bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021–12–19
37 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. CLN7 is an organellar chloride channel regulating lysosomal function.
  2. Efficient progranulin exit from the ER requires its interaction with prosaposin, a Surf4 cargo.
  3. Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.
  4. AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins.
  5. Cryo-EM structure of mouse TRPML2 in lipid nanodiscs.
  6. p53-mediated AKT and mTOR inhibition requires RFX7 and DDIT4 and depends on nutrient abundance.
  7. Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy.
  8. p38 regulates the tumor suppressor PDCD4 via the TSC-mTORC1 pathway.
  9. Rushika M. Perera: Lysosomes unveil what cancer cells hide.
  10. TFEB Dependent Autophagy-Lysosomal Pathway: An Emerging Pharmacological Target in Sepsis.
  11. AAV-mediated gene therapy for galactosialidosis: A long-term safety and efficacy study.
  12. Sterol uptake by the NPC system in eukaryotes: A Saccharomyces cerevisiae perspective.
  13. Mechanisms of Non-Vesicular Exchange of Lipids at Membrane Contact Sites: Of Shuttles, Tunnels and, Funnels.
  14. Automated Assays to Identify Modulators of Transcription Factor EB Translocation and Autophagy.
  15. Autophagy in healthy aging and disease.
  16. AP-3 shows off its flexibility for the cryo-EM camera.
  17. Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells.
  18. Increased expression of complement components in tuberous sclerosis complex and focal cortical dysplasia type 2B brain lesions.
  19. ER proteins decipher the tubulin code to regulate organelle distribution.
  20. Damage of the cardiovascular system in lysosomal storage disease - mucopolysaccharidosis.
  21. TFE3-Mediated Autophagy is Involved in Dopaminergic Neurodegeneration in Parkinson's Disease.
  22. Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner.
  23. Regulatory role of cathepsin L in induction of nuclear laminopathy in Alzheimer's disease.
  24. Differences in gene expression patterns, revealed by RNA-seq analysis, between various Sanfilippo and Morquio disease subtypes.
  25. Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation.
  26. Discovery of a potent FKBP38 agonist that ameliorates HFD-induced hyperlipidemia via mTOR/P70S6K/SREBPs pathway.
  27. Novel subtype of mucopolysaccharidosis caused by arylsulfatase K (ARSK) deficiency.
  28. Roles of mTOR in thoracic aortopathy understood by complex intracellular signaling interactions.
  29. V-ATPase V0a1 promotes Weibel-Palade body biogenesis through the regulation of membrane fission.
  30. Syntaxin 12 and COMMD3 are new factors that function with VPS33B in the biogenesis of platelet α-granules.
  31. APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes.
  32. When deficient lysosomes make the liver fatty and the arteries greasy: how to treat, whom and when ?
  33. LC-MS lipidomics of renal biopsies for the diagnosis of Fabry disease.
  34. Smurf1 silencing restores PTEN expression that ameliorates progression of human glioblastoma and sensitizes tumor cells to mTORC1/C2 inhibitor Torin1.
  35. Regulation of Hook1-mediated endosomal sorting of clathrin-independent cargo by γ-taxilin.
  36. Case Report: A Novel LAMP2 Splice-Altering Mutation Causes Cardiac-Only Danon Disease.
  37. CD82 protects against glaucomatous axonal transport deficits via mTORC1 activation in mice.
  1. Sci Adv. 2021 Dec 17. 7(51): eabj9608
    CLN7 is an organellar chloride channel regulating lysosomal function.
    Yayu Wang, Wenping Zeng, Bingqian Lin, Yichuan Yao, Canjun Li, Wenqi Hu, Haotian Wu, Jiamin Huang, Mei Zhang, Tian Xue, Dejian Ren, Lili Qu, Chunlei Cang.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/sciadv.abj9608
  2. J Cell Biol. 2022 Feb 07. pii: e202104044. [Epub ahead of print]221(2):
    Efficient progranulin exit from the ER requires its interaction with prosaposin, a Surf4 cargo.
    Swathi Devireddy, Shawn M Ferguson.
      Progranulin is a lysosomal protein whose haploinsufficiency causes frontotemporal dementia, while homozygous loss of progranulin causes neuronal ceroid lipofuscinosis, a lysosomal storage disease. The sensitivity of cells to progranulin deficiency raises important questions about how cells coordinate intracellular trafficking of progranulin to ensure its efficient delivery to lysosomes. In this study, we discover that progranulin interactions with prosaposin, another lysosomal protein, first occur within the lumen of the endoplasmic reticulum (ER) and are required for the efficient ER exit of progranulin. Mechanistically, we identify an interaction between prosaposin and Surf4, a receptor that promotes loading of lumenal cargos into COPII-coated vesicles, and establish that Surf4 is critical for the efficient export of progranulin and prosaposin from the ER. Collectively, this work demonstrates that a network of interactions occurring early in the secretory pathway promote the ER exit and subsequent lysosomal delivery of newly translated progranulin and prosaposin.
    DOI:  https://doi.org/10.1083/jcb.202104044
  3. PLoS Biol. 2021 Dec;19(12): e3001480
    Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.
    Adamantios Mamais, Jillian H Kluss, Luis Bonet-Ponce, Natalie Landeck, Rebekah G Langston, Nathan Smith, Alexandra Beilina, Alice Kaganovich, Manik C Ghosh, Laura Pellegrini, Ravindran Kumaran, Ioannis Papazoglou, George R Heaton, Rina Bandopadhyay, Nunziata Maio, Changyoun Kim, Matthew J LaVoie, David C Gershlick, Mark R Cookson.
      Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.
    DOI:  https://doi.org/10.1371/journal.pbio.3001480
  4. iScience. 2021 Dec 17. 24(12): 103521
    AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins.
    Tobias Becker, Cedric Cappel, Francesco Di Matteo, Giovanna Sonsalla, Ewelina Kaminska, Fabio Spada, Silvia Cappello, Markus Damme, Pavel Kielkowski.
      Protein AMPylation is a posttranslational modification with an emerging role in neurodevelopment. In metazoans two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques. However, the function of AMPylation remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. We show that protein AMPylation is likely a posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, gel-based separation of modified and unmodified proteins, and an activity assay, we determine that the modified, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation correlates with its catalytic activity. Together, our findings indicate that AMPylation is a so far unknown lysosomal posttranslational modification connected to neuronal differentiation and it may provide a molecular rationale behind lysosomal storage diseases and neurodegeneration.
    Keywords:  Cell biology; Classification Description; Molecular biology; Neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2021.103521
  5. J Biol Chem. 2021 Dec 13. pii: S0021-9258(21)01296-5. [Epub ahead of print] 101487
    Cryo-EM structure of mouse TRPML2 in lipid nanodiscs.
    Xiaojing Song, Jian Li, Miao Tian, Huaiyi Zhu, Xiaohui Hu, Yuting Zhang, Yanru Cao, Heyang Ye, Peter J McCormick, Bo Zeng, Yang Fu, Jingjing Duan, Jin Zhang.
      In mammalians, transient receptor potential mucolipin ion channels (TRPMLs) exhibit variable permeability to cations such as Ca2+, Fe2+, Zn2+, and Na+, and can be activated by the phosphoinositide PI(3,5)P2 in the endolysosomal system. Loss or dysfunction of TRPMLs has been implicated in lysosomal storage disorders, infectious diseases, and metabolic diseases. TRPML2 has recently been identified as a mechanosensitive and hypotonicity-sensitive channel in endolysosomal organelles, which distinguishes it from TRPML1 and TRPML3. However, the molecular and gating mechanism of TRPML2 remains elusive. Here, we present the cryo-EM structure of the full-length mouse TRPML2 in lipid nanodiscs at 3.14 Å resolution. The TRPML2 homo-tetramer structure at pH 7.4 in the apo state reveals an inactive conformation and some unique features of the extracytosolic/luminal domain and voltage sensor-like domain that have implications for the ion-conducting pathway. This structure enables new comparisons between the different subgroups of TRPML channels with available structures and provides structural insights into the conservation and diversity of TRPML channels. These comparisons have broad implications for understanding a variety of molecular mechanisms of TRPMLs in different pH conditions, including with and without bound agonists and antagonists.
    DOI:  https://doi.org/10.1016/j.jbc.2021.101487
  6. Oncogene. 2021 Dec 14.
    p53-mediated AKT and mTOR inhibition requires RFX7 and DDIT4 and depends on nutrient abundance.
    Luis Coronel, David Häckes, Katjana Schwab, Konstantin Riege, Steve Hoffmann, Martin Fischer.
      In recent years the tumor suppressor p53 has been increasingly recognized as a potent regulator of the cell metabolism and for its ability to inhibit the critical pro-survival kinases AKT and mTOR. The mechanisms through which p53 controls AKT and mTOR, however, are largely unclear. Here, we demonstrate that p53 activates the metabolic regulator DDIT4 indirectly through the regulatory factor X 7 (RFX7). We provide evidence that DDIT4 is required for p53 to inhibit mTOR complex 2 (mTORC2)-dependent AKT activation. Most strikingly, we also find that the DDIT4 regulator RFX7 is required for p53-mediated inhibition of mTORC1 and AKT. Our results suggest that AMPK activation plays no role and p53-mediated AKT inhibition is not critical for p53-mediated mTORC1 inhibition. Moreover, using recently developed physiological cell culture media we uncover that basal p53 and RFX7 activity can play a critical role in restricting mTORC1 activity under physiological nutrient conditions, and we propose a nutrient-dependent model for p53-RFX7-mediated mTORC1 inhibition. These results establish RFX7 and its downstream target DDIT4 as essential effectors in metabolic control elicited by p53.
    DOI:  https://doi.org/10.1038/s41388-021-02147-z
  7. Brain Commun. 2021 ;3(4): fcab245
    Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy.
    Italian Undiagnosed Diseases Network
      The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.
    Keywords:  Caenorhabditis elegans disease modelling; V-ATPase; epileptic encephalopathy; lysosomal disease; organelle acidification
    DOI:  https://doi.org/10.1093/braincomms/fcab245
  8. Cell Stress. 2021 Dec;5(12): 176-182
    p38 regulates the tumor suppressor PDCD4 via the TSC-mTORC1 pathway.
    Clarissa Braun, Karl Katholnig, Christopher Kaltenecker, Monika Linke, Nyamdelger Sukhbaatar, Markus Hengstschläger, Thomas Weichhart.
      Programmed cell death protein 4 (PDCD4) exerts critical functions as tumor suppressor and in immune cells to regulate inflammatory processes. The phosphoinositide 3-kinase (PI3K) promotes degradation of PDCD4 via mammalian target of rapamycin complex 1 (mTORC1). However, additional pathways that may regulate PDCD4 expression are largely ill-defined. In this study, we have found that activation of the mitogen-activated protein kinase p38 promoted degradation of PDCD4 in macrophages and fibroblasts. Mechanistically, we identified a pathway from p38 and its substrate MAP kinase-activated protein kinase 2 (MK2) to the tuberous sclerosis complex (TSC) to regulate mTORC1-dependent degradation of PDCD4. Moreover, we provide evidence that TSC1 and TSC2 regulate PDCD4 expression via an additional mechanism independent of mTORC1. These novel data extend our knowledge of how PDCD4 expression is regulated by stress- and nutrient-sensing pathways.
    Keywords:  MK2; PDCD4; TSC1; TSC2; cancer; mTORC1; macrophages; p38; rapamycin
    DOI:  https://doi.org/10.15698/cst2021.12.260
  9. J Cell Biol. 2022 Jan 03. pii: e202112049. [Epub ahead of print]221(1):
    Rushika M. Perera: Lysosomes unveil what cancer cells hide.
    Lucia Morgado-Palacin.
      Rushika M. Perera studies how pancreatic cancer cells use autophagy and the lysosome to adapt to stress.
    DOI:  https://doi.org/10.1083/jcb.202112049
  10. Front Pharmacol. 2021 ;12 794298
    TFEB Dependent Autophagy-Lysosomal Pathway: An Emerging Pharmacological Target in Sepsis.
    Xin Liu, Xinchuan Zheng, Yongling Lu, Qian Chen, Jiang Zheng, Hong Zhou.
      Sepsis is a life-threatening syndrome induced by aberrant host response towards infection. The autophagy-lysosomal pathway (ALP) plays a fundamental role in maintaining cellular homeostasis and conferring organ protection. However, this pathway is often impaired in sepsis, resulting in dysregulated host response and organ dysfunction. Transcription factor EB (TFEB) is a master modulator of the ALP. TFEB promotes both autophagy and lysosomal biogenesis via transcriptional regulation of target genes bearing the coordinated lysosomal expression and regulation (CLEAR) motif. Recently, increasing evidences have linked TFEB and the TFEB dependent ALP with pathogenetic mechanisms and therapeutic implications in sepsis. Therefore, this review describes the existed knowledge about the mechanisms of TFEB activation in regulating the ALP and the evidences of their protection against sepsis, such as immune modulation and organ protection. In addition, TFEB activators with diversified pharmacological targets are summarized, along with recent advances of their potential therapeutic applications in treating sepsis.
    Keywords:  TFEB; TFEB activators; autophagy-lysosomal pathway; immunity; inflammation; sepsis
    DOI:  https://doi.org/10.3389/fphar.2021.794298
  11. Mol Ther Methods Clin Dev. 2021 Dec 10. 23 644-658
    AAV-mediated gene therapy for galactosialidosis: A long-term safety and efficacy study.
    Huimin Hu, Rosario Mosca, Elida Gomero, Diantha van de Vlekkert, Yvan Campos, Leigh E Fremuth, Scott A Brown, Jason A Weesner, Ida Annunziata, Alessandra d'Azzo.
      AAV-mediated gene therapy holds promise for the treatment of lysosomal storage diseases (LSDs), some of which are already in clinical trials. Yet, ultra-rare subtypes of LSDs, such as some glycoproteinoses, have lagged. Here, we report on a long-term safety and efficacy preclinical study conducted in the murine model of galactosialidosis, a glycoproteinosis caused by a deficiency of protective protein/cathepsin A (PPCA). One-month-old Ctsa -/- mice were injected intravenously with a high dose of a self-complementary AAV2/8 vector expressing human CTSA in the liver. Treated mice, examined up to 12 months post injection, appeared grossly indistinguishable from their wild-type littermates. Sustained expression of scAAV2/8-CTSA in the liver resulted in the release of the therapeutic precursor protein in circulation and its widespread uptake by cells in visceral organs and the brain. Increased cathepsin A activity resolved lysosomal vacuolation throughout the affected organs and sialyl-oligosacchariduria. No signs of hyperplasia or inflammation were detected in the liver up to a year of age. Clinical chemistry panels, blood cell counts, and T cell immune responses were normal in all treated animals. These results warrant a close consideration of this gene therapy approach for the treatment of galactosialidosis, an orphan disease with no cure in sight.
    Keywords:  AAV-mediated gene therapy; galactosialidosis; lysosomal multienzyme complex; lysosomal storage disease; protective protein cathepsin A
    DOI:  https://doi.org/10.1016/j.omtm.2021.10.007
  12. FEBS Lett. 2021 Dec 12.
    Sterol uptake by the NPC system in eukaryotes: A Saccharomyces cerevisiae perspective.
    Mikael B L Winkler, Lynette Nel, Kelly M Frain, Emil Dedic, Esben Olesen, Bjørn Panyella Pedersen.
      Sterols are an essential component of membranes in all eukaryotic cells and the precursor of multiple indispensable cellular metabolites. After endocytotic uptake, sterols are integrated into the lysosomal membrane by the Niemann-Pick type C (NPC) system before redistribution to other membranes. The process is driven by two proteins that, together, compose the NPC system: the lysosomal sterol shuttle protein NPC2 and the membrane protein NPC1 (named NCR1 in fungi), which integrates sterols into the lysosomal membrane. The Saccharomyces cerevisiae NPC system provides a compelling model to study the molecular mechanism of sterol integration into membranes and sterol homeostasis. This review summarizes recent advances in the field, and by interpreting available structural data, we propose a unifying conceptual model for sterol loading, transfer and transport by NPC proteins.
    Keywords:  NCR1; NPC system; NPC1; NPC2; Niemann-Pick type C Disease; cholesterol/sterol uptake; lysosome/vacuole; molecular mechanism; structural biology
    DOI:  https://doi.org/10.1002/1873-3468.14253
  13. Front Cell Dev Biol. 2021 ;9 784367
    Mechanisms of Non-Vesicular Exchange of Lipids at Membrane Contact Sites: Of Shuttles, Tunnels and, Funnels.
    Pascal F Egea.
      Eukaryotic cells are characterized by their exquisite compartmentalization resulting from a cornucopia of membrane-bound organelles. Each of these compartments hosts a flurry of biochemical reactions and supports biological functions such as genome storage, membrane protein and lipid biosynthesis/degradation and ATP synthesis, all essential to cellular life. Acting as hubs for the transfer of matter and signals between organelles and throughout the cell, membrane contacts sites (MCSs), sites of close apposition between membranes from different organelles, are essential to cellular homeostasis. One of the now well-acknowledged function of MCSs involves the non-vesicular trafficking of lipids; its characterization answered one long-standing question of eukaryotic cell biology revealing how some organelles receive and distribute their membrane lipids in absence of vesicular trafficking. The endoplasmic reticulum (ER) in synergy with the mitochondria, stands as the nexus for the biosynthesis and distribution of phospholipids (PLs) throughout the cell by contacting nearly all other organelle types. MCSs create and maintain lipid fluxes and gradients essential to the functional asymmetry and polarity of biological membranes throughout the cell. Membrane apposition is mediated by proteinaceous tethers some of which function as lipid transfer proteins (LTPs). We summarize here the current state of mechanistic knowledge of some of the major classes of LTPs and tethers based on the available atomic to near-atomic resolution structures of several "model" MCSs from yeast but also in Metazoans; we describe different models of lipid transfer at MCSs and analyze the determinants of their specificity and directionality. Each of these systems illustrate fundamental principles and mechanisms for the non-vesicular exchange of lipids between eukaryotic membrane-bound organelles essential to a wide range of cellular processes such as at PL biosynthesis and distribution, lipid storage, autophagy and organelle biogenesis.
    Keywords:  autophagy; lipid distribution; lipid transfer protein; lipid-droplet; membrane asymmetry; membrane contact site; mitochondria-attached membranes; organelle
    DOI:  https://doi.org/10.3389/fcell.2021.784367
  14. Assay Drug Dev Technol. 2021 Dec 13.
    Automated Assays to Identify Modulators of Transcription Factor EB Translocation and Autophagy.
    Anaelle da Costa, Christèle Picoli, Franck Mouthon, Mathieu Charvériat.
      Autophagy is a process leading to the degradation of cellular material, in organelles called lysosomes, to supply energy or generate building blocks for the synthesis of new materials. Over the past decades, its role has been evidenced in several indications, notably in neurodegenerative disorders and orphan diseases called lysosomal storage disorders and its modulation is largely envisioned as a therapeutic avenue to alleviate the symptoms and reverse the clinical courses of these indications. Identifying new chemical classes and drugs is, hence, of huge importance. In this study, we developed automated assays to assess the potential efficacy of chemical compounds on different steps of autophagy, notably its induction through the localization of a largely involved transcription factor, transcription factor EB (TFEB). These assays were then used to screen a collection of 1,520 approved drugs. This study led to the identification of five candidate hits modulating autophagy and TFEB subcellular localization. Our results suggest the repurposing potential of already approved drugs in central nervous system disorders with lysosomal storage impairments.
    Keywords:  LC3; TFEB; autophagy; lysosome
    DOI:  https://doi.org/10.1089/adt.2021.119
  15. Nat Aging. 2021 Aug;1(8): 634-650
    Autophagy in healthy aging and disease.
    Yahyah Aman, Tomas Schmauck-Medina, Malene Hansen, Richard I Morimoto, Anna Katharina Simon, Ivana Bjedov, Konstantinos Palikaras, Anne Simonsen, Terje Johansen, Nektarios Tavernarakis, David C Rubinsztein, Linda Partridge, Guido Kroemer, John Labbadia, Evandro F Fang.
      Autophagy is a fundamental cellular process that eliminates molecules and subcellular elements, including nucleic acids, proteins, lipids and organelles, via lysosome-mediated degradation to promote homeostasis, differentiation, development and survival. While autophagy is intimately linked to health, the intricate relationship among autophagy, aging and disease remains unclear. This Review examines several emerging features of autophagy and postulates how they may be linked to aging as well as to the development and progression of disease. In addition, we discuss current preclinical evidence arguing for the use of autophagy modulators as suppressors of age-related pathologies such as neurodegenerative diseases. Finally, we highlight key questions and propose novel research avenues that will likely reveal new links between autophagy and the hallmarks of aging. Understanding the precise interplay between autophagy and the risk of age-related pathologies across organisms will eventually facilitate the development of clinical applications that promote long-term health.
    DOI:  https://doi.org/10.1038/s43587-021-00098-4
  16. J Biol Chem. 2021 Dec 10. pii: S0021-9258(21)01301-6. [Epub ahead of print] 101491
    AP-3 shows off its flexibility for the cryo-EM camera.
    Todd R Graham.
      The tetrameric adaptor protein AP-3 is critical for transport of proteins to lysosomes and lysosome-related organelles. Structures of homologous adaptors AP-1 and AP-2 have revealed a closed-to-open conformational change upon membrane recruitment and phosphoinositide binding. Recently, Schoppe and colleagues reported the first cryo-EM structures of AP-3 from budding yeast and described remarkably flexible solution structures that are all in the open conformation. The apparent lack of a closed conformational state, the first such description in the literature, allows AP-3 to be more reliant on cargo interaction for its initial membrane recruitment compared to AP-1.
    DOI:  https://doi.org/10.1016/j.jbc.2021.101491
  17. Cell Mol Life Sci. 2021 Dec 18.
    Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells.
    Julia Sirés-Campos, Ana Lambertos, Cédric Delevoye, Graça Raposo, Dorothy C Bennett, Elena Sviderskaya, Celia Jiménez-Cervantes, Conchi Olivares, José Carlos García-Borrón.
      Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III-IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.
    Keywords:  Lysosome-related organelles; Mahogunin Ring Finger 1 (MGRN1); Melanin; Melanosomal pH; Mucolipin 3 (MCOLN3); Tyrosinase
    DOI:  https://doi.org/10.1007/s00018-021-04053-9
  18. Epilepsia. 2021 Dec 14.
    Increased expression of complement components in tuberous sclerosis complex and focal cortical dysplasia type 2B brain lesions.
    Victoria-Elisabeth Gruber, Mark J Luinenburg, Katrin Colleselli, Verena Endmayr, Jasper J Anink, Till S Zimmer, Floor Jansen, Peter Gosselaar, Roland Coras, Theresa Scholl, Ingmar Blumcke, José Pimentel, Johannes A Hainfellner, Romana Höftberger, Karl Rössler, Martha Feucht, Jackelien van Scheppingen, Eleonora Aronica, Angelika Mühlebner.
       OBJECTIVE: Increasing evidence supports the contribution of inflammatory mechanisms to the neurological manifestations of epileptogenic developmental pathologies linked to mammalian target of rapamycin (mTOR) pathway dysregulation (mTORopathies), such as tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD). In this study, we aimed to investigate the expression pattern and cellular distribution of the complement factors C1q and C3 in resected cortical tissue of clinically well-characterized patients with TSC and FCD2B.
    METHODS: We applied immunohistochemistry in TSC (n = 29) and FCD2B (n = 32) samples and compared them to autopsy and biopsy controls (n = 27). Furthermore, protein expression was observed via Western blot, and for descriptive colocalization studies immunofluorescence double labeling was performed.
    RESULTS: Protein expression for C3 was significantly upregulated in TSC and FCD2B white and gray matter lesions compared to controls. Staining of the synaptic vesicle protein synaptophysin showed a remarkable increase in the white matter of both TSC and FCD2B. Furthermore, confocal imaging revealed colocalization of complement factors with astroglial, microglial, neuronal, and abnormal cells in various patterns.
    SIGNIFICANCE: Our results demonstrate that the prominent activation of the complement pathway represents a common pathological hallmark of TSC and FCD2B, suggesting that complement overactivation may play a role in these mTORopathies.
    Keywords:  complement; cortical development; epilepsy; focal cortical dysplasia; inflammation; tuberous sclerosis complex
    DOI:  https://doi.org/10.1111/epi.17139
  19. Nature. 2021 Dec 15.
    ER proteins decipher the tubulin code to regulate organelle distribution.
    Pengli Zheng, Christopher J Obara, Ewa Szczesna, Jonathon Nixon-Abell, Kishore K Mahalingan, Antonina Roll-Mecak, Jennifer Lippincott-Schwartz, Craig Blackstone.
      Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.
    DOI:  https://doi.org/10.1038/s41586-021-04204-9
  20. Pol Merkur Lekarski. 2021 Dec 16. 49(294): 448-452
    Damage of the cardiovascular system in lysosomal storage disease - mucopolysaccharidosis.
    Sergiy Mykolayovych Pyvovar, Iuriy Stepanovych Rudyk, Tetiana Valentynivna Lozykc.
      The analysis of literature data reflecting the issues of the pathology of the cardiovascular system in mucopolysaccharidosis are presented. It was found out that heart and vessels damage is one of the cardinal signs of this pathology, often leading to death. Cardiac pathology is recorded in all types of mucopolysaccharidosis, but it is most significant for patients with three clinical variants of Hurler syndrome, Hunter, and Maroteaux-Lamy syndromes. Typical signs of damage to the cardiovascular system in mucopolysaccharidosis are thickening of the valves with the development of their dysfunction (while the severity of damage to the left-sided valves is more pronounced), myocardial hypertrophy, conduction disturbance, coronary artery disease, arterial hypertension. Many researchers emphasize the difficulties of clinical and functional examination of the cardiovascular system in patients with mucopolysaccharidosis, which is due to the presence of physical and intellectual limitations in patients, ands a gradual increase in symptoms. For the treatment of cardiovascular pathology at mucopolysaccharidosis, medical and surgical methods are used, including enzyme replacement therapy and stem cell transplantation.
    Keywords:  cardiovascular system; diagnostics; lysosomal storage disease; mucopolysaccharidosis; treatment
  21. Front Cell Dev Biol. 2021 ;9 761773
    TFE3-Mediated Autophagy is Involved in Dopaminergic Neurodegeneration in Parkinson's Disease.
    Xin He, Yue Xie, Qiongping Zheng, Zeyu Zhang, Shanshan Ma, Junyu Li, Mingtao Li, Qiaoying Huang.
      Impairment of autophagy has been strongly implicated in the progressive loss of nigral dopaminergic neurons in Parkinson's disease (PD). Transcription factor E3 (TFE3), an MiTF/TFE family transcription factor, has been identified as a master regulator of the genes that are associated with lysosomal biogenesis and autophagy. However, whether TFE3 is involved in parkinsonian neurodegeneration remains to be determined. In this study, we found decreased TFE3 expression in the nuclei of the dopaminergic neurons of postmortem human PD brains. Next, we demonstrated that TFE3 knockdown led to autophagy dysfunction and neurodegeneration of dopaminergic neurons in mice, implying that reduction of nuclear TFE3 may contribute to autophagy dysfunction-mediated cell death in PD. Further, we showed that enhancement of autophagy by TFE3 overexpression dramatically reversed autophagy downregulation and dopaminergic neurons loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. Taken together, these findings demonstrate that TFE3 plays an essential role in maintaining autophagy and the survival of dopaminergic neurons, suggesting TFE3 activation may serve as a promising strategy for PD therapy.
    Keywords:  MPTP; Parkinson’s disease; TFE3; autophagy; dopaminergic neurons
    DOI:  https://doi.org/10.3389/fcell.2021.761773
  22. EMBO J. 2021 Dec 17. e109360
    Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner.
    Md Murad Khan, Seowon Lee, Sergio Couoh-Cardel, Rebecca A Oot, Hyunmin Kim, Stephan Wilkens, Soung-Hun Roh.
      The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V1 -ATPase and Vo proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted Vo and mutant V1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V1 to Vo provides sufficient free energy to overcome Vo autoinhibition. Moreover, the structures suggest that the unequal spacing of Vo 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V1 and Vo motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.
    Keywords:  Cryo-electron microscopy; Oxr1p; TLDc domain; reversible disassembly; vacuolar ATPase
    DOI:  https://doi.org/10.15252/embj.2021109360
  23. Aging Cell. 2021 Dec 14. e13531
    Regulatory role of cathepsin L in induction of nuclear laminopathy in Alzheimer's disease.
    Md Imamul Islam, Pandian Nagakannan, Tetiana Shcholok, Fabio Contu, Sabine Mai, Benedict C Albensi, Marc R Del Bigio, Jun-Feng Wang, Md Golam Sharoar, Riqiang Yan, Il-Seon Park, Eftekhar Eftekharpour.
      Experimental and clinical therapies in the field of Alzheimer's disease (AD) have focused on elimination of extracellular amyloid beta aggregates or prevention of cytoplasmic neuronal fibrillary tangles formation, yet these approaches have been generally ineffective. Interruption of nuclear lamina integrity, or laminopathy, is a newly identified concept in AD pathophysiology. Unraveling the molecular players in the induction of nuclear lamina damage may lead to identification of new therapies. Here, using 3xTg and APP/PS1 mouse models of AD, and in vitro model of amyloid beta42 (Aβ42) toxicity in primary neuronal cultures and SH-SY5Y neuroblastoma cells, we have uncovered a key role for cathepsin L in the induction of nuclear lamina damage. The applicability of our findings to AD pathophysiology was validated in brain autopsy samples from patients. We report that upregulation of cathepsin L is an important process in the induction of nuclear lamina damage, shown by lamin B1 cleavage, and is associated with epigenetic modifications in AD pathophysiology. More importantly, pharmacological targeting and genetic knock out of cathepsin L mitigated Aβ42 induced lamin B1 degradation and downstream structural and molecular changes. Affirming these findings, overexpression of cathepsin L alone was sufficient to induce lamin B1 cleavage. The proteolytic activity of cathepsin L on lamin B1 was confirmed using mass spectrometry. Our research identifies cathepsin L as a newly identified lamin B1 protease and mediator of laminopathy observed in AD. These results uncover a new aspect in the pathophysiology of AD that can be pharmacologically prevented, raising hope for potential therapeutic interventions.
    Keywords:  acetylation; amyloid beta; chromatin; histone; lysosomal membrane permeabilization; methylation; nuclear lamina; super-resolution microscopy
    DOI:  https://doi.org/10.1111/acel.13531
  24. Gene. 2021 Dec 08. pii: S0378-1119(21)00685-5. [Epub ahead of print]812 146090
    Differences in gene expression patterns, revealed by RNA-seq analysis, between various Sanfilippo and Morquio disease subtypes.
    Karolina Wiśniewska, Lidia Gaffke, Karolina Krzelowska, Grzegorz Węgrzyn, Karolina Pierzynowska.
      Mucopolysaccharidoses (MPS) are genetic disorders that affect up to 1 in 25,000 births. They are caused by dysfunctions of lysosomal hydrolases that degrade glycosaminoglycans (GAGs) which accumulate in cells, damaging their proper functioning. There are 7 types of MPS, distinguished by the kind of accumulated GAG and the defective enzyme, which differ significantly in the course of the disease. Despite the storage of the same GAGs, two of them (MPS III and IV) are divided into subtypes. While the course of MPS IV A and B is similar, the variability between MPS III A, B, C and D is high. This suggests that there are additional aspects that could influence the course of the disease. Therefore, the aim of this study was to determine differences of patterns of gene expression between all MPS III and IV subtypes. Transcriptomic studies, carried out with dermal fibroblasts from patients with all MPS III and IV subtypes, showed a significant variation in the gene expression pattern between individual MPS III subtypes, in contrast to MPS IV. Detailed analysis of transcripts with altered expression levels between MPS III subtypes indicated that these transcripts are mainly involved in maintaining the proper structure of connective tissue (COL4A1, COL4A2, COMP) and the structure of ribosomes (RPL10, RPL23, RPLP2). The results presented in this study indicate a significant role of genetic factors in the diversified course of MPS III subtypes.
    Keywords:  Cellular processes; Diseases’ course; Genetic factors; Morquio syndrome; Mucopolysaccharidosis; Sanfilippo syndrome; Transcriptomics
    DOI:  https://doi.org/10.1016/j.gene.2021.146090
  25. Front Immunol. 2021 ;12 789142
    Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation.
    Solvey Pollmann, David Scharnetzki, Dominique Manikowski, Malte Lenders, Eva Brand.
      Fabry disease (FD) is an X-linked multisystemic lysosomal storage disease due to a deficiency of α-galactosidase A (GLA/AGAL). Progressive cellular accumulation of the AGAL substrate globotriaosylceramide (Gb3) leads to endothelial dysfunction. Here, we analyzed endothelial function in vivo and in vitro in an AGAL-deficient genetic background to identify the processes underlying this small vessel disease. Arterial stiffness and endothelial function was prospectively measured in five males carrying GLA variants (control) and 22 FD patients under therapy. AGAL-deficient endothelial cells (EA.hy926) and monocytes (THP1) were used to analyze endothelial glycocalyx structure, function, and underlying inflammatory signals. Glycocalyx thickness and small vessel function improved significantly over time (p<0.05) in patients treated with enzyme replacement therapy (ERT, n=16) and chaperones (n=6). AGAL-deficient endothelial cells showed reduced glycocalyx and increased monocyte adhesion (p<0.05). In addition, increased expression of angiopoietin-2, heparanase and NF-κB was detected (all p<0.05). Incubation of wild-type endothelial cells with pathological globotriaosylsphingosine concentrations resulted in comparable findings. Treatment of AGAL-deficient cells with recombinant AGAL (p<0.01), heparin (p<0.01), anti-inflammatory (p<0.001) and antioxidant drugs (p<0.05), and a specific inhibitor (razuprotafib) of angiopoietin-1 receptor (Tie2) (p<0.05) improved glycocalyx structure and endothelial function in vitro. We conclude that chronic inflammation, including the release of heparanases, appears to be responsible for the degradation of the endothelial glycocalyx and may explain the endothelial dysfunction in FD. This process is partially reversible by FD-specific and anti-inflammatory treatment, such as targeted protective Tie2 treatment.
    Keywords:  Fabry disease (FD); NF-κB; angiopoietin-1-receptor; endothelial dysfunction; globotriaosylsphingosine (lyso-Gb3); heparanase
    DOI:  https://doi.org/10.3389/fimmu.2021.789142
  26. Acta Pharm Sin B. 2021 Nov;11(11): 3542-3552
    Discovery of a potent FKBP38 agonist that ameliorates HFD-induced hyperlipidemia via mTOR/P70S6K/SREBPs pathway.
    Ping-Ting Xiao, Zhi-Shen Xie, Yu-Jia Kuang, Shi-Yu Liu, Chun Zeng, Ping Li, E-Hu Liu.
      The mammalian target of rapamycin (mTOR)-sterol regulatory element-binding proteins (SREBPs) signaling promotes lipogenesis. However, mTOR inhibitors also displayed a significant side effect of hyperlipidemia. Thus, it is essential to develop mTOR-specific inhibitors to inhibit lipogenesis. Here, we screened the endogenous inhibitors of mTOR, and identified that FKBP38 as a vital regulator of lipid metabolism. FKBP38 decreased the lipid content in vitro and in vivo via suppression of the mTOR/P70S6K/SREBPs pathway. 3,5,6,7,8,3',4'-Heptamethoxyflavone (HMF), a citrus flavonoid, was found to target FKBP38 to suppress the mTOR/P70S6K/SREBPs pathway, reduce lipid level, and potently ameliorate hyperlipidemia and insulin resistance in high fat diet (HFD)-fed mice. Our findings suggest that pharmacological intervention by targeting FKBP38 to suppress mTOR/P70S6K/SREBPs pathway is a potential therapeutic strategy for hyperlipidemia, and HMF could be a leading compound for development of anti-hyperlipidemia drugs.
    Keywords:  3,5,6,7,8,3ʹ,4ʹ-heptamethoxyflavone; FKBP38; Hyperlipidemia; SREBP; mTOR
    DOI:  https://doi.org/10.1016/j.apsb.2021.03.031
  27. J Med Genet. 2021 Dec 16. pii: jmedgenet-2021-108061. [Epub ahead of print]
    Novel subtype of mucopolysaccharidosis caused by arylsulfatase K (ARSK) deficiency.
    Sarah Verheyen, Jasmin Blatterer, Michael R Speicher, Gandham SriLakshmi Bhavani, Geert-Jan Boons, Mai-Britt Ilse, Dominik Andrae, Jens Sproß, Frédéric Maxime Vaz, Susanne G Kircher, Laura Posch-Pertl, Daniela Baumgartner, Torben Lübke, Hitesh Shah, Ali Al Kaissi, Katta M Girisha, Barbara Plecko.
       BACKGROUND: Mucopolysaccharidoses (MPS) are monogenic metabolic disorders that significantly affect the skeleton. Eleven enzyme defects in the lysosomal degradation of glycosaminoglycans (GAGs) have been assigned to the known MPS subtypes (I-IX). Arylsulfatase K (ARSK) is a recently characterised lysosomal hydrolase involved in GAG degradation that removes the 2-O-sulfate group from 2-sulfoglucuronate. Knockout of Arsk in mice was consistent with mild storage pathology, but no human phenotype has yet been described.
    METHODS: In this study, we report four affected individuals of two unrelated consanguineous families with homozygous variants c.250C>T, p.(Arg84Cys) and c.560T>A, p.(Leu187Ter) in ARSK, respectively. Functional consequences of the two ARSK variants were assessed by mutation-specific ARSK constructs derived by site-directed mutagenesis, which were ectopically expressed in HT1080 cells. Urinary GAG excretion was analysed by dimethylene blue and electrophoresis, as well as liquid chromatography/mass spectrometry (LC-MS)/MS analysis.
    RESULTS: The phenotypes of the affected individuals include MPS features, such as short stature, coarse facial features and dysostosis multiplex. Reverse phenotyping in two of the four individuals revealed additional cardiac and ophthalmological abnormalities. Mild elevation of dermatan sulfate was detected in the two subjects investigated by LC-MS/MS. Human HT1080 cells expressing the ARSK-Leu187Ter construct exhibited absent protein levels by western blot, and cells with the ARSK-Arg84Cys construct showed markedly reduced enzyme activity in an ARSK-specific enzymatic assay against 2-O-sulfoglucuronate-containing disaccharides as analysed by C18-reversed-phase chromatography followed by MS.
    CONCLUSION: Our work provides a detailed clinical and molecular characterisation of a novel subtype of mucopolysaccharidosis, which we suggest to designate subtype X.
    Keywords:  genetics; human genetics; orthopedics; pediatrics; phenotype
    DOI:  https://doi.org/10.1136/jmedgenet-2021-108061
  28. PLoS Comput Biol. 2021 Dec 13. 17(12): e1009683
    Roles of mTOR in thoracic aortopathy understood by complex intracellular signaling interactions.
    Ana C Estrada, Linda Irons, Bruno V Rego, Guangxin Li, George Tellides, Jay D Humphrey.
      Thoracic aortopathy-aneurysm, dissection, and rupture-is increasingly responsible for significant morbidity and mortality. Advances in medical genetics and imaging have improved diagnosis and thus enabled earlier prophylactic surgical intervention in many cases. There remains a pressing need, however, to understand better the underlying molecular and cellular mechanisms with the hope of finding robust pharmacotherapies. Diverse studies in patients and mouse models of aortopathy have revealed critical changes in multiple smooth muscle cell signaling pathways that associate with disease, yet integrating information across studies and models has remained challenging. We present a new quantitative network model that includes many of the key smooth muscle cell signaling pathways and validate the model using a detailed data set that focuses on hyperactivation of the mechanistic target of rapamycin (mTOR) pathway and its inhibition using rapamycin. We show that the model can be parameterized to capture the primary experimental findings both qualitatively and quantitatively. We further show that simulating a population of cells by varying receptor reaction weights leads to distinct proteomic clusters within the population, and that these clusters emerge due to a bistable switch driven by positive feedback in the PI3K/AKT/mTOR signaling pathway.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009683
  29. Elife. 2021 Dec 14. pii: e71526. [Epub ahead of print]10
    V-ATPase V0a1 promotes Weibel-Palade body biogenesis through the regulation of membrane fission.
    Yasuo Yamazaki, Yuka Eura, Koichi Kokame.
      Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.71526
  30. Blood. 2021 Dec 14. pii: blood.2021012056. [Epub ahead of print]
    Syntaxin 12 and COMMD3 are new factors that function with VPS33B in the biogenesis of platelet α-granules.
    Andrea L Ambrosio, Hallie P Febvre, Santiago Mauro Di Pietro.
      Platelet a-granules regulate hemostasis and myriad other physiological processes but their biogenesis is unclear. Mutations in only three proteins are known to cause a-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized a-granule proteins through megakaryocyte endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Stx12, a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12 deficient megakaryocytes display reduced a-granule numbers and overall levels of a-granule proteins, thus revealing Stx12 as new component of the a-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in megakaryocytes contains COMMD3, one of ten COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced a-granule numbers and overall levels of a-granule proteins, establishing the COMMD3/CCC complex as a new factor in a-granule biogenesis. Furthermore, P-Selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-Selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in a-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of a-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in megakaryocytes to make a-granules.
    DOI:  https://doi.org/10.1182/blood.2021012056
  31. Stem Cell Reports. 2021 Nov 30. pii: S2213-6711(21)00588-9. [Epub ahead of print]
    APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes.
    Sherida M de Leeuw, Aron W T Kirschner, Karina Lindner, Ruslan Rust, Vanessa Budny, Witold E Wolski, Anne-Claude Gavin, Roger M Nitsch, Christian Tackenberg.
      The apolipoprotein E4 (APOE4) variant is the strongest genetic risk factor for Alzheimer disease (AD), while the APOE2 allele is protective. A major question is how different APOE genotypes affect the physiology of astrocytes, the main APOE-producing brain cells. Here, we differentiated human APOE-isogenic induced pluripotent stem cells (iPSCs) (APOE4, E3, E2, and APOE knockout [APOE-KO]) to functional "iAstrocytes". Mass-spectrometry-based proteomic analysis showed genotype-dependent reductions of cholesterol and lipid metabolic and biosynthetic pathways (reduction: APOE4 >E3 >E2). Cholesterol efflux and biosynthesis were reduced in APOE4 iAstrocytes, while subcellular localization of cholesterol in lysosomes was elevated. An increase in immunoregulatory proteomic pathways (APOE4 >E3 >E2) was accompanied by elevated cytokine release in APOE4 cells (APOE4 >E3 >E2 >KO). Activation of iAstrocytes exacerbated proteomic changes and cytokine secretion mostly in APOE4 iAstrocytes, while APOE2 and APOE-KO iAstrocytes were least affected. Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced β-amyloid uptake, while APOE2 iAstrocytes show opposing effects.
    Keywords:  APOE; Alzheimer disease; Aβ; astrocytes; cholesterol; homeostasis; iPSCs; inflammation; isogenic; lipid metabolism; proteomics
    DOI:  https://doi.org/10.1016/j.stemcr.2021.11.007
  32. J Hepatol. 2021 Dec 13. pii: S0168-8278(21)02240-6. [Epub ahead of print]
    When deficient lysosomes make the liver fatty and the arteries greasy: how to treat, whom and when ?
    Florence Lacaille.
      
    DOI:  https://doi.org/10.1016/j.jhep.2021.12.002
  33. J Mass Spectrom Adv Clin Lab. 2021 Nov;22 71-78
    LC-MS lipidomics of renal biopsies for the diagnosis of Fabry disease.
    Hoda Safari Yazd, Sina Feizbakhsh Bazargani, Christine A Vanbeek, Kelli King-Morris, Coy Heldermon, Mark S Segal, William L Clapp, Timothy J Garrett.
       Introduction: Lipidomics analysis or lipid profiling is a system-based analysis of all lipids in a sample to provide a comprehensive understanding of lipids within a biological system. In the last few years, lipidomics has made it possible to better understand the metabolic processes associated with several rare disorders and proved to be a powerful tool for their clinical investigation. Fabry disease is a rare X-linked lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A (α-GAL A). This deficiency results in the progressive accumulation of glycosphingolipids, mostly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), as well as galabiosylceramide (Ga2) and their isoforms/analogs in the vascular endothelium, nerves, cardiomyocytes, renal glomerular podocytes, and biological fluids.
    Objectives: The primary objective of this study was to evaluate lipidomic signatures in renal biopsies to help understand variations in Fabry disease markers that could be used in future diagnostic tests.
    Methods: Lipidomic analysis was performed by ultra-high pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) on kidney biopsies that were left over after clinical pathology analysis to diagnose Fabry disease.
    Results: We employed UHPLC-HRMS lipidomics analysis on the renal biopsy of a patient suspicious for Fabry disease. Our result confirmed α-GAL A enzyme activity declined in this patient since a Ga2-related lipid biomarker was substantially higher in the patient's renal tissue biopsy compared with two controls. This suggests this patient has a type of LSD that could be non-classical Fabry disease.
    Conclusion: This study shows that lipidomics analysis is a valuable tool for rare disorder diagnosis, which can be conducted on leftover tissue samples without disrupting normal patient care.
    Keywords:  CAN, Acetonitrile; CDH, Cerebrodihexoside; Chcl3, Chloroform; Cnvs, Copy Number Variants; EIC, Extracted Ion Chromatogram; ERT, Enzyme Replacement Therapy; GLA, Glactosidase Alpha; Ga2, Galabiosylceramide; Gb3, Globotriaosylceramide; IPA, 2-Propanol; LC/MS, Liquid Chromatography-Mass Spectrometry; LSD, Lysosomal Storage Disorder; Lyso-Gb3, Globotriaosylsphingosine; MS/MS, Tandem Mass Spectrometry; Meoh, Methanol; ND, Not Detected; OCT, Optimal Cutting Temperature; SECIM, Southeast Center for Integrated Metabolomics; SRM, Selected Reaction Monitoring; UHPLC-HRMS, Ultra-High Pressure Liquid Chromatography-High-Resolution Mass Spectrometry; α-GAL A, α-Galactosidase A
    DOI:  https://doi.org/10.1016/j.jmsacl.2021.11.004
  34. iScience. 2021 Dec 17. 24(12): 103528
    Smurf1 silencing restores PTEN expression that ameliorates progression of human glioblastoma and sensitizes tumor cells to mTORC1/C2 inhibitor Torin1.
    Qin Xia, Wenxuan Li, Sakhawat Ali, Mengchuan Xu, Yang Li, Shengzhen Li, Xinyi Meng, Liqun Liu, Lei Dong.
      Amplification of ubiquitin E3 ligase Smurf1 promotes degradation of PTEN leading to hyperactivation of the Akt/mTORC1 pathway. However, inhibitors of this pathway have not hitherto yielded promising results in clinical studies because of strong drug resistance. Here, we investigated Smurf1 expression in various glioblastoma (GB) cell lines and patient tissues. The therapeutic efficacy of Smurf1 silencing and Torin1 treatment was assessed in GB cells and orthotopic mouse model. We found Smurf1 loss elevates PTEN levels that interrupt the epidermal growth factor receptor pathway activity. Cotreatment with Smurf1 silencing and mTORC1/C2 inhibitor Torin1 remarkably decreased phosphorylation of Akt, and mTORC1 downstream targets 4EBP1 and S6K resulting in synergistic inhibitory effects. Smurf1 knockdown in orthotopic GB mouse model impaired tumor growth and enhanced cytotoxicity of Torin1. Together, these findings suggest a rational combination of Smurf1 inhibition and Torin1 as a promising new avenue to circumvent PI3K/Akt pathway-driven tumor progression and drug resistance.
    Keywords:  Molecular biology; Oncology
    DOI:  https://doi.org/10.1016/j.isci.2021.103528
  35. J Cell Sci. 2021 Dec 13. pii: jcs.258849. [Epub ahead of print]
    Regulation of Hook1-mediated endosomal sorting of clathrin-independent cargo by γ-taxilin.
    Satoru Higashi, Tomohiko Makiyama, Hiroshi Sakane, Satoru Nogami, Hiromichi Shirataki.
      In clathrin-independent endocytosis, Hook1, a microtubule- and cargo-tethering protein, participates in sorting of cargo proteins such as CD98 and CD147 into recycling endosomes. However, the molecular mechanism that regulates Hook1-mediated endosomal sorting is not fully understood. Here, we found that γ-taxilin is a novel regulator of Hook1-mediated endosomal sorting. γ-Taxilin depletion promoted both CD98-positive tubular formation and CD98 recycling. Conversely, overexpression of γ-taxilin inhibited the CD98-positive tubular formation. Depletion of Hook1, or Rab10 or Rab22a (which are both involved in Hook1-mediated endosomal sorting), attenuated the effect of γ-taxilin depletion on the CD98-positive tubular formation. γ-Taxilin depletion promoted CD147-mediated spreading of HeLa cells, suggesting that γ-taxilin may be a pivotal player in various cellular functions in which Hook1-mediated cargo proteins are involved. γ-Taxilin bound to the C-terminal region of Hook1 and inhibited its interaction with CD98; the latter interaction is necessary for sorting CD98. We suggest that γ-taxilin negatively regulates the sorting of Hook1-mediated cargo proteins into recycling endosomes by interfering with the interactions between Hook1 and the cargo proteins.
    Keywords:  Clathrin-independent endocytosis; Endosomal sorting; Hook1; Recycling endosome; Taxilin
    DOI:  https://doi.org/10.1242/jcs.258849
  36. Front Cardiovasc Med. 2021 ;8 763240
    Case Report: A Novel LAMP2 Splice-Altering Mutation Causes Cardiac-Only Danon Disease.
    Zongzhe Li, Fei Ma, Rui Li, Zhichao Xiao, Hesong Zeng, Dao Wen Wang.
      Danon disease (DD) is a rare glycogen storage lysosomal disorder caused by mutations in the LAMP2 gene. Patients with DD are usually characterized clinically by severe multisystem syndromes. We describe a specific family with a novel pathogenic splice-altering mutation in the LAMP2 gene (c.741+2T>C) with cardiac-only symptoms (frequent ventricular tachycardia, intraventricular block, and hypertrophic cardiomyopathy). Minigene assays were used to evaluate the consequence of the splice-site mutation in the LAMP2 gene. The results showed that the c.741+2T>C mutation led to extra 6-bp preservation of intron 5 at the junction between exons 5 and 6 during transcriptional processing of the mRNA, which creates a stop codon and truncated the LAMP2 protein to 248-amino-acid residues. The mutant LAMP2 protein was predicted to have a conformational change, lacks the important transmembrane domain, and subsequent protein destabilization.
    Keywords:  Danon disease; LAMP2; genetic diagnosis; splicing mutation; targeted sequencing
    DOI:  https://doi.org/10.3389/fcvm.2021.763240
  37. Cell Death Dis. 2021 Dec 11. 12(12): 1149
    CD82 protects against glaucomatous axonal transport deficits via mTORC1 activation in mice.
    Meng Ye, Jingqiu Huang, Qianxue Mou, Jing Luo, Yuanyuan Hu, Xiaotong Lou, Ke Yao, Bowen Zhao, Qiming Duan, Xing Li, Hong Zhang, Yin Zhao.
      Glaucoma is a leading cause of irreversible blindness worldwide and is characterized by progressive optic nerve degeneration and retinal ganglion cell loss. Axonal transport deficits have been demonstrated to be the earliest crucial pathophysiological changes underlying axonal degeneration in glaucoma. Here, we explored the role of the tetraspanin superfamily member CD82 in an acute ocular hypertension model. We found a transient downregulation of CD82 after acute IOP elevation, with parallel emergence of axonal transport deficits. The overexpression of CD82 with an AAV2/9 vector in the mouse retina improved optic nerve axonal transport and ameliorated subsequent axon degeneration. Moreover, the CD82 overexpression stimulated optic nerve regeneration and restored vision in a mouse optic nerve crush model. CD82 exerted a protective effect through the upregulation of TRAF2, which is an E3 ubiquitin ligase, and activated mTORC1 through K63-linked ubiquitylation and intracellular repositioning of Raptor. Therefore, our study offers deeper insight into the tetraspanin superfamily and demonstrates a potential neuroprotective strategy in glaucoma treatment.
    DOI:  https://doi.org/10.1038/s41419-021-04445-6
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