bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒08‒29
38 papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.
  2. Progranulin associates with Rab2 and is involved in autophagosome-lysosome fusion in Gaucher disease.
  3. Lysosomal agents inhibit store-operated Ca2+ entry.
  4. Alkaline intracellular pHi (pHi) activates AMPK-mTORC2 signaling to promote cell survival during growth factor limitation.
  5. Microglial lysosome dysfunction contributes to white matter pathology and TDP-43 proteinopathy in GRN-associated FTD.
  6. The Yoga of Rag GTPases: Dynamic structural poses confer amino acid sensing by mTORC1.
  7. Cordycepin inhibits cell senescence by ameliorating lysosomal dysfunction and inducing autophagy through the AMPK and mTOR-p70S6K pathway.
  8. mTORC1 Promotes ARID1A Degradation and Oncogenic Chromatin Remodeling in Hepatocellular Carcinoma.
  9. NDST3 deacetylates α-tubulin and suppresses V-ATPase assembly and lysosomal acidification.
  10. Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays.
  11. The role of Niemann-Pick type C2 in zebrafish embryonic development.
  12. LC3B phosphorylation: autophagosome's ticket for a ride toward the cell nucleus.
  13. Myelin Defects in Niemann-Pick Type C Disease: Mechanisms and Possible Therapeutic Perspectives.
  14. Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism.
  15. The Energy Status of Astrocytes Is the Achilles' Heel of eIF2B-Leukodystrophy.
  16. Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo.
  17. Isolation of autophagic fractions from mouse liver for biochemical analyses.
  18. Proteomic Analysis of Niemann-Pick Type C Hepatocytes Reveals Potential Therapeutic Targets for Liver Damage.
  19. Target of Rapamycin Complex 1 (TORC1), Protein Kinase A (PKA) and Cytosolic pH Regulate a Transcriptional Circuit for Lipid Droplet Formation.
  20. Endolysosome and autophagy dysfunction in Alzheimer disease.
  21. Glucocerebrosidase mutations: A paradigm for neurodegeneration pathways.
  22. CLN6 deficiency causes selective changes in the lysosomal protein composition.
  23. Targeting Lysosomes to Reverse Hydroquinone-Induced Autophagy Defects and Oxidative Damage in Human Retinal Pigment Epithelial Cells.
  24. Catabolism of lysosome-related organelles in color-changing spiders supports intracellular turnover of pigments.
  25. Release of Iron-Loaded Ferritin in Sodium Iodate-Induced Model of Age Related Macular Degeneration: An In-Vitro and In-Vivo Study.
  26. Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans.
  27. Genetic reduction of mTOR extends lifespan in a mouse model of Hutchinson-Gilford Progeria syndrome.
  28. AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.
  29. Cathepsin L, a Target of Hypoxia-Inducible Factor-1-α, Is Involved in Melanosome Degradation in Melanocytes.
  30. Enhanced Activity of Exportin-1/CRM1 in Neurons Contributes to Autophagy Dysfunction and Senescent Features in Old Mouse Brain.
  31. Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments.
  32. Role of Nuclear Factor of Activated T Cells (NFAT) Pathway in Regulating Autophagy and Inflammation in Retinal Pigment Epithelial Cells.
  33. Phosphorylation of Androgen Receptor by mTORC1 Promotes Liver Steatosis and Tumorigenesis.
  34. C5orf51 is a component of the MON1-CCZ1 complex and controls RAB7A localization and stability during mitophagy.
  35. Epigenetic inactivation of the autophagy-lysosomal system in appendix in Parkinson's disease.
  36. Loss of tyrosine catabolic enzyme HPD promotes glutamine anaplerosis through mTOR signaling in liver cancer.
  37. Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.
  38. High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins.
  1. Cell. 2021 Aug 25. pii: S0092-8674(21)00944-2. [Epub ahead of print]
    Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.
    Todd Logan, Matthew J Simon, Anil Rana, Gerald M Cherf, Ankita Srivastava, Sonnet S Davis, Ray Lieh Yoon Low, Chi-Lu Chiu, Meng Fang, Fen Huang, Akhil Bhalla, Ceyda Llapashtica, Rachel Prorok, Michelle E Pizzo, Meredith E K Calvert, Elizabeth W Sun, Jennifer Hsiao-Nakamoto, Yashas Rajendra, Katrina W Lexa, Devendra B Srivastava, Bettina van Lengerich, Junhua Wang, Yaneth Robles-Colmenares, Do Jin Kim, Joseph Duque, Melina Lenser, Timothy K Earr, Hoang Nguyen, Roni Chau, Buyankhishig Tsogtbaatar, Ritesh Ravi, Lukas L Skuja, Hilda Solanoy, Howard J Rosen, Bradley F Boeve, Adam L Boxer, Hilary W Heuer, Mark S Dennis, Mihalis S Kariolis, Kathryn M Monroe, Laralynne Przybyla, Pascal E Sanchez, Rene Meisner, Dolores Diaz, Kirk R Henne, Ryan J Watts, Anastasia G Henry, Kannan Gunasekaran, Giuseppe Astarita, Jung H Suh, Joseph W Lewcock, Sarah L DeVos, Gilbert Di Paolo.
      GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
    Keywords:  GBA; LBPA; galectin-3; lipidomics; lipids; lipofuscin; lysobisphosphatidic acid; lysosome; metabolomics; neurodegenerative disease
    DOI:  https://doi.org/10.1016/j.cell.2021.08.002
  2. J Mol Med (Berl). 2021 Aug 27.
    Progranulin associates with Rab2 and is involved in autophagosome-lysosome fusion in Gaucher disease.
    Xiangli Zhao, Rossella Liberti, Jinlong Jian, Wenyu Fu, Aubryanna Hettinghouse, Ying Sun, Chuan-Ju Liu.
      Progranulin (PGRN) is a key regulator of lysosomes, and its deficiency has been linked to various lysosomal storage diseases (LSDs), including Gaucher disease (GD), one of the most common LSD. Here, we report that PGRN plays a previously unrecognized role in autophagy within the context of GD. PGRN deficiency is associated with the accumulation of LC3-II and p62 in autophagosomes of GD animal model and patient fibroblasts, resulting from the impaired fusion of autophagosomes and lysosomes. PGRN physically interacted with Rab2, a critical molecule in autophagosome-lysosome fusion. Additionally, a fragment of PGRN containing the Grn E domain was required and sufficient for binding to Rab2. Furthermore, this fragment significantly ameliorated PGRN deficiency-associated impairment of autophagosome-lysosome fusion and autophagic flux. These findings not only demonstrate that PGRN is a crucial mediator of autophagosome-lysosome fusion but also provide new evidence indicating PGRN's candidacy as a molecular target for modulating autophagy in GD and other LSDs in general. KEY MESSAGES : PGRN acts as a crucial factor involved in autophagosome-lysosome fusion in GD. PGRN physically interacts with Rab2, a molecule in autophagosome-lysosome fusion. A 15-kDa C-terminal fragment of PGRN is required and sufficient for binding to Rab2. This PGRN derivative ameliorates PGRN deficiency-associated impairment of autophagy. This study provides new insights into autophagy and may develop novel therapy for GD.
    Keywords:  Autophagosome-lysosome fusion; Autophagy; Gaucher disease; Progranulin; Rab2
    DOI:  https://doi.org/10.1007/s00109-021-02127-6
  3. J Cell Sci. 2021 Jan 15. pii: jcs248658. [Epub ahead of print]134(2):
    Lysosomal agents inhibit store-operated Ca2+ entry.
    Anthony J Morgan, Antony Galione.
      Pharmacological manipulation of lysosome membrane integrity or ionic movements is a key strategy for probing lysosomal involvement in cellular processes. However, we have found an unexpected inhibition of store-operated Ca2+ entry (SOCE) by these agents. Dipeptides [glycyl-L-phenylalanine 2-naphthylamide (GPN) and L-leucyl-L-leucine methyl ester] that are inducers of lysosomal membrane permeabilization (LMP) uncoupled endoplasmic reticulum Ca2+-store depletion from SOCE by interfering with Stim1 oligomerization and/or Stim1 activation of Orai. Similarly, the K+/H+ ionophore, nigericin, that rapidly elevates lysosomal pH, also inhibited SOCE in a Stim1-dependent manner. In contrast, other strategies for manipulating lysosomes (bafilomycin A1, lysosomal re-positioning) had no effect upon SOCE. Finally, the effects of GPN on SOCE and Stim1 was reversed by a dynamin inhibitor, dynasore. Our data show that lysosomal agents not only release Ca2+ from stores but also uncouple this release from the normal recruitment of Ca2+ influx.
    Keywords:  Ca2+; GPN; LMP; Lysosome; Orai1; Stim1
    DOI:  https://doi.org/10.1242/jcs.248658
  4. J Biol Chem. 2021 Aug 18. pii: S0021-9258(21)00903-0. [Epub ahead of print] 101100
    Alkaline intracellular pHi (pHi) activates AMPK-mTORC2 signaling to promote cell survival during growth factor limitation.
    D Kazyken, S I Lentz, D C Fingar.
      mTOR complex 2 (mTORC2) signaling controls cell metabolism, promotes cell survival, and contributes to tumorigenesis, yet its upstream regulation remains poorly defined. While considerable evidence supports the prevailing view that amino acids activate mTOR complex 1 (mTORC1) but not mTORC2, several studies reported paradoxical activation of mTORC2 signaling by amino acids. We noted that after amino acid starvation of cells in culture, addition of an amino acid solution increased mTORC2 signaling. Interestingly, we found the pH of the amino acid solution to be alkaline, ∼pH 10. These observations led us to discover and demonstrate here that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF1-AM) coupled with live-cell imaging, we demonstrate that culturing cells in media at alkaline pH induces a rapid rise in pHi, which increases mTORC2 catalytic activity and downstream signaling to the pro-growth and -survival kinase Akt. Alkaline pHi also activates AMPK, a canonical sensor of energetic stress. Functionally, alkaline pHi attenuates mTOR- and AMPK-mediated apoptosis caused by growth factor withdrawal. Collectively, these findings reveal that alkaline pHi increases mTORC2- and AMPK-mediated signaling to promote cell survival during conditions of growth factor limitation, analogous to the demonstrated ability of energetic stress to activate AMPK-mTORC2 and promote cell survival. As elevated pHi represents an under-appreciated hallmark of cancer cells, we propose that alkaline pHi stress sensing by AMPK-mTORC2 may contribute to tumorigenesis by enabling cancer cells at the core of a growing tumor to evade apoptosis and survive.
    Keywords:  AMPK; Akt; intracellular pH (pHi); mTORC2
    DOI:  https://doi.org/10.1016/j.jbc.2021.101100
  5. Cell Rep. 2021 Aug 24. pii: S2211-1247(21)01015-9. [Epub ahead of print]36(8): 109581
    Microglial lysosome dysfunction contributes to white matter pathology and TDP-43 proteinopathy in GRN-associated FTD.
    Yanwei Wu, Wei Shao, Tiffany W Todd, Jimei Tong, Mei Yue, Shunsuke Koga, Monica Castanedes-Casey, Ariston L Librero, Chris W Lee, Ian R Mackenzie, Dennis W Dickson, Yong-Jie Zhang, Leonard Petrucelli, Mercedes Prudencio.
      Loss-of-function mutations in the progranulin gene (GRN), which encodes progranulin (PGRN), are a major cause of frontotemporal dementia (FTD). GRN-associated FTD is characterized by TDP-43 inclusions and neuroinflammation, but how PGRN loss causes disease remains elusive. We show that Grn knockout (KO) mice have increased microgliosis in white matter and an accumulation of myelin debris in microglial lysosomes in the same regions. Accumulation of myelin debris is also observed in white matter of patients with GRN-associated FTD. In addition, our findings also suggest that PGRN insufficiency in microglia leads to impaired lysosomal-mediated clearance of myelin debris. Finally, Grn KO mice that are deficient in cathepsin D (Ctsd), a key lysosomal enzyme, have augmented myelin debris and increased neuronal TDP-43 pathology. Together, our data strongly imply that PGRN loss affects microglial activation and lysosomal function, resulting in the accumulation of myelin debris and contributing to TDP-43 pathology.
    Keywords:  FTD; GRN; PGRN; TDP-43; granulin; lysosome; microglia; myelin; progranulin; white matter
    DOI:  https://doi.org/10.1016/j.celrep.2021.109581
  6. J Biol Chem. 2021 Aug 19. pii: S0021-9258(21)00906-6. [Epub ahead of print] 101103
    The Yoga of Rag GTPases: Dynamic structural poses confer amino acid sensing by mTORC1.
    Diane C Fingar.
      Heterodimeric Rag GTPases play a critical role in relaying fluctuating levels of cellular amino acids to the sensor mTOR complex 1 (mTORC1). Important mechanistic questions remain unresolved, however, regarding how guanine nucleotide binding enables Rag GTPases to transition dynamically between distinct Yoga-like structural poses that control activation state. Egri et al. identify a critical interdomain hydrogen bond within RagA and RagC that stabilizes their GDP-bound states. They demonstrate that this long-distance interaction controls Rag structure and function to confer appropriate amino acid sensing by mTORC1.
    DOI:  https://doi.org/10.1016/j.jbc.2021.101103
  7. FEBS Open Bio. 2021 Aug 27.
    Cordycepin inhibits cell senescence by ameliorating lysosomal dysfunction and inducing autophagy through the AMPK and mTOR-p70S6K pathway.
    Shi Qi Zuo, Can Li, Yi Lun Liu, Yue Hao Tan, Xing Wan, Tian Xu, Qiang Li, Li Wang, Yong Li Wu, Feng Mei Deng, Bin Tang.
      Cell senescence is closely related to autophagy. In this article, we identified a natural nucleoside analogue, cordycepin, that has the ability to significantly improve lysosomal function, enhance the activity of the lysosomal representative protease cathepsin B (CTSB), and promote the expression of the functional protein lysosomal-associated membrane protein 2 (LAMP2) on the lysosomal membrane. Cordycepin then restores the damaged autophagy level of aging cells by activating the classic AMPK and mTOR-p70S6K signaling pathways, thus inhibiting cell senescence in an H2 O2 -induced stress-induced premature senescence (SIPS) cell model. This study provides new theoretical support for the further development of cordycepin and clinical antiaging drugs to inhibit cell senescence and suggests that the regulatory mechanisms of lysosomes in senescent cells should be considered when treating age-related diseases.
    Keywords:  AMPK signaling pathways; autophagy; cell senescence; cordycepin; lysosomal function; lysosomal protease
    DOI:  https://doi.org/10.1002/2211-5463.13263
  8. Cancer Res. 2021 Aug 24. pii: canres.0206.2021. [Epub ahead of print]
    mTORC1 Promotes ARID1A Degradation and Oncogenic Chromatin Remodeling in Hepatocellular Carcinoma.
    Shanshan Zhang, Yu-Feng Zhou, Jian Cao, Stephen K Burley, Hui-Yun Wang, X F Steven Zheng.
      The SWI/SNF chromatin remodeling complexes control accessibility of chromatin to transcriptional and co-regulatory machineries. Chromatin remodeling plays important roles in normal physiology and diseases, particularly cancer. The ARID1A-containing SWI/SNF complex is commonly mutated and thought to be a key tumor suppressor in hepatocellular carcinoma (HCC), but its regulation in response to oncogenic signals remains poorly understood. mTOR is a conserved central controller of cell growth and an oncogenic driver of HCC. Remarkably, cancer mutations in mTOR and SWI/SNF complex are mutually exclusive in human HCC tumors, suggesting that they share a common oncogenic function. Here we report that mTOR complex 1 (mTORC1) interact with ARID1A and regulates ubiquitination and proteasomal degradation of ARID1A protein. The mTORC1-ARID1A axis promoted oncogenic chromatin remodeling and YAP-dependent transcription, thereby enhancing liver cancer cell growth in vitro and tumor development in vivo. Conversely, excessive ARID1A expression counteracted AKT-driven liver tumorigenesis in vivo. Moreover, dysregulation of this axis conferred resistance to mTOR-targeted therapies. These findings demonstrate that the ARID1A-SWI/SNF complex is a regulatory target for oncogenic mTOR signaling, which is important for mTORC1-driven hepatocarcinogenesis with implications for therapeutic interventions in HCC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-0206
  9. EMBO J. 2021 Aug 25. e107204
    NDST3 deacetylates α-tubulin and suppresses V-ATPase assembly and lysosomal acidification.
    Qing Tang, Mingming Liu, Yang Liu, Ran-Der Hwang, Tao Zhang, Jiou Wang.
      Lysosomes are key organelles maintaining cellular homeostasis in health and disease. Here, we report the identification of N-deacetylase and N-sulfotransferase 3 (NDST3) as a potent regulator of lysosomal functions through an unbiased genetic screen. NDST3 constitutes a new member of the histone deacetylase (HDAC) family and catalyzes the deacetylation of α-tubulin. Loss of NDST3 promotes assembly of the V-ATPase holoenzyme on the lysosomal membrane and thereby increases the acidification of the organelle. NDST3 is downregulated in tissues and cells from patients carrying the C9orf72 hexanucleotide repeat expansion linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Deficiency in C9orf72 decreases the level of NDST3, and downregulation of NDST3 exacerbates the proteotoxicity of poly-dipeptides generated from the C9orf72 hexanucleotide repeats. These results demonstrate a previously unknown regulatory mechanism through which microtubule acetylation regulates lysosomal activities and suggest that NDST3 could be targeted to modulate microtubule and lysosomal functions in relevant diseases.
    Keywords:  N-deacetylase and N-sulfotransferase 3; V-ATPase assembly; amyotrophic lateral sclerosis; lysosomal acidification; microtubule acetylation
    DOI:  https://doi.org/10.15252/embj.2020107204
  10. Int J Mol Sci. 2021 Aug 16. pii: 8766. [Epub ahead of print]22(16):
    Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays.
    Raef Shams, Yoshihiro Ito, Hideyuki Miyatake.
      The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB-GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein-protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60-167) or M-heat domain (∆M, aa 967-1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240-1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148-2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.
    Keywords:  G-Protein; RHEB; allosteric activation; binding kinetics; kinase domain; mTORC1
    DOI:  https://doi.org/10.3390/ijms22168766
  11. Development. 2021 Apr 01. pii: dev194258. [Epub ahead of print]148(7):
    The role of Niemann-Pick type C2 in zebrafish embryonic development.
    Wei-Chia Tseng, Ana J Johnson Escauriza, Chon-Hwa Tsai-Morris, Benjamin Feldman, Ryan K Dale, Christopher A Wassif, Forbes D Porter.
      Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.
    Keywords:  Cholesterol; Niemann–Pick type C; Niemann–Pick type C2; Notch3; Npc2; Zebrafish
    DOI:  https://doi.org/10.1242/dev.194258
  12. Autophagy. 2021 Aug 23. 1-3
    LC3B phosphorylation: autophagosome's ticket for a ride toward the cell nucleus.
    Jose L Nieto-Torres, Sandra E Encalada, Malene Hansen.
      Macroautophagy/autophagy is a multi-step process that leads to cargo degradation via the fusion of hydrolases-containing lysosomes with cargo-loaded autophagosomes. For this process to occur, autophagosomes are directionally transported by molecular motors toward the nucleus, where they fuse with lysosomes for cargo degradation. The molecular basis for this regulation, including the cell machinery required for this directional transport, has not been fully identified. Using a combination of proteomic and live-imaging approaches in mammalian cells, including primary neurons, we describe that the phosphorylation of the autophagosome protein Atg8/LC3B by the Hippo kinase STK4/MST1, an event we previously reported to be required for autophagy completion, reduces the binding of the transport-related protein FYCO1 to MAP1LC3B/LC3B. This event in turn allows the proficient microtubule-based transport of autophagosomes toward the perinuclear area, thus facilitating the contact of autophagosomes with lysosomes. In the absence of LC3B phosphorylation, autophagosomes undergo aberrant transport including increased movement toward the cell periphery resulting in reduced autophagosome-lysosome colocalization. Thus, LC3B phosphorylation modulates the directional transport of autophagosomes to meet with lysosomes in the perinuclear area, a crucial event in ensuring autophagic degradation of cargo.
    Keywords:  Atg8; FYCO1; LC3B; STK4; MST1; autophagosome transport; phosphorylation
    DOI:  https://doi.org/10.1080/15548627.2021.1961073
  13. Int J Mol Sci. 2021 Aug 17. pii: 8858. [Epub ahead of print]22(16):
    Myelin Defects in Niemann-Pick Type C Disease: Mechanisms and Possible Therapeutic Perspectives.
    Antonietta Bernardo, Chiara De Nuccio, Sergio Visentin, Alberto Martire, Luisa Minghetti, Patrizia Popoli, Antonella Ferrante.
      Niemann-Pick type C (NPC) disease is a wide-spectrum clinical condition classified as a neurovisceral disorder affecting mainly the liver and the brain. It is caused by mutations in one of two genes, NPC1 and NPC2, coding for proteins located in the lysosomes. NPC proteins are deputed to transport cholesterol within lysosomes or between late endosome/lysosome systems and other cellular compartments, such as the endoplasmic reticulum and plasma membrane. The first trait of NPC is the accumulation of unesterified cholesterol and other lipids, like sphingosine and glycosphingolipids, in the late endosomal and lysosomal compartments, which causes the blockade of autophagic flux and the impairment of mitochondrial functions. In the brain, the main consequences of NPC are cerebellar neurodegeneration, neuroinflammation, and myelin defects. This review will focus on myelin defects and the pivotal importance of cholesterol for myelination and will offer an overview of the molecular targets and the pharmacological strategies so far proposed, or an object of clinical trials for NPC. Finally, it will summarize recent data on a new and promising pharmacological perspective involving A2A adenosine receptor stimulation in genetic and pharmacological NPC dysmyelination models.
    Keywords:  A2AR; Niemann–Pick type C disease; adenosine; cholesterol; mitochondrial impairment; myelination; oligodendrocytes
    DOI:  https://doi.org/10.3390/ijms22168858
  14. Int J Mol Sci. 2021 Aug 20. pii: 9001. [Epub ahead of print]22(16):
    Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism.
    Bernadette Breiden, Konrad Sandhoff.
      Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed.
    Keywords:  ASM inhibitors; Niemann-Pick disease; acid sphingomyelinase deficiency (ASMD); depression; lipid accumulation; lysosomal phospholipase C; membrane lipids; regulation; sphingomyelin; topology
    DOI:  https://doi.org/10.3390/ijms22169001
  15. Cells. 2021 Jul 22. pii: 1858. [Epub ahead of print]10(8):
    The Energy Status of Astrocytes Is the Achilles' Heel of eIF2B-Leukodystrophy.
    Melisa Herrero, Maron Daw, Andrea Atzmon, Orna Elroy-Stein.
      Translation initiation factor 2B (eIF2B) is a master regulator of global protein synthesis in all cell types. The mild genetic Eif2b5(R132H) mutation causes a slight reduction in eIF2B enzymatic activity which leads to abnormal composition of mitochondrial electron transfer chain complexes and impaired oxidative phosphorylation. Previous work using primary fibroblasts isolated from Eif2b5(R132H/R132H) mice revealed that owing to increased mitochondrial biogenesis they exhibit normal cellular ATP level. In contrast to fibroblasts, here we show that primary astrocytes isolated from Eif2b5(R132H/R132H) mice are unable to compensate for their metabolic impairment and exhibit chronic state of low ATP level regardless of extensive adaptation efforts. Mutant astrocytes are hypersensitive to oxidative stress and to further energy stress. Moreover, they show migration deficit upon exposure to glucose starvation. The mutation in Eif2b5 prompts reactive oxygen species (ROS)-mediated inferior ability to stimulate the AMP-activated protein kinase (AMPK) axis, due to a requirement to increase the mammalian target of rapamycin complex-1 (mTORC1) signalling in order to enable oxidative glycolysis and generation of specific subclass of ROS-regulating proteins, similar to cancer cells. The data disclose the robust impact of eIF2B on metabolic and redox homeostasis programs in astrocytes and point at their hyper-sensitivity to mutated eIF2B. Thereby, it illuminates the central involvement of astrocytes in Vanishing White Matter Disease (VWMD), a genetic neurodegenerative leukodystrophy caused by homozygous hypomorphic mutations in genes encoding any of the 5 subunits of eIF2B.
    Keywords:  AMPK; ROS; astrocytes; eIF2B-leukodystrophy; energy stress; impaired mitochondria function; mTOR; oxidative stress; translation regulation
    DOI:  https://doi.org/10.3390/cells10081858
  16. Neurobiol Dis. 2021 Aug 19. pii: S0969-9961(21)00236-9. [Epub ahead of print] 105487
    Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo.
    Federica Albanese, Daniela Mercatelli, Luca Finetti, Giulia Lamonaca, Sara Pizzi, Derya R Shimshek, Giovanni Bernacchia, Michele Morari.
      Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 α-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 α-synuclein inclusion in striatal and nigral neurons. No genotype differences in ALP markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/LC3-I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice. LC3B puncta accumulation and pSer129 α-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G2019S knock-in mice. We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy.
    Keywords:  Autophagy; Chaperone-mediated autophagy; Chloroquine; G2019S LRRK2; Glucocerebrosidase; LC3; MLi-2; Parkinson's disease; TFEB; pSer129 α-synuclein
    DOI:  https://doi.org/10.1016/j.nbd.2021.105487
  17. STAR Protoc. 2021 Sep 17. 2(3): 100730
    Isolation of autophagic fractions from mouse liver for biochemical analyses.
    Henrietta Bains, Rajat Singh.
      Isolation of autophagosomes, autolysosomes, and lysosomes allows mechanistic studies into the pathophysiology of autophagy-a lysosomal quality control pathway. Here, we outline a Nycodenz density gradient ultracentrifugation approach for high-yield isolation of autophagic fractions from mouse liver. These fractions can be used for immunoblotting, transmission electron microscopy, and proteomic and lipidomic analyses. For complete details on the use and execution of this protocol, please refer to Toledo et al. (2018).
    Keywords:  cell biology; cell separation/fractionation; metabolism
    DOI:  https://doi.org/10.1016/j.xpro.2021.100730
  18. Cells. 2021 Aug 21. pii: 2159. [Epub ahead of print]10(8):
    Proteomic Analysis of Niemann-Pick Type C Hepatocytes Reveals Potential Therapeutic Targets for Liver Damage.
    Elisa Balboa, Tamara Marín, Juan Esteban Oyarzún, Pablo S Contreras, Robert Hardt, Thea van den Bosch, Alejandra R Alvarez, Boris Rebolledo-Jaramillo, Andres D Klein, Dominic Winter, Silvana Zanlungo.
      Niemann-Pick type C disease (NPCD) is a lysosomal storage disorder caused by mutations in the NPC1 gene. The most affected tissues are the central nervous system and liver, and while significant efforts have been made to understand its neurological component, the pathophysiology of the liver damage remains unclear. In this study, hepatocytes derived from wild type and Npc1-/- mice were analyzed by mass spectrometry (MS)-based proteomics in conjunction with bioinformatic analysis. We identified 3832 proteins: 416 proteins had a p-value smaller than 0.05, of which 37% (n = 155) were considered differentially expressed proteins (DEPs), 149 of them were considered upregulated, and 6 were considered downregulated. We focused the analysis on pathways related to NPC pathogenic mechanisms, finding that the most significant changes in expression levels occur in proteins that function in the pathways of liver damage, lipid metabolism, and inflammation. Moreover, in the group of DEPs, 30% (n = 47) were identified as lysosomal proteins and 7% (n = 10) were identified as mitochondrial proteins. Importantly, we found that lysosomal DEPs, including CTSB/D/Z, LIPA, DPP7 and GLMP, and mitocondrial DEPs, AKR1B10, and VAT1 had been connected with liver fibrosis, damage, and steatosis in previous studies, validiting our dataset. Our study found potential therapeutic targets for the treatment of liver damage in NPCD.
    Keywords:  Niemann Pick type C disease; liver damage; lysosomal storage disorder; mass spectrometry; proteomic analysis
    DOI:  https://doi.org/10.3390/cells10082159
  19. Int J Mol Sci. 2021 Aug 20. pii: 9017. [Epub ahead of print]22(16):
    Target of Rapamycin Complex 1 (TORC1), Protein Kinase A (PKA) and Cytosolic pH Regulate a Transcriptional Circuit for Lipid Droplet Formation.
    Vitor Teixeira, Telma S Martins, William A Prinz, Vítor Costa.
      Lipid droplets (LDs) are ubiquitous organelles that fulfill essential roles in response to metabolic cues. The identification of several neutral lipid synthesizing and regulatory protein complexes have propelled significant advance on the mechanisms of LD biogenesis in the endoplasmic reticulum (ER). However, our understanding of signaling networks, especially transcriptional mechanisms, regulating membrane biogenesis is very limited. Here, we show that the nutrient-sensing Target of Rapamycin Complex 1 (TORC1) regulates LD formation at a transcriptional level, by targeting DGA1 expression, in a Sit4-, Mks1-, and Sfp1-dependent manner. We show that cytosolic pH (pHc), co-regulated by the plasma membrane H+-ATPase Pma1 and the vacuolar ATPase (V-ATPase), acts as a second messenger, upstream of protein kinase A (PKA), to adjust the localization and activity of the major transcription factor repressor Opi1, which in turn controls the metabolic switch between phospholipid metabolism and lipid storage. Together, this work delineates hitherto unknown molecular mechanisms that couple nutrient availability and pHc to LD formation through a transcriptional circuit regulated by major signaling transduction pathways.
    Keywords:  cell signaling; lipid droplet; membrane biogenesis; nutrient; transcription
    DOI:  https://doi.org/10.3390/ijms22169017
  20. Autophagy. 2021 Aug 25. 1-2
    Endolysosome and autophagy dysfunction in Alzheimer disease.
    Christy Hung, Frederick J Livesey.
      Abnormalities of the neuronal endolysosome and macroautophagy/autophagy system are an early and prominent feature of Alzheimer disease (AD). SORL1 is notable as a gene in which mutations are causal for a rare, autosomal dominant form of AD, and also variants that increase the risk of developing the common form of late-onset AD. In our recent study, we used patient-derived stem cells and CRISPR engineering to study the effects of SORL1 mutations on the endolysosome and autophagy system in human forebrain neurons. SORL1 mutations causal for monogenic AD are typically truncating mutations, and we found, using stem cells generated from an individual with dementia due to a heterozygous SORL1 truncation mutation, that this class of mutation results in SORL1 haploinsufficiency. Reducing SORL1 protein by half results in disrupted endosomal trafficking in patient-derived neurons, which we confirmed by studying the endolysosomal system in isogenic CRISPR-engineered SORL1 heterozygous null neurons. We also found that SORL1 homozygous null neurons develop more severe phenotypes, with endosome abnormalities, lysosome dysfunction and defects in the degradative phase of autophagy. Endolysosome and autophagy defects in SORL1 mutant neurons are dependent on APP, a key AD gene, as they are rescued by extracellular antisense oligonucleotides that reduce APP protein.
    Keywords:  Alzheimer’s disease; autophagy; endosome; live-cell imaging; lysosome
    DOI:  https://doi.org/10.1080/15548627.2021.1963630
  21. Free Radic Biol Med. 2021 Aug 24. pii: S0891-5849(21)00473-1. [Epub ahead of print]
    Glucocerebrosidase mutations: A paradigm for neurodegeneration pathways.
    Sophia R L Vieira, Anthony H V Schapira.
      Biallelic (homozygous or compound heterozygous) glucocerebrosidase gene (GBA) mutations cause Gaucher disease, whereas heterozygous mutations are numerically the most important genetic risk factor for Parkinson disease (PD) and are associated with the development of other synucleinopathies, notably Dementia with Lewy Bodies. This phenomenon is not limited to GBA, with converging evidence highlighting further examples of autosomal recessive disease genes increasing neurodegeneration risk in heterozygous mutation carriers. Nevertheless, despite extensive research, the cellular mechanisms by which mutations in GBA, encoding lysosomal enzyme β-glucocerebrosidase (GCase), predispose to neurodegeneration remain incompletely understood. Alpha-synuclein (A-SYN) accumulation, autophagic lysosomal dysfunction, mitochondrial abnormalities, ER stress and neuroinflammation have been proposed as candidate pathogenic pathways in GBA-linked PD. The observation of GCase and A-SYN interactions in PD initiated the development and evaluation of GCase-targeted therapeutics in PD clinical trials.
    Keywords:  Alpha-synuclein; Ambroxol; Autosomal recessive; Dementia with lewy bodies; Gaucher disease; Glucocerebrosidase; Neurodegeneration; Parkinson disease; Synucleinopathies
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.08.230
  22. Proteomics. 2021 Aug 25. e2100043
    CLN6 deficiency causes selective changes in the lysosomal protein composition.
    Andreas Tuermer, Simone Mausbach, Edgar Kaade, Markus Damme, Marc Sylvester, Volkmar Gieselmann, Melanie Thelen.
      Neuronal ceroid lipofuscinoses (NCLs) collectively account for the highest prevalence of inherited neurodegenerative diseases in childhood. This disease group is classified by the deposition of similar autofluorescence storage material in lysosomes that is accompanied by seizures, blindness and premature mortality in later disease stages. Defects in several genes affecting various proteins lead to NCL, one of them being CLN6, a transmembrane protein resident in the endoplasmic reticulum. Dysfunctionality of CLN6 causes variant late infantile NCL (vLINCL). The function of CLN6 and how its deficiency affects lysosomal integrity remains unknown. In this work, we performed a comparative proteomic analysis of isolated lysosomal fractions from liver tissue of nclf mice, a natural mouse model displaying a similar disease course than its human counterpart. We could identify a drastic reduction in the protein amounts of selected lysosomal proteins, amongst them several members of the NCL protein family. Most of these proteins were N-glycosylated, soluble hydrolases and their reduction in protein levels was verified by western blotting and enzymatic assays. Hereby we could directly link Cln6 dysfunction to changes in the lysosomal compartment and to other NCL forms. This article is protected by copyright. All rights reserved.
    Keywords:  Cln6; lysosome; Neuronal ceroid lipofuscinosis
    DOI:  https://doi.org/10.1002/pmic.202100043
  23. Int J Mol Sci. 2021 Aug 22. pii: 9042. [Epub ahead of print]22(16):
    Targeting Lysosomes to Reverse Hydroquinone-Induced Autophagy Defects and Oxidative Damage in Human Retinal Pigment Epithelial Cells.
    Samuel Abokyi, Sze-Wan Shan, Christie Hang-I Lam, Kirk Patrick Catral, Feng Pan, Henry Ho-Lung Chan, Chi-Ho To, Dennis Yan-Yin Tse.
      In age-related macular degeneration (AMD), hydroquinone (HQ)-induced oxidative damage in retinal pigment epithelium (RPE) is believed to be an early event contributing to dysregulation of inflammatory cytokines and vascular endothelial growth factor (VEGF) homeostasis. However, the roles of antioxidant mechanisms, such as autophagy and the ubiquitin-proteasome system, in modulating HQ-induced oxidative damage in RPE is not well-understood. This study utilized an in-vitro AMD model involving the incubation of human RPE cells (ARPE-19) with HQ. In comparison to hydrogen peroxide (H2O2), HQ induced fewer reactive oxygen species (ROS) but more oxidative damage as characterized by protein carbonyl levels, mitochondrial dysfunction, and the loss of cell viability. HQ blocked the autophagy flux and increased proteasome activity, whereas H2O2 did the opposite. Moreover, the lysosomal membrane-stabilizing protein LAMP2 and cathepsin D levels declined with HQ exposure, suggesting loss of lysosomal membrane integrity and function. Accordingly, HQ induced lysosomal alkalization, thereby compromising the acidic pH needed for optimal lysosomal degradation. Pretreatment with MG132, a proteasome inhibitor and lysosomal stabilizer, upregulated LAMP2 and autophagy and prevented HQ-induced oxidative damage in wildtype RPE cells but not cells transfected with shRNA against ATG5. This study demonstrated that lysosomal dysfunction underlies autophagy defects and oxidative damage induced by HQ in human RPE cells and supports lysosomal stabilization with the proteasome inhibitor MG132 as a potential remedy for oxidative damage in RPE and AMD.
    Keywords:  age-related macular degeneration; autophagy; hydroquinone; lysosomal alkalization; oxidative stress; ubiquitin-proteasome system (UPS)
    DOI:  https://doi.org/10.3390/ijms22169042
  24. Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2103020118. [Epub ahead of print]118(35):
    Catabolism of lysosome-related organelles in color-changing spiders supports intracellular turnover of pigments.
    Florent Figon, Ilse Hurbain, Xavier Heiligenstein, Sylvain Trépout, Arnaud Lanoue, Kadda Medjoubi, Andrea Somogyi, Cédric Delevoye, Graça Raposo, Jérôme Casas.
      Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and second, how their catabolism is intracellularly processed. Using scanning transmission electron microscopy, electron tomography, and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.
    Keywords:  endosome; melanosome; mimicry; ommochrome; pigment granule
    DOI:  https://doi.org/10.1073/pnas.2103020118
  25. Antioxidants (Basel). 2021 Aug 05. pii: 1253. [Epub ahead of print]10(8):
    Release of Iron-Loaded Ferritin in Sodium Iodate-Induced Model of Age Related Macular Degeneration: An In-Vitro and In-Vivo Study.
    Ajay Ashok, Suman Chaudhary, Aaron S Wise, Neil A Rana, Dallas McDonald, Alexander E Kritikos, Ewald Lindner, Neena Singh.
      To evaluate the role of iron in sodium iodate (NaIO3)-induced model of age-related macular degeneration (AMD) in ARPE-19 cells in-vitro and in mouse models in-vivo. ARPE-19 cells, a human retinal pigment epithelial cell line, was exposed to 10 mM NaIO3 for 24 h, and the expression and localization of major iron modulating proteins was evaluated by Western blotting (WB) and immunostaining. Synthesis and maturation of cathepsin-D (cat-D), a lysosomal enzyme, was evaluated by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) and WB, respectively. For in-vivo studies, C57BL/6 mice were injected with 40 mg/kg mouse body weight of NaIO3 intraperitoneally, and their retina was evaluated after 3 weeks as above. NaIO3 induced a 10-fold increase in ferritin in ARPE-19 cells, which co-localized with LC3II, an autophagosomal marker, and LAMP-1, a lysosomal marker. A similar increase in ferritin was noted in retinal lysates and retinal sections of NaIO3-injected mice by WB and immunostaining. Impaired synthesis and maturation of cat-D was also noted. Accumulated ferritin was loaded with iron, and released from retinal pigmented epithelial (RPE) cells in Perls' and LAMP-1 positive vesicles. NaIO3 impairs lysosomal degradation of ferritin by decreasing the transcription and maturation of cat-D in RPE cells. Iron-loaded ferritin accumulates in lysosomes and is released in lysosomal membrane-enclosed vesicles to the extracellular milieu. Accumulation of ferritin in RPE cells and fusion of ferritin-containing vesicles with adjacent photoreceptor cells is likely to create an iron overload, compromising their viability. Moreover, reduced activity of cat-D is likely to promote accumulation of other cellular debris in lysosomal vesicles, contributing to AMD-like pathology.
    Keywords:  age-related macular degeneration; exosomes; ferritin; lysosomes; sodium iodate
    DOI:  https://doi.org/10.3390/antiox10081253
  26. Diagnostics (Basel). 2021 Jul 27. pii: 1347. [Epub ahead of print]11(8):
    Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans.
    Nivethitha Arunkumar, Dung Chi Vu, Shaukat Khan, Hironori Kobayashi, Thi Bich Ngoc Can, Tsubasa Oguni, Jun Watanabe, Misa Tanaka, Seiji Yamaguchi, Takeshi Taketani, Yasuhiko Ago, Hidenori Ohnishi, Sampurna Saikia, José V Álvarez, Shunji Tomatsu.
      Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.
    Keywords:  enzyme assay; glycosaminoglycans; mucolipidosis; mucopolysaccharidoses; newborn screening
    DOI:  https://doi.org/10.3390/diagnostics11081347
  27. Aging Cell. 2021 Aug 28. e13457
    Genetic reduction of mTOR extends lifespan in a mouse model of Hutchinson-Gilford Progeria syndrome.
    Wayne A Cabral, Urraca L Tavarez, Indeevar Beeram, Diana Yeritsyan, Yoseph D Boku, Michael A Eckhaus, Ara Nazarian, Michael R Erdos, Francis S Collins.
      Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder most notably characterized by cardiovascular disease and premature death from myocardial infarction or stroke. The majority of cases are caused by a de novo single nucleotide mutation in the LMNA gene that activates a cryptic splice donor site, resulting in production of a toxic form of lamin A with a 50 amino acid internal deletion, termed progerin. We previously reported the generation of a transgenic murine model of progeria carrying a human BAC harboring the common mutation, G608G, which in the single-copy state develops features of HGPS that are limited to the vascular system. Here, we report the phenotype of mice bred to carry two copies of the BAC, which more completely recapitulate the phenotypic features of HGPS in skin, adipose, skeletal, and vascular tissues. We further show that genetic reduction of the mechanistic target of rapamycin (mTOR) significantly extends lifespan in these mice, providing a rationale for pharmacologic inhibition of the mTOR pathway in the treatment of HGPS.
    Keywords:  S6 Kinase; lamin A/C; laminopathies; mTOR; progeria
    DOI:  https://doi.org/10.1111/acel.13457
  28. FEBS Open Bio. 2021 Aug 24.
    AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.
    Seho Cha, Won-Il Seo, Ha-Na Woo, Hee Jong Kim, Steven Hyun Seung Lee, Jin Kim, Jun-Sub Choi, Keerang Park, Joo Yong Lee, Beom Jun Lee, Heuiran Lee.
      Expanding on previous demonstrations of the therapeutic effects of adeno-associated virus (AAV) carrying small-hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)-stimulated endothelial cells were treated with AAV2-shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2-shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HUVECs) and decreased downstream signaling cascades of mTOR complex 1 (mTORC1) and mTORC2 under VEGF treatment. Moreover, the angiogenic potential of VEGF was significantly inhibited by AAV2-shmTOR, which preserved endothelial integrity by maintaining tight junctions between HUVECs. These data thus support previous in vivo studies and provide evidence that AAV2-shmTOR induces therapeutic effects by inhibiting the neovascularization of endothelial cells.
    Keywords:  adeno-associated virus; angiogenesis; endothelial cells; migration; proliferation; retinal vascular disorder; small-hairpin mTOR; tight junction
    DOI:  https://doi.org/10.1002/2211-5463.13281
  29. Int J Mol Sci. 2021 Aug 10. pii: 8596. [Epub ahead of print]22(16):
    Cathepsin L, a Target of Hypoxia-Inducible Factor-1-α, Is Involved in Melanosome Degradation in Melanocytes.
    Ji Young Kim, Eun Jung Lee, Yuri Ahn, Sujin Park, Yu Jeong Bae, Tae Gyun Kim, Sang Ho Oh.
      Hypoxic conditions induce the activation of hypoxia-inducible factor-1α (HIF-1α) to restore the supply of oxygen to tissues and cells. Activated HIF-1α translocates into the nucleus and binds to hypoxia response elements to promote the transcription of target genes. Cathepsin L (CTSL) is a lysosomal protease that degrades cellular proteins via the endolysosomal pathway. In this study, we attempted to determine if CTSL is a hypoxia responsive target gene of HIF-1α, and decipher its role in melanocytes in association with the autophagic pathway. The results of our luciferase reporter assay showed that the expression of CTSL is transcriptionally activated through the binding of HIF1-α at its promoter. Under autophagy-inducing starvation conditions, HIF-1α and CTSL expression is highly upregulated in melan-a cells. The mature form of CTSL is closely involved in melanosome degradation through lysosomal activity upon autophagosome-lysosome fusion. The inhibition of conversion of pro-CTSL to mature CTSL leads to the accumulation of gp100 and tyrosinase in addition to microtubule-associated protein 1 light chain 3 (LC3) II, due to decreased lysosomal activity in the autophagic pathway. In conclusion, we have identified that CTSL, a novel target of HIF-1α, participates in melanosome degradation in melanocytes through lysosomal activity during autophagosome-lysosome fusion.
    Keywords:  Cathepsin L; autophagy; hypoxia-inducible factor-1-alpha; melanosome
    DOI:  https://doi.org/10.3390/ijms22168596
  30. Oxid Med Cell Longev. 2021 ;2021 6682336
    Enhanced Activity of Exportin-1/CRM1 in Neurons Contributes to Autophagy Dysfunction and Senescent Features in Old Mouse Brain.
    Elisa Gorostieta-Salas, Daniel Moreno-Blas, Cristian Gerónimo-Olvera, Bulmaro Cisneros, Felipe A Court, Susana Castro-Obregón.
      Brain aging is characterized by dysfunctional autophagy and cellular senescence, among other features. While autophagy can either promote or suppress cellular senescence in proliferating cells, in postmitotic cells, such as neurons, autophagy impairment promotes cellular senescence. CRM1 (exportin-1/XPO1) exports hundreds of nuclear proteins into the cytoplasm, including the transcription factors TFEB (the main inducer of autophagy and lysosomal biogenesis genes) and STAT3, another autophagy modulator. It appears that CRM1 is a modulator of aging-associated senescence and autophagy, because pharmacological inhibition of CRM1 improved autophagic degradation in flies, by increasing nuclear TFEB levels, and because enhanced CRM1 activity is mechanistically linked to senescence in fibroblasts from Hutchinson-Gilford progeria syndrome patients and old healthy individuals; furthermore, the exogenous overexpression of CRM1 induced senescence in normal fibroblasts. In this work, we tested the hypothesis that impaired autophagic flux during brain aging occurs due to CRM1 accumulation in the brain. We found that CRM1 levels and activity increased in the hippocampus and cortex during physiological aging, which resulted in a decrease of nuclear TFEB and STAT3. Consistent with an autophagic flux impairment, we observed accumulation of the autophagic receptor p62/SQSTM1 in neurons of old mice, which correlated with increased neuronal senescence. Using an in vitro model of neuronal senescence, we demonstrate that CRM1 inhibition improved autophagy flux and reduced SA-β-gal activity by restoring TFEB nuclear localization. Collectively, our data suggest that enhanced CRM1-mediated export of proteins during brain aging perturbs neuronal homeostasis, contributing to autophagy impairment, and neuronal senescence.
    DOI:  https://doi.org/10.1155/2021/6682336
  31. Int J Mol Sci. 2021 Aug 06. pii: 8450. [Epub ahead of print]22(16):
    Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments.
    Yoko Ito, Nicolas Esnay, Louise Fougère, Matthieu Pierre Platre, Fabrice Cordelières, Yvon Jaillais, Yohann Boutté.
      A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant's early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.
    Keywords:  endosomes; multivesicular bodies; phosphatidylinositol-3-phosphate; trans-Golgi network; vacuole; very-long chain fatty acids
    DOI:  https://doi.org/10.3390/ijms22168450
  32. Int J Mol Sci. 2021 Aug 12. pii: 8684. [Epub ahead of print]22(16):
    Role of Nuclear Factor of Activated T Cells (NFAT) Pathway in Regulating Autophagy and Inflammation in Retinal Pigment Epithelial Cells.
    Hsuan-Yeh Pan, Ashley V Ladd, Manas R Biswal, Mallika Valapala.
      Nuclear factor of activated T cells (NFAT) family of transcription factors are substrates of calcineurin and play an important role in integrating Ca2+ signaling with a variety of cellular functions. Of the five NFAT proteins (NFAT1-5), NFAT1-4 are subject to dephosphorylation and activation by calcineurin, a Ca2+-calmodulin-dependent phosphatase. Increased levels of intracellular Ca2+ activates calcineurin, which in turn dephosphorylates and promotes nuclear translocation of NFAT. We investigated the functions of NFAT proteins in the retinal pigment epithelial cells (RPE). Our results show that NFAT-mediated luciferase activity was induced upon treatment with the bacterial endotoxin, lipopolysaccharide (LPS) and treatment with the NFAT peptide inhibitor, MAGPHPVIVITGPHEE (VIVIT) decreased LPS-induced NFAT luciferase activity. LPS-induced activation of NFAT-regulated cytokines (IL-6 and IL-8) is inhibited by treatment of cells with VIVIT. We also investigated the effects of NFAT signaling on the autophagy pathway. Our results show that inhibition of NFAT with VIVIT in cells deprived of nutrients resulted in cytosolic retention of transcription Factor EB (TFEB), decreased expression of TFEB-regulated coordinated Lysosomal Expression and Regulation CLEAR network genes and decreased starvation-induced autophagy flux in the RPE cells. In summary, these studies suggest that the NFAT pathway plays an important role in the regulation of autophagy and inflammation in the RPE.
    Keywords:  autophagy; calcineurin; lysosomal function; nuclear factor of activated in T cells (NFAT); retinal pigment epithelium (RPE); transcription factor EB
    DOI:  https://doi.org/10.3390/ijms22168684
  33. Hepatology. 2021 Aug 26.
    Phosphorylation of Androgen Receptor by mTORC1 Promotes Liver Steatosis and Tumorigenesis.
    Qian-Nan Ren, Hong Zhang, Chao-Yue Sun, Yu-Feng Zhou, Xue-Feng Yang, Jian-Wu Long, Xiao-Xing Li, Shi-Juan Mai, Mei-Yin Zhang, Hui-Zhong Zhang, Hai-Qiang Mai, Min-Shan Chen, X F Steven Zheng, Hui-Yun Wang.
      Androgen receptor (AR) has been reported to play an important role in the development and progression of man's prostate cancer. Hepatocellular carcinoma (HCC) is also male-dominant, but the role of AR in HCC remains poorly understood. Here we show that mTORC1 interacts with hepatic AR and phosphorylates it at S96 in response to nutrient and mitogenic stimuli in HCC cells. S96 phosphorylation promotes the stability, nuclear localization and transcriptional activity of AR, which enhances de novo lipogenesis and proliferation in hepatocytes and induces liver steatosis and hepatocarcinogenesis in mice independently and cooperatively with androgen. Furthermore, high ARS96 phosphorylation is observed in human liver steatotic and HCC tissues and is associated with overall survival and disease-free survival, which has been proven as an independent survival predictor for HCC patients. IN CONCLUSION: AR S96 phosphorylation by mTORC1drives liver steatosis and HCC development and progression independently and cooperatively with androgen, which not only explains why HCC is man-biased but also provides a target molecule for prevention and treatment of HCC and a potential survival predictor in HCC patients.
    Keywords:  Androgen receptor (AR); Fatty acid metabolism; Hepatocellular carcinoma (HCC); Phosphorylation; mTOR
    DOI:  https://doi.org/10.1002/hep.32120
  34. Autophagy. 2021 Aug 25. 1-12
    C5orf51 is a component of the MON1-CCZ1 complex and controls RAB7A localization and stability during mitophagy.
    Bing-Ru Yan, Taoyingnan Li, Etienne Coyaud, Estelle M N Laurent, Jonathan St-Germain, Yuhuan Zhou, Peter K Kim, Brian Raught, John H Brumell.
      Depolarized mitochondria can be degraded via mitophagy, a selective form of autophagy. The RAB GTPase RAB7A was recently shown to play a key role in this process. RAB7A regulates late endocytic trafficking under normal growth conditions but is translocated to the mitochondrial surface following depolarization. However, how RAB7A activity is regulated during mitophagy is not understood. Here, using a proximity-dependent biotinylation approach (miniTurbo), we identified C5orf51 as a specific interactor of GDP-locked RAB7A. C5orf51 also interacts with the RAB7A guanine nucleotide exchange factor (GEF) complex members MON1 and CCZ1. In the absence of C5orf51, localization of RAB7A on depolarized mitochondria is compromised and the protein is degraded by the proteasome. Furthermore, depletion of C5orf51 also inhibited ATG9A recruitment to depolarized mitochondria. Together, these results indicate that C5orf51 is a positive regulator of RAB7A in its shuttling between late endosomes and mitochondria to enable mitophagy.Abbreviations: ATG9A: autophagy related 9A; Baf A1: bafilomycin A1; BioID: proximity-dependent biotin identification; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CCZ1: CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; DQ-BSA: dye quenched-bovine serum albumin; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; KO: knockout; LRPPRC: leucine rich pentatricopeptide repeat containing; MG132: carbobenzoxy-Leu-Leu-leucinal; MON1: MON1 homolog, secretory trafficking associated; mtDNA: mitochondrial DNA; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RMC1: regulator of MON1-CCZ1; TBC1D15: TBC1 domain family member 15; TBC1D17: TBC1 domain family member 17; TOMM20: translocase of outer mitochondrial membrane 20; WDR91: WD repeat domain 91; WT: wild type.
    Keywords:  Autophagy; C5orf51; RAB7A; guanine nucleotide exchange factor; mitophagy
    DOI:  https://doi.org/10.1080/15548627.2021.1960116
  35. Nat Commun. 2021 Aug 26. 12(1): 5134
    Epigenetic inactivation of the autophagy-lysosomal system in appendix in Parkinson's disease.
    Juozas Gordevicius, Peipei Li, Lee L Marshall, Bryan A Killinger, Sean Lang, Elizabeth Ensink, Nathan C Kuhn, Wei Cui, Nazia Maroof, Roberta Lauria, Christina Rueb, Juliane Siebourg-Polster, Pierre Maliver, Jared Lamp, Irving Vega, Fredric P Manfredsson, Markus Britschgi, Viviane Labrie.
      The gastrointestinal tract may be a site of origin for α-synuclein pathology in idiopathic Parkinson's disease (PD). Disruption of the autophagy-lysosome pathway (ALP) may contribute to α-synuclein aggregation. Here we examined epigenetic alterations in the ALP in the appendix by deep sequencing DNA methylation at 521 ALP genes. We identified aberrant methylation at 928 cytosines affecting 326 ALP genes in the appendix of individuals with PD and widespread hypermethylation that is also seen in the brain of individuals with PD. In mice, we find that DNA methylation changes at ALP genes induced by chronic gut inflammation are greatly exacerbated by α-synuclein pathology. DNA methylation changes at ALP genes induced by synucleinopathy are associated with the ALP abnormalities observed in the appendix of individuals with PD specifically involving lysosomal genes. Our work identifies epigenetic dysregulation of the ALP which may suggest a potential mechanism for accumulation of α-synuclein pathology in idiopathic PD.
    DOI:  https://doi.org/10.1038/s41467-021-25474-x
  36. Cell Rep. 2021 Aug 24. pii: S2211-1247(21)01055-X. [Epub ahead of print]36(8): 109617
    Loss of tyrosine catabolic enzyme HPD promotes glutamine anaplerosis through mTOR signaling in liver cancer.
    Man Tong, Tin-Lok Wong, Hongzhi Zhao, Yuanyuan Zheng, Yu-Nong Xie, Cheuk-Hin Li, Lei Zhou, Noélia Che, Jing-Ping Yun, Kwan Man, Terence Kin-Wah Lee, Zongwei Cai, Stephanie Ma.
      The liver plays central roles in coordinating different metabolic processes, such as the catabolism of amino acids. In this study, we identify a loss of tyrosine catabolism and a concomitant increase in serum tyrosine levels during liver cancer development. Liver cells with disordered tyrosine catabolism, as exemplified by the suppression of a tyrosine catabolic enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD), display augmented tumorigenic and proliferative potentials. Metabolomics profiling and isotope tracing reveal the metabolic reliance of HPD-silenced cells on glutamine, coupled with increased tricarboxylic acid cycle metabolites and their associated amino acid pools. Mechanistically, HPD silencing reduces ketone bodies, which regulate the proliferative and metabolic phenotypes via the AMPK/mTOR/p70S6 kinase pathway and mTOR-dependent glutaminase (GLS) activation. Collectively, our results demonstrate a metabolic link between tyrosine and glutamine metabolism, which could be exploited as a potentially promising anticancer therapy for liver cancer.
    Keywords:  HPD; glutamine metabolism; liver cancer; mTOR signaling; tyrosine catabolism
    DOI:  https://doi.org/10.1016/j.celrep.2021.109617
  37. Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2100500118. [Epub ahead of print]118(35):
    Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.
    Vera A van der Weijden, Jochen T Bick, Stefan Bauersachs, Anna B Rüegg, Thomas B Hildebrandt, Frank Goeritz, Katarina Jewgenow, Pieter Giesbertz, Hannelore Daniel, Emilie Derisoud, Pascale Chavatte-Palmer, Rupert M Bruckmaier, Barbara Drews, Susanne E Ulbrich.
      Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.
    Keywords:  European roe deer (Capreolus capreolus); embryo development; embryonic diapause
    DOI:  https://doi.org/10.1073/pnas.2100500118
  38. Methods Mol Biol. 2021 ;2293 27-43
    High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins.
    Ashwini K Mishra, David G Lambright.
      Measurement of intrinsic as well as GTPase-activating Protein (GAP) catalyzed GTP hydrolysis is central to understanding the molecular mechanism and function of GTPases in diverse cellular processes. For the Rab GTPase family, which comprises at least 60 distinct proteins in humans, putative GAPs have been identified from both eukaryotic organisms and pathogenic bacteria. A major obstacle has involved identification of target substrates and determination of the specificity for the Rab family. Here, we describe a sensitive, high-throughput method to quantitatively profile GAP activity for Rab GTPases in microplate format based on detection of inorganic phosphate released after GTP hydrolysis. The method takes advantage of a well-characterized fluorescent phosphate sensor, requires relatively low protein concentrations, and can, in principle, be applied to any GAP-GTPase system.
    Keywords:  GAP assay; GAP reaction; GTP hydrolysis; GTPase; High-throughput; PBP-MDCC; Phosphate; Phosphate-binding protein; Rab GTPase
    DOI:  https://doi.org/10.1007/978-1-0716-1346-7_3
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