in targeting and degrading mutant p53 proteins, exploring the potential strategies of targeting mutant p53 in cancer by autophagy.
    Keywords:  TP53; autophagy; cancer; chaperone-mediated autophagy; mutant p53
  25. J Biol Chem. 2021 Feb 17. pii: S0021-9258(21)00194-0. [Epub ahead of print] 100421
      Intracellular organelles do not, as thought for a long time, act in isolation but are dynamically tethered together by entire machines responsible for inter-organelle trafficking and positioning. Among the proteins responsible for tethering is the family of vesicle-associated proteins (VAPs) that appear in all eukaryotes and are localized primarily in the endoplasmic reticulum. The major functional role of VAP proteins is to tether the endoplasmic reticulum with different organelles, and regulate lipid metabolism and transport. VAP proteins have gained increasing attention because of their role in human pathology where they contribute to infections by viruses and bacteria, and participate in neurodegeneration. In this review, we discuss the structure, evolution and functions of VAP proteins, focusing more specifically on VAP-B for its relationship with amyotrophic lateral sclerosis and other neurodegenerative diseases.
    Keywords:  ALS; VAMP; endoplasmic reticulum; tethering proteins
  26. Biochim Biophys Acta Mol Basis Dis. 2021 Feb 19. pii: S0925-4439(21)00035-1. [Epub ahead of print] 166102
      Mitophagy is defective in several neurodegenerative diseases, including Ataxia Telangiectasia (A-T). However, the molecular mechanism underlying defective mitophagy in A-T is unknown. Literature indicates that damaged mitochondria are transported to the perinuclear region prior to their removal via mitophagy. Our previous work has indicated that conjugation of SUMO2 (Small Ubiquitin-like Modifier 2) to mitofusins (Mfns) may be necessary for congression of mitochondria into SUMO2-/ubiquitin-/LC3-positive compact structures resembling mito-aggresomes at the perinuclear region in CCCP-treated HEK293 cells. Here, we demonstrate that Mfns are SUMOylated, and mitochondria are transported to the perinuclear region; however, mitochondria fail to congress into mito-aggresome-like structures in CCCP-treated A-T cells. Defect in mitochondrial congression is causally related to constitutively elevated ISG15 (Interferon-Stimulated Gene 15), an antagonist of the ubiquitin pathway, in A-T cells. Suppression of the ISG15 pathway restores mitochondrial congression, reduce oxidative stress, and level of unhealthy mitochondria, which is suggestive of restoration of mitophagy in A-T cells. ISG15 is also constitutively elevated and mitophagy is defective in Amytrophic Lateral Sclerosis (ALS). The constitutively elevated ISG15 pathway therefore appears to be a common unifying biochemical mechanism underlying defective mitophagy in neurodegenerative disorders thus, implying the broader significance of our findings, and suggest the potential role of ISG15 inhibitors in their treatment.
    Keywords:  26S proteasome; Ataxia Telangiectasia; Autophagy; Interferon-Stimulated Gene 15 (ISG15); Mitofusin; Mitophagy; Ubiquitin
  27. Oxid Med Cell Longev. 2021 ;2021 8811935
      Acute pancreatitis (AP) is a common disorder with significant hospital admission and mortality. Due to the unclarified pathological mechanism, there is still no effective and specific treatment for AP. Recently, autophagy has been found to be closely related with occurrence and development of AP, which is crucial in determining its severity and outcomes. Emerging evidence indicates that autophagy can be regulated and influenced by microRNAs and organelles, including mitochondria, endoplasmic reticulum and lysosome, through various ways in AP. Of note, the complex interplays and close relationships among autophagy, microRNA and organelles in AP are vital for figuring out pathogenesis but not clear yet. Thus, this review summarizes the role of autophagy in the pathological mechanism of AP, especially the relationship between impaired autophagy and organelles, and discusses the regulatory mechanism of microRNA on autophagy, which could offer new insights into understanding the pathogenesis of AP and developing new potential therapeutic targets against AP.
  28. Cancer Med. 2021 Feb 24.
      Tumor cell-intrinsic programmed death-ligand 1 (PD-L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. We show here that BC cell-intrinsic PD-L1 signals in mouse MB49 and human RT4, UM-UC3, and UM-UC-14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α-PD-L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell-intrinsic PD-L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune-independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell-intrinsic PD-L1 signals also promoted basal and stress-induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell-intrinsic PD-L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis-platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell-intrinsic PD-L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof-of-concept, we showed that the autophagy inhibitor chloroquine improved cis-platinum treatment efficacy in vivo, with greater efficacy in PD-L1 null versus PD-L1-replete BC.
    Keywords:  PD-L1; autophagy; bladder cancer; chemotherapy; mTOR
  29. Front Aging Neurosci. 2020 ;12 630743
      Brain aging is characterized by a time-dependent decline of tissue integrity and function, and it is a major risk for neurodegenerative diseases and brain cancer. Chaperone-mediated autophagy (CMA) is a selective form of autophagy specialized in protein degradation, which is based on the individual translocation of a cargo protein through the lysosomal membrane. Regulation of processes such as proteostasis, cellular energetics, or immune system activity has been associated with CMA, indicating its pivotal role in tissue homeostasis. Since first studies associating Parkinson's disease (PD) to CMA dysfunction, increasing evidence points out that CMA is altered in both physiological and pathological brain aging. In this review article, we summarize the current knowledge regarding the impact of CMA during aging in brain physiopathology, highlighting the role of CMA in neurodegenerative diseases and glioblastoma, the most common and aggressive brain tumor in adults.
    Keywords:  CMA; LAMP2; glioblastoma; neurodegenerative diseases; physiological aging
  30. Plant Cell. 2020 Dec 03. pii: koaa022. [Epub ahead of print]
      During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity.
  31. Biochim Biophys Acta Gen Subj. 2021 Feb 23. pii: S0304-4165(21)00044-1. [Epub ahead of print] 129886
      BACKGROUND: In most sexually reproducing organisms, mitochondrial DNA (mtDNA) is inherited maternally.SCOPE OF REVIEW: In this review, we summarise recent knowledge on how paternal mitochondria and their mtDNA are selectively eliminated from embryos.
    MAJOR CONCLUSIONS: Studies based on Caenorhabditis elegans have revealed that paternal mitochondria and their mtDNA are selectively degraded in embryos via mitophagy. Thus, mitophagy functions as the mechanisms of maternal inheritance of mtDNA. The mitophagy of paternal mitochondria is conserved in other species, and the underlying molecular mechanisms have begun to be elucidated. In addition to mitophagy, autophagy-independent digestion of paternal mtDNA before and after fertilization serves as another mechanism for maternal inheritance of mtDNA.
    GENERAL SIGNIFICANCE: Maternal inheritance of mtDNA is strictly controlled via multistep mechanisms. These studies also demonstrate a physiological role of mitophagy during animal development.
    Keywords:  Allophagy; Fertilization; Maternal inheritance; Mitochondria; Mitochondrial DNA; Mitophagy
  32. Cell Mol Life Sci. 2021 Feb 23.
      Preservation of mitochondrial quality is paramount for cellular homeostasis. The integrity of mitochondria is guarded by the balanced interplay between anabolic and catabolic mechanisms. The removal of bio-energetically flawed mitochondria is mediated by the process of mitophagy; the impairment of which leads to the accumulation of defective mitochondria which signal the activation of compensatory mechanisms to the nucleus. This process is known as the mitochondrial retrograde response (MRR) and is enacted by Reactive Oxygen Species (ROS), Calcium (Ca2+), ATP, as well as imbalanced lipid and proteostasis. Central to this mitochondria-to-nucleus signalling are the transcription factors (e.g. the nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB) which drive the expression of genes to adapt the cell to the compromised homeostasis. An increased degree of cellular proliferation is among the consequences of the MRR and as such, engagement of mitochondrial-nuclear communication is frequently observed in cancer. Mitophagy and the MRR are therefore interlinked processes framed to, respectively, prevent or compensate for mitochondrial defects.In this review, we discuss the available knowledge on the interdependency of these processes and their contribution to cell signalling in cancer.
    Keywords:  Cell signalling and Cancer; Mitochondrial retrograde response; Mitophagy
  33. Int J Biol Sci. 2021 ;17(2): 390-401
      Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by phosphorylated TDP-43 (pTDP-43)-positive inclusions in neurons and glial cells. However, the pathogenic mechanism that underlies ALS remains largely unknown. To investigate the effects of autophagy deficiency in the formation and spreading of pathological TDP-43 along corticospinal tract axons, TDP-43 preformed fibrils (PFFs) were prepared and unilaterally injected into the fifth layer of the left primary motor cortex (M1) or the left anterior horn of the seventh cervical spinal cord segment (C7) of Atg5+/- mice. After the injection of TDP-43 PFFs, the elevated levels of pTDP-43 were present in several pyramidal tract-associated regions of Atg5+/- mice. Additionally, the occurrence of spontaneous potentials detected by electromyography demonstrates evidence of lower motor neuron dysfunction in M1-TDP-43 PFFs-injected Atg5+/- mice, and prolonged central motor conduction time detected by motor evoked potentials provides evidence of upper motor neuron dysfunction in C7-TDP-43 PFFs-injected Atg5+/- mice. These results show that injection of TDP-43 PFFs into the M1 or C7 of Atg5+/- mice induces the spreading of pathological TDP-43 along corticospinal tract axons in both an anterograde and retrograde manner. Importantly, TDP-43 PFFs-injected Atg5+/- mice also display ALS-like motor dysfunction. Taken together, our findings provide direct evidence that TDP-43 PFFs-injected Atg5+/- mice exhibited ALS-like neuropathology and motor phenotypes, suggesting that autophagy deficiency promotes the formation and spreading of pathological TDP-43 in vivo.
    Keywords:  Atg5+/- mice; TAR DNA-binding protein 43 (TDP-43); amyotrophic lateral sclerosis (ALS); autophagy; preformed fibrils (PFFs)