bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021–05–02
forty-five papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons.
  2. Elevated mRNA expression and defective processing of cathepsin D in HeLa cells lacking the mannose 6-phosphate pathway.
  3. Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes.
  4. Epstein-Barr Virus LMP1 Modulates the CD63 Interactome.
  5. Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.
  6. Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells.
  7. Current Approaches and Future Directions for the Treatment of mTORopathies.
  8. Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T.
  9. Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes.
  10. The Role of Exosomes in Lysosomal Storage Disorders.
  11. Mitochondria Turnover and Lysosomal Function in Hematopoietic Stem Cell Metabolism.
  12. Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle.
  13. Suppression of lysosomal acid alpha-glucosidase impacts the modulation of TFEB translocation in pancreatic cancer.
  14. Interstitial lung disease in lysosomal storage disorders.
  15. S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux.
  16. The Unique Phenotype of Lipid-Laden Macrophages.
  17. PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
  18. STED imaging the dynamic of lysosomes by dually fluorogenic Si-rhodamine.
  19. Convergent and Divergent Mechanisms of Epileptogenesis in mTORopathies.
  20. Modeling Sialidosis with Neural Precursor Cells Derived from Patient-Derived Induced Pluripotent Stem Cells.
  21. 4E-BP2-dependent translation in cerebellar Purkinje cells controls spatial memory but not autism-like behaviors.
  22. Biomarkers in Fabry Disease. Implications for Clinical Diagnosis and Follow-up.
  23. Targeting TFE3 Protects Against Lysosomal Malfunction-Induced Pyroptosis in Random Skin Flaps via ROS Elimination.
  24. A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling.
  25. Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.
  26. The Oncogene Transcription Factor EB Regulates Vascular Functions.
  27. Murine Models of Lysosomal Storage Diseases Exhibit Differences in Brain Protein Aggregation and Neuroinflammation.
  28. Connectivity Map Analysis of a Single-Cell RNA-Sequencing -Derived Transcriptional Signature of mTOR Signaling.
  29. An ω-3, but Not an ω-6 Polyunsaturated Fatty Acid Decreases Membrane Dipole Potential and Stimulates Endo-Lysosomal Escape of Penetratin.
  30. Phosphate restriction impairs mTORC1 signaling leading to increased bone marrow adipose tissue and decreased bone in growing mice.
  31. A comprehensive analysis of RAS-effector interactions reveals interaction hotspots and new binding partners.
  32. Rictor, an essential component of mTOR complex 2, undergoes caspase-mediated cleavage during apoptosis induced by multiple stimuli.
  33. A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets.
  34. Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes.
  35. PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism.
  36. Lysosome transport interrupted.
  37. mTORC1 Inhibition in Metastatic Breast Cancer Patients Negatively Affects Peripheral NK Cell Maturation and Number.
  38. Brain-specific functions of the endocytic machinery.
  39. The Clinical Significance and Potential Role of Cathepsin S in IgA Nephropathy.
  40. In Silico Analysis of the Molecular-Level Impact of SMPD1 Variants on Niemann-Pick Disease Severity.
  41. Increased Alveolar Heparan Sulphate and Reduced Pulmonary Surfactant Amount and Function in the Mucopolysaccharidosis IIIA Mouse.
  42. Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology.
  43. Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development.
  44. Genetic removal of p70 S6K1 corrects coding sequence length-dependent alterations in mRNA translation in fragile X syndrome mice.
  45. Fabry Disease and the Heart: A Comprehensive Review.
  1. Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00350-8. [Epub ahead of print]35(4): 109034
    Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons.
    Pearl P Y Lie, Dun-Sheng Yang, Philip Stavrides, Chris N Goulbourne, Ping Zheng, Panaiyur S Mohan, Anne M Cataldo, Ralph A Nixon.
      Lysosomal trafficking and maturation in neurons remain poorly understood and are unstudied in vivo despite high disease relevance. We generated neuron-specific transgenic mice to track vesicular CTSD acquisition, acidification, and traffic within the autophagic-lysosomal pathway in vivo, revealing that mature lysosomes are restricted from axons. Moreover, TGN-derived transport carriers (TCs), not lysosomes, supply lysosomal components to axonal organelles. Ultrastructurally distinctive TCs containing TGN and lysosomal markers enter axons, engaging autophagic vacuoles and late endosomes. This process is markedly upregulated in dystrophic axons of Alzheimer models. In cultured neurons, most axonal LAMP1 vesicles are weakly acidic TCs that shuttle lysosomal components bidirectionally, conferring limited degradative capability to retrograde organelles before they mature fully to lysosomes within perikarya. The minor LAMP1 subpopulation attaining robust acidification are retrograde Rab7+ endosomes/amphisomes, not lysosomes. Restricted lysosome entry into axons explains the unique lysosome distribution in neurons and their vulnerability toward neuritic dystrophy in disease.
    Keywords:  LAMP1; acidification; autophagic vacuole; axonal transport; late endosome; lysosome; post-Golgi traffic; transport carrier
    DOI:  https://doi.org/10.1016/j.celrep.2021.109034
  2. FEBS Open Bio. 2021 May 01.
    Elevated mRNA expression and defective processing of cathepsin D in HeLa cells lacking the mannose 6-phosphate pathway.
    Lin Liu, Balraj Doray.
      Disruption of the mannose 6-phosphate (M-6-P) pathway in HeLa cells by inactivation of the GNPTAB gene, which encodes the α/β subunits of GlcNAc-1-phosphotransferase, results in missorting of newly synthesized lysosomal acid hydrolases to the cell culture media instead of transport to the endolysosomal system. We previously demonstrated that the majority of the lysosomal aspartyl protease, cathepsin D, is secreted in these GNPTAB-/- HeLa cells. However, the intracellular content of cathepsin D in these cells was still greater than that of WT HeLa cells which retained most of the protease, indicating a marked elevation of cathepsin D expression in response to abrogation of the M-6-P pathway. Here, we demonstrate that HeLa cells lacking GlcNAc-1-phosphotransferase show a 5-fold increase in cathepsin D mRNA expression over control cells, accounting for the increase of cathepsin D at the protein level. Further, we show that this increase at the mRNA level occurs independent of the transcription factors TFEB and TFE3. The intracellular cathepsin D can still be trafficked to lysosomes in the absence of the M-6-P pathway, but fails to undergo proteolytic processing into the fully mature heavy and light chains. Uptake experiments performed by feeding GNPTAB-/- HeLa cells with various phosphorylated cathepsins reveal that only cathepsin B is capable of partially restoring cleavage, providing evidence for a role for cathepsin B in the proteolytic processing of cathepsin D.
    Keywords:  GlcNAc-1-phosphotransferase; cathepsin B; cathepsin D; cathepsin L; lysosomes; mannose 6-phosphate pathway
    DOI:  https://doi.org/10.1002/2211-5463.13169
  3. J Biol Chem. 2021 Apr 22. pii: S0021-9258(21)00492-0. [Epub ahead of print] 100703
    Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes.
    Michael C Jaskolka, Maureen Tarsio, Anne M Smardon, Md Murad Khan, Patricia M Kane.
      The vacuolar H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid, reversible disassembly of the peripheral V1 domain from the integral membrane Vo domain, accompanied by release of the V1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H+-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V1 complexes. Both purified RAVE and RAVE-V1 contain substoichiometric levels of subunit C. RAVE-V1 binds tightly to expressed subunit C in vitro but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V1 interacts with the Vo subunit Vph1NT in vitro. RAVE-V1 complexes, like isolated V1, have no MgATPase activity, suggesting that RAVE cannot reverse V1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V1 carrying a mutant subunit H incapable of inhibition with Vo complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.
    Keywords:  RAVE; assembly; lysosomal acidification; proton pump; regulation; vacuolar ATPase; vacuole; yeast
    DOI:  https://doi.org/10.1016/j.jbc.2021.100703
  4. Viruses. 2021 Apr 15. pii: 675. [Epub ahead of print]13(4):
    Epstein-Barr Virus LMP1 Modulates the CD63 Interactome.
    Mujeeb Cheerathodi, Dingani Nkosi, Allaura S Cone, Sara B York, David G Meckes.
      Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains; it is associated with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in the cellular trafficking of different proteins, EV cargo sorting, and vesicle formation. We have previously shown that CD63 is important in LMP1 trafficking to EVs, and this also affects LMP1-mediated intracellular signaling including MAPK/ERK, NF-κB, and mTOR activation. Using the BioID method combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as a network of proximal interacting proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism, and transportation. The CD63-only interactome was enriched in Rab GTPases, SNARE proteins, and sorting nexins, while adding LMP1 into the interactome increased the presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of protein enrichment from protein localization and vesicle-mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTOR, Nedd4 L, and PP2A, indicating the formation of a multiprotein complex with CD63, thereby potentially regulating LMP1-dependent mTOR signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into the network of partners required for endocytic trafficking and extracellular vesicle cargo sorting, formation, and secretion.
    Keywords:  CD63; Epstein-Barr virus; Herpesvirus; autophagy; exosomes; extracellular vesicles; interactions; latent membrane protein 1; mTOR; mass spectrometry; proteomics; signaling; tetraspanin
    DOI:  https://doi.org/10.3390/v13040675
  5. Int J Mol Sci. 2021 Apr 19. pii: 4220. [Epub ahead of print]22(8):
    Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.
    Graciela Argüello, Elisa Balboa, Pablo J Tapia, Juan Castro, María José Yañez, Pamela Mattar, Rodrigo Pulgar, Silvana Zanlungo.
      Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in β-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.
    Keywords:  Niemann–Pick C; TFEB; cholesterol; genistein; lysosomal storage diseases; lysosome clearance
    DOI:  https://doi.org/10.3390/ijms22084220
  6. Sci Rep. 2021 Apr 26. 11(1): 8927
    Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells.
    Alice Dupont Juhl, Christian W Heegaard, Stephan Werner, Gerd Schneider, Kathiresan Krishnan, Douglas F Covey, Daniel Wüstner.
      Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.
    DOI:  https://doi.org/10.1038/s41598-021-87876-7
  7. Dev Neurosci. 2021 Apr 28. 1-16
    Current Approaches and Future Directions for the Treatment of mTORopathies.
    Vasiliki Karalis, Helen S Bateup.
      The mechanistic target of rapamycin (mTOR) is a kinase at the center of an evolutionarily conserved signaling pathway that orchestrates cell growth and metabolism. mTOR responds to an array of intra- and extracellular stimuli and in turn controls multiple cellular anabolic and catabolic processes. Aberrant mTOR activity is associated with numerous diseases, with particularly profound impact on the nervous system. mTOR is found in two protein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2), which are governed by different upstream regulators and have distinct cellular actions. Mutations in genes encoding for mTOR regulators result in a collection of neurodevelopmental disorders known as mTORopathies. While these disorders can affect multiple organs, neuropsychiatric conditions such as epilepsy, intellectual disability, and autism spectrum disorder have a major impact on quality of life. The neuropsychiatric aspects of mTORopathies have been particularly challenging to treat in a clinical setting. Current therapeutic approaches center on rapamycin and its analogs, drugs that are administered systemically to inhibit mTOR activity. While these drugs show some clinical efficacy, adverse side effects, incomplete suppression of mTOR targets, and lack of specificity for mTORC1 or mTORC2 may limit their utility. An increased understanding of the neurobiology of mTOR and the underlying molecular, cellular, and circuit mechanisms of mTOR-related disorders will facilitate the development of improved therapeutics. Animal models of mTORopathies have helped unravel the consequences of mTOR pathway mutations in specific brain cell types and developmental stages, revealing an array of disease-related phenotypes. In this review, we discuss current progress and potential future directions for the therapeutic treatment of mTORopathies with a focus on findings from genetic mouse models.
    Keywords:  Epilepsy; Neurodevelopmental disorders; PTEN; Rapamycin; Raptor; Rictor; Tuberous sclerosis complex; mTORC1; mTORC2; mTORopathy
    DOI:  https://doi.org/10.1159/000515672
  8. Int J Mol Sci. 2021 Apr 13. pii: 4009. [Epub ahead of print]22(8):
    Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T.
    Maik Liedtke, Christin Völkner, Alexandra V Jürs, Franziska Peter, Michael Rabenstein, Andreas Hermann, Moritz J Frech.
      Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.
    Keywords:  Niemann-Pick type C2 (NP-C2); autophagy; cholesterol; filipin; hiPSC; oxidative stress; patch clamp
    DOI:  https://doi.org/10.3390/ijms22084009
  9. Toxins (Basel). 2021 Apr 09. pii: 272. [Epub ahead of print]13(4):
    Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes.
    Masahiro Nagahama, Keiko Kobayashi, Sadayuki Ochi, Masaya Takehara.
      Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.
    Keywords:  C. botulinum C2 toxin; cathepsin B; internalization
    DOI:  https://doi.org/10.3390/toxins13040272
  10. Biomolecules. 2021 Apr 15. pii: 576. [Epub ahead of print]11(4):
    The Role of Exosomes in Lysosomal Storage Disorders.
    Adenrele M Gleason, Elizabeth G Woo, Cindy McKinney, Ellen Sidransky.
      Exosomes, small membrane-bound organelles formed from endosomal membranes, represent a heterogenous source of biological and pathological biomarkers capturing the metabolic status of a cell. Exosomal cargo, including lipids, proteins, mRNAs, and miRNAs, can either act as inter-cellular messengers or are shuttled for autophagic/lysosomal degradation. Most cell types in the central nervous system (CNS) release exosomes, which serve as long and short distance communicators between neurons, astrocytes, oligodendrocytes, and microglia. Lysosomal storage disorders are diseases characterized by the accumulation of partially or undigested cellular waste. The exosomal content in these diseases is intrinsic to each individual disorder. Emerging research indicates that lysosomal dysfunction enhances exocytosis, and hence, in lysosomal disorders, exosomal secretion may play a role in disease pathogenesis. Furthermore, the unique properties of exosomes and their ability to carry cargo between adjacent cells and organs, and across the blood-brain barrier, make them attractive candidates for use as therapeutic delivery vehicles. Thus, understanding exosomal content and function may have utility in the treatment of specific lysosomal storage disorders. Since lysosomal dysfunction and the deficiency of at least one lysosomal enzyme, glucocerebrosidase, is associated with the development of parkinsonism, the study and use of exosomes may contribute to an improved understanding of Parkinson disease, potentially leading to new therapeutics.
    Keywords:  Gaucher disease; Parkinson disease; endocytic pathways; exosomes; lysosomal storage disorder; lysosomes; neurodegenerative disease
    DOI:  https://doi.org/10.3390/biom11040576
  11. Int J Mol Sci. 2021 Apr 28. pii: 4627. [Epub ahead of print]22(9):
    Mitochondria Turnover and Lysosomal Function in Hematopoietic Stem Cell Metabolism.
    Makiko Mochizuki-Kashio, Hiroko Shiozaki, Toshio Suda, Ayako Nakamura-Ishizu.
      Hematopoietic stem cells (HSCs) reside in a hypoxic microenvironment that enables glycolysis-fueled metabolism and reduces oxidative stress. Nonetheless, metabolic regulation in organelles such as the mitochondria and lysosomes as well as autophagic processes have been implicated as essential for the determination of HSC cell fate. This review encompasses the current understanding of anaerobic metabolism in HSCs as well as the emerging roles of mitochondrial metabolism and lysosomal regulation for hematopoietic homeostasis.
    Keywords:  ROS; autophagy; folliculin; hematopoietic stem cells; lysosome; mitochondria
    DOI:  https://doi.org/10.3390/ijms22094627
  12. Nutrients. 2021 Apr 09. pii: 1236. [Epub ahead of print]13(4):
    Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle.
    Lacee J Laufenberg, Kristen T Crowell, Charles H Lang.
      The Ragulator protein complex is critical for directing the Rag GTPase proteins and mTORC1 to the lysosome membrane mediating amino acid-stimulated protein synthesis. As there is a lack of evidence on alcohol's effect on the Rag-Ragulator complex as a possible mechanism for the development of alcoholic skeletal muscle wasting, the aim of our study was to examine alterations in various protein-protein complexes in the Rag-Ragulator pathway produced acutely by feeding and how these are altered by alcohol under in vivo conditions. Mice (C57Bl/6; adult males) were fasted, and then provided rodent chow for 30 min ("refed") or remained food-deprived ("fasted"). Mice subsequently received ethanol (3 g/kg ethanol) or saline intraperitoneally, and hindlimb muscles were collected 1 h thereafter for analysis. Refeeding-induced increases in myofibrillar and sarcoplasmic protein synthesis, and mTOR and S6K1 phosphorylation, were prevented by alcohol. This inhibition was not associated with a differential rise in the intracellular leucine concentration or plasma leucine or insulin levels. Alcohol increased the amount of the Sestrin1•GATOR2 complex in the fasted state and prevented the refeeding-induced decrease in Sestrin1•GATOR2 seen in control mice. Alcohol antagonized the increase in the RagA/C•Raptor complex formation seen in the refed state. Alcohol antagonized the increase in Raptor with immunoprecipitated LAMPTOR1 (part of the Ragulator complex) after refeeding and decreased the association of RagC with LAMPTOR1. Finally, alcohol increased the association of the V1 domain of v-ATPase with LAMPTOR1 and prevented the refeeding-induced decrease in v-ATPase V1 with LAMPTOR1. Overall, these data demonstrate that acute alcohol intake disrupts multiple protein-protein complexes within the Rag-Ragulator complex, which are associated with and consistent with the concomitant decline in nutrient-stimulated muscle protein synthesis under in vivo conditions.
    Keywords:  anabolic resistance; leucine: LAMPTOR1: V-ATPase; mTORC1; protein synthesis
    DOI:  https://doi.org/10.3390/nu13041236
  13. Cancer Sci. 2021 May 01.
    Suppression of lysosomal acid alpha-glucosidase impacts the modulation of TFEB translocation in pancreatic cancer.
    Ryoga Hamura, Yoshihiro Shirai, Yohta Shimada, Nobuhiro Saito, Tomohiko Taniai, Takashi Horiuchi, Naoki Takada, Yumi Kanegae, Toru Ikegami, Toya Ohashi, Katsuhiko Yanaga.
      Lysosomal degradation plays a crucial role in the metabolism of biological macromolecules supplied by autophagy. Recent evidence demonstrated the regulation of the autophagy-lysosome system, which contributes to intracellular homeostasis, chemoresistance, and tumor progression, as a promising therapeutic approach for pancreatic cancer (PC). However, the details of lysosomal catabolic function in PC cells is not fully elucidated. In this study, we show evidence that suppression of acid alpha-glucosidase (GAA), one of the lysosomal enzymes, improves chemosensitivity and exerts apoptotic effects on PC cells through the disturbance of expression of the transcription factor EB. The levels of lysosomal enzyme were elevated by gemcitabine in PC cells. In particular, the levels of GAA were responsive to gemcitabine in a dose- and time-dependent manner. Small interfering RNA against the GAA gene (siGAA) suppressed cell proliferation and promoted apoptosis in gemcitabine-treated PC cells. In untreated PC cells, we observed accumulation of depolarized mitochondria. Gene therapy using adenoviral vector carrying short hairpin RNA against the GAA gene increased the number of apoptotic cells and decreased the tumor growth in xenograft model mice. These results indicate that GAA is one of the key targets to improve the efficacy of gemcitabine and develop novel therapies for PC.
    Keywords:  GAA; Lysosome; TFEB translocation; gene therapy; pancreatic cancer
    DOI:  https://doi.org/10.1111/cas.14921
  14. Eur Respir Rev. 2021 Jun 30. pii: 200363. [Epub ahead of print]30(160):
    Interstitial lung disease in lysosomal storage disorders.
    Raphaël Borie, Bruno Crestani, Alice Guyard, Olivier Lidove.
      Lysosomes are intracellular organelles that are responsible for degrading and recycling macromolecules. Lysosomal storage diseases (LSDs) are a group of inherited diseases caused by mutations affecting genes that encode the function of the lysosomal enzymes. Three LSDs are associated with lung involvement and/or interstitial lung disease (ILD): Gaucher disease (GD); Niemann-Pick disease, also known as acid sphingomyelinase deficiency (ASMD); and Fabry disease (FD). In GD and in ASMD, analysis of bronchoalveolar lavage fluid and lung biopsy can be informative, showing foamy cells. In GD, ILD is rare. Enzyme replacement therapy (ERT) has been available since 1991 and has greatly changed the natural history of GD, with pulmonary failure and death reported before the ERT era. In ASMD, ILD is frequent and is usually associated with spleen enlargement, low platelet cell count and low level of high-density lipoprotein-cholesterol. Results of ERT are promising regarding preliminary results of olipudase alfa in paediatric and adult ASMD populations. The most frequent respiratory manifestation in FD is COPD-like symptoms regardless of smoking habit and dyspnoea due to congestive heart failure. Early diagnosis of these three LSDs is crucial to prevent irreversible organ damage. Early initiation of ERT can, at least in part, prevent organ failure.
    DOI:  https://doi.org/10.1183/16000617.0363-2020
  15. Cells. 2021 Apr 17. pii: 929. [Epub ahead of print]10(4):
    S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux.
    Aleksandra Hać, Karolina Pierzynowska, Anna Herman-Antosiewicz.
      Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.
    Keywords:  S6 kinase 1 (S6K1); autophagic flux; autophagosome-lysosome fusion; lysosome; serum deprivation; sulforaphane; tubulin acetylation
    DOI:  https://doi.org/10.3390/cells10040929
  16. Int J Mol Sci. 2021 Apr 14. pii: 4039. [Epub ahead of print]22(8):
    The Unique Phenotype of Lipid-Laden Macrophages.
    Marco van Eijk, Johannes M F G Aerts.
      Macrophages are key multi-talented cells of the innate immune system and are equipped with receptors involved in damage and pathogen recognition with connected immune response guiding signaling systems. In addition, macrophages have various systems that are involved in the uptake of extracellular and intracellular cargo. The lysosomes in macrophages play a central role in the digestion of all sorts of macromolecules and the entry of nutrients to the cytosol, and, thus, the regulation of endocytic processes and autophagy. Simplistically viewed, two macrophage phenotype extremes exist. On one end of the spectrum, the classically activated pro-inflammatory M1 cells are present, and, on the other end, alternatively activated anti-inflammatory M2 cells. A unique macrophage population arises when lipid accumulation occurs, either caused by flaws in the catabolic machinery, which is observed in lysosomal storage disorders, or as a result of an acquired condition, which is found in multiple sclerosis, obesity, and cardiovascular disease. The accompanying overload causes a unique metabolic activation phenotype, which is discussed here, and, consequently, a unifying phenotype is proposed.
    Keywords:  GPNMB; Gaucher disease; TREM-2; adipose tissue; foam cell; macrophage; multiple sclerosis; obesity
    DOI:  https://doi.org/10.3390/ijms22084039
  17. J Cell Sci. 2022 Mar 01. pii: jcs255026. [Epub ahead of print]135(5):
    PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
    Yuan Gao, Juan Xiong, Qing-Zhu Chu, Wei-Ke Ji.
      Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and late endosomes/lysosomes (LE/lys) are emerging as critical hubs for diverse cellular events, and changes in their extents are linked to severe neurological diseases. While recent studies show that the synaptotagmin-like mitochondrial-lipid-binding (SMP) domain-containing protein PDZD8 may mediate the formation of ER-LE/lys MCSs, the cellular functions of PDZD8 remain largely elusive. Here, we attempt to investigate the lipid transfer activities of PDZD8 and the extent to which its cellular functions depend on its lipid transfer activities. In accordance with recent studies, we demonstrate that PDZD8 is a protrudin (ZFYVE27)-interacting protein and that PDZD8 acts as a tether at ER-LE/lys MCSs. Furthermore, we discover that the SMP domain of PDZD8 binds glycerophospholipids and ceramides both in vivo and in vitro, and that the SMP domain can transport lipids between membranes in vitro. Functionally, PDZD8 is required for LE/lys positioning and neurite outgrowth, which is dependent on the lipid transfer activity of the SMP domain.
    Keywords:  Endoplasmic reticulum; Late endosomes; Lipid transfer; Lysosomes; Membrane contact sites; PDZD8
    DOI:  https://doi.org/10.1242/jcs.255026
  18. Chemistry. 2021 Apr 26.
    STED imaging the dynamic of lysosomes by dually fluorogenic Si-rhodamine.
    Mengting Fan, Haiyan An, Chuanfeng Wang, Shuhui Huo, Ting Wang, Xiaoyan Cui, Dazhi Zhang.
      Super-resolution microscopy (SRM) imaging of the finite subcellular structures and subtle bioactivities inside organelles delivers abundant cellular information with high fidelity to unravel the intricate biological processes. An ideal fluorescent probe with precise control of fluorescence is critical in SRM technique like stimulated emission depletion (STED). Si-rhodamine was decorated with both targeting group and H + -receptor, affording the dually fluorogenic Si-rhodamine in which the NIR fluorescence was efficiently controlled by the coalescent of spirolactone-zwitterion equilibrium and PeT mechanism. The dually fluorogenic characters of the probe offer a perfect mutual enhancement in sensitivity, specificity and spatial resolution. Strong fluorescence only released in the existence of targeting protein at acidic lysosomal pH, ensured precisely tracking the dynamic of lysosomal structure and pH in living cells by STED.
    Keywords:  Fluorogenic; STED; Si-rhodamine; lysosome *
    DOI:  https://doi.org/10.1002/chem.202100623
  19. Front Neuroanat. 2021 ;15 664695
    Convergent and Divergent Mechanisms of Epileptogenesis in mTORopathies.
    Lena H Nguyen, Angélique Bordey.
      Hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) due to mutations in genes along the PI3K-mTOR pathway and the GATOR1 complex causes a spectrum of neurodevelopmental disorders (termed mTORopathies) associated with malformation of cortical development and intractable epilepsy. Despite these gene variants' converging impact on mTORC1 activity, emerging findings suggest that these variants contribute to epilepsy through both mTORC1-dependent and -independent mechanisms. Here, we review the literature on in utero electroporation-based animal models of mTORopathies, which recapitulate the brain mosaic pattern of mTORC1 hyperactivity, and compare the effects of distinct PI3K-mTOR pathway and GATOR1 complex gene variants on cortical development and epilepsy. We report the outcomes on cortical pyramidal neuronal placement, morphology, and electrophysiological phenotypes, and discuss some of the converging and diverging mechanisms responsible for these alterations and their contribution to epileptogenesis. We also discuss potential therapeutic strategies for epilepsy, beyond mTORC1 inhibition with rapamycin or everolimus, that could offer personalized medicine based on the gene variant.
    Keywords:  GATOR1 complex; cortical development; epilepsy; focal cortical dysplasia; in utero electroporation; mTOR; neuron migration; tuberous sclerosis complex
    DOI:  https://doi.org/10.3389/fnana.2021.664695
  20. Int J Mol Sci. 2021 Apr 22. pii: 4386. [Epub ahead of print]22(9):
    Modeling Sialidosis with Neural Precursor Cells Derived from Patient-Derived Induced Pluripotent Stem Cells.
    Binna Seol, Young-Dae Kim, Yee Sook Cho.
      Sialidosis, caused by a genetic deficiency of the lysosomal sialidase gene (NEU1), is a systemic disease involving various tissues and organs, including the nervous system. Understanding the neurological dysfunction and pathology associated with sialidosis remains a challenge, partially due to the lack of a human model system. In this study, we have generated two types of induced pluripotent stem cells (iPSCs) with sialidosis-specific NEU1G227R and NEU1V275A/R347Q mutations (sialidosis-iPSCs), and further differentiated them into neural precursor cells (iNPCs). Characterization of NEU1G227R- and NEU1V275A/R347Q- mutated iNPCs derived from sialidosis-iPSCs (sialidosis-iNPCs) validated that sialidosis-iNPCs faithfully recapitulate key disease-specific phenotypes, including reduced NEU1 activity and impaired lysosomal and autophagic function. In particular, these cells showed defective differentiation into oligodendrocytes and astrocytes, while their neuronal differentiation was not notably affected. Importantly, we found that the phenotypic defects of sialidosis-iNPCs, such as impaired differentiation capacity, could be effectively rescued by the induction of autophagy with rapamycin. Our results demonstrate the first use of a sialidosis-iNPC model with NEU1G227R- and NEU1V275A/R347Q- mutation(s) to study the neurological defects of sialidosis, particularly those related to a defective autophagy-lysosome pathway, and may help accelerate the development of new drugs and therapeutics to combat sialidosis and other LSDs.
    Keywords:  NEU1; induced pluripotent stem cell; lysosomal storage disease; neural cell model; sialidosis
    DOI:  https://doi.org/10.3390/ijms22094386
  21. Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00352-1. [Epub ahead of print]35(4): 109036
    4E-BP2-dependent translation in cerebellar Purkinje cells controls spatial memory but not autism-like behaviors.
    Mehdi Hooshmandi, Vinh Tai Truong, Eviatar Fields, Riya Elizabeth Thomas, Calvin Wong, Vijendra Sharma, Ilse Gantois, Patricia Soriano Roque, Kleanthi Chalkiadaki, Neil Wu, Anindyo Chakraborty, Soroush Tahmasebi, Masha Prager-Khoutorsky, Nahum Sonenberg, Aparna Suvrathan, Alanna J Watt, Christos G Gkogkas, Arkady Khoutorsky.
      Recent studies have demonstrated that selective activation of mammalian target of rapamycin complex 1 (mTORC1) in the cerebellum by deletion of the mTORC1 upstream repressors TSC1 or phosphatase and tensin homolog (PTEN) in Purkinje cells (PCs) causes autism-like features and cognitive deficits. However, the molecular mechanisms by which overactivated mTORC1 in the cerebellum engenders these behaviors remain unknown. The eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) is a central translational repressor downstream of mTORC1. Here, we show that mice with selective ablation of 4E-BP2 in PCs display a reduced number of PCs, increased regularity of PC action potential firing, and deficits in motor learning. Surprisingly, although spatial memory is impaired in these mice, they exhibit normal social interaction and show no deficits in repetitive behavior. Our data suggest that, downstream of mTORC1/4E-BP2, there are distinct cerebellar mechanisms independently controlling social behavior and memory formation.
    Keywords:  4E-BP2; Purkinje cells; autism spectrum disorders; motor learning; spatial memory
    DOI:  https://doi.org/10.1016/j.celrep.2021.109036
  22. J Clin Med. 2021 Apr 13. pii: 1664. [Epub ahead of print]10(8):
    Biomarkers in Fabry Disease. Implications for Clinical Diagnosis and Follow-up.
    Clara Carnicer-Cáceres, Jose Antonio Arranz-Amo, Cristina Cea-Arestin, Maria Camprodon-Gomez, David Moreno-Martinez, Sara Lucas-Del-Pozo, Marc Moltó-Abad, Ariadna Tigri-Santiña, Irene Agraz-Pamplona, Jose F Rodriguez-Palomares, Jorge Hernández-Vara, Mar Armengol-Bellapart, Mireia Del-Toro-Riera, Guillem Pintos-Morell.
      Fabry disease (FD) is a lysosomal storage disorder caused by deficient alpha-galactosidase A activity in the lysosome due to mutations in the GLA gene, resulting in gradual accumulation of globotriaosylceramide and other derivatives in different tissues. Substrate accumulation promotes different pathogenic mechanisms in which several mediators could be implicated, inducing multiorgan lesions, mainly in the kidney, heart and nervous system, resulting in clinical manifestations of the disease. Enzyme replacement therapy was shown to delay disease progression, mainly if initiated early. However, a diagnosis in the early stages represents a clinical challenge, especially in patients with a non-classic phenotype, which prompts the search for biomarkers that help detect and predict the evolution of the disease. We have reviewed the mediators involved in different pathogenic mechanisms that were studied as potential biomarkers and can be easily incorporated into clinical practice. Some accumulation biomarkers seem to be useful to detect non-classic forms of the disease and could even improve diagnosis of female patients. The combination of such biomarkers with some response biomarkers, may be useful for early detection of organ injury. The incorporation of some biomarkers into clinical practice may increase the capacity of detection compared to that currently obtained with the established diagnostic markers and provide more information on the progression and prognosis of the disease.
    Keywords:  Gb3; biomarkers; cardiomyopathy; chronic kidney disease; classic phenotype; fabry disease; inflammatory response; late-onset phenotype; lyso-Gb3; vasculopathy
    DOI:  https://doi.org/10.3390/jcm10081664
  23. Front Cell Dev Biol. 2021 ;9 643996
    Targeting TFE3 Protects Against Lysosomal Malfunction-Induced Pyroptosis in Random Skin Flaps via ROS Elimination.
    Jiafeng Li, Junsheng Lou, Gaoxiang Yu, Yijie Chen, Ruiheng Chen, Zhuliu Chen, Chenyu Wu, Jian Ding, Yu Xu, Jingtao Jiang, Huazi Xu, Xuwei Zhu, Weiyang Gao, Kailiang Zhou.
      Increasing evidence indicates that pyroptosis, a new type of programmed cell death, may participate in random flap necrosis and play an important role. ROS-induced lysosome malfunction is an important inducement of pyroptosis. Transcription factor E3 (TFE3) exerts a decisive effect in oxidative metabolism and lysosomal homeostasis. We explored the effect of pyroptosis in random flap necrosis and discussed the effect of TFE3 in modulating pyroptosis. Histological analysis via hematoxylin-eosin staining, immunohistochemistry, general evaluation of flaps, evaluation of tissue edema, and laser Doppler blood flow were employed to determine the survival of the skin flaps. Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays were used to calculate the expressions of pyroptosis, oxidative stress, lysosome function, and the AMPK-MCOLN1 signaling pathway. In cell experiments, HUVEC cells were utilized to ensure the relationship between TFE3, reactive oxygen species (ROS)-induced lysosome malfunction and cell pyroptosis. Our results indicate that pyroptosis exists in the random skin flap model and oxygen and glucose deprivation/reperfusion cell model. In addition, NLRP3-mediated pyroptosis leads to necrosis of the flaps. Moreover, we also found that ischemic flaps can augment the accumulation of ROS, thereby inducing lysosomal malfunction and finally initiating pyroptosis. Meanwhile, we observed that TFE3 levels are interrelated with ROS levels, and overexpression and low expression of TFE3 levels can, respectively, inhibit and promote ROS-induced lysosomal dysfunction and pyroptosis during in vivo and in vitro experiments. In conclusion, we found the activation of TFE3 in random flaps is partially regulated by the AMPK-MCOLN1 signal pathway. Taken together, TFE3 is a key regulator of ROS-induced pyroptosis in random skin flaps, and TFE3 may be a promising therapeutic target for improving random flap survival.
    Keywords:  AMPK-MCOLN1 signaling pathway; ROS; TFE3; pyroptosis; random skin flap
    DOI:  https://doi.org/10.3389/fcell.2021.643996
  24. Front Mol Biosci. 2021 ;8 646574
    A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling.
    Stephen J Fairweather, Shoko Okada, Gregory Gauthier-Coles, Kiran Javed, Angelika Bröer, Stefan Bröer.
      Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of Xenopus laevis oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in Xe nopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.
    Keywords:  GC-MS; SNAT2; Xenopus laevis oocytes; amino acid signaling; amino acid transporters; mTORC1 signaling; metabolomics; slc38a2
    DOI:  https://doi.org/10.3389/fmolb.2021.646574
  25. Cancers (Basel). 2021 Apr 21. pii: 2004. [Epub ahead of print]13(9):
    Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.
    Prabhu Thirusangu, Christopher L Pathoulas, Upasana Ray, Yinan Xiao, Julie Staub, Ling Jin, Ashwani Khurana, Viji Shridhar.
      We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.
    Keywords:  CTSL; LMP; MOMP; autophagy; ovarian cancer; quinacrine
    DOI:  https://doi.org/10.3390/cancers13092004
  26. Front Physiol. 2021 ;12 640061
    The Oncogene Transcription Factor EB Regulates Vascular Functions.
    Gabriella Doronzo, Elena Astanina, Federico Bussolino.
      Transcription factor EB (TFEB) represents an emerging player in vascular biology. It belongs to the bHLH-leucine zipper transcription factor microphthalmia family, which includes microphthalmia-associated transcription factor, transcription factor E3 and transcription factor EC, and is known to be deregulated in cancer. The canonical transcriptional pathway orchestrated by TFEB adapts cells to stress in all kinds of tissues by supporting lysosomal and autophagosome biogenesis. However, emerging findings highlight that TFEB activates other genetic programs involved in cell proliferation, metabolism, inflammation and immunity. Here, we first summarize the general principles and mechanisms by which TFEB activates its transcriptional program. Then, we analyze the current knowledge of TFEB in the vascular system, placing particular emphasis on its regulatory role in angiogenesis and on the involvement of the vascular unit in inflammation and atherosclerosis.
    Keywords:  angiogenesis; autophagy; cell cycle; embryo; inflammation
    DOI:  https://doi.org/10.3389/fphys.2021.640061
  27. Biomedicines. 2021 Apr 21. pii: 446. [Epub ahead of print]9(5):
    Murine Models of Lysosomal Storage Diseases Exhibit Differences in Brain Protein Aggregation and Neuroinflammation.
    Jennifer Clarke, Can Kayatekin, Catherine Viel, Lamya Shihabuddin, Sergio Pablo Sardi.
      Genetic, epidemiological and experimental evidence implicate lysosomal dysfunction in Parkinson's disease (PD) and related synucleinopathies. Investigate several mouse models of lysosomal storage diseases (LSDs) and evaluate pathologies reminiscent of synucleinopathies. We obtained brain tissue from symptomatic mouse models of Gaucher, Fabry, Sandhoff, Niemann-Pick A (NPA), Hurler, Pompe and Niemann-Pick C (NPC) diseases and assessed for the presence of Lewy body-like pathology (proteinase K-resistant α-synuclein and tau aggregates) and neuroinflammation (microglial Iba1 and astrocytic GFAP) by immunofluorescence. All seven LSD models exhibited evidence of proteinopathy and/or inflammation in the central nervous system (CNS). However, these phenotypes were divergent. Gaucher and Fabry mouse models displayed proteinase K-resistant α-synuclein and tau aggregates but no neuroinflammation; whereas Sandhoff, NPA and NPC showed marked neuroinflammation and no overt proteinopathy. Pompe disease animals uniquely displayed widespread distribution of tau aggregates accompanied by moderate microglial activation. Hurler mice also demonstrated proteinopathy and microglial activation. The present study demonstrated additional links between LSDs and pathogenic phenotypes that are hallmarks of synucleinopathies. The data suggest that lysosomal dysregulation can contribute to brain region-specific protein aggregation and induce widespread neuroinflammation in the brain. However, only a few LSD models examined exhibited phenotypes consistent with synucleinopathies. While no model can recapitulate the complexity of PD, they can enable the study of specific pathways and mechanisms contributing to disease pathophysiology. The present study provides evidence that there are existing, previously unutilized mouse models that can be employed to study pathogenic mechanisms and gain insights into potential PD subtypes, helping to determine if they are amenable to pathway-specific therapeutic interventions.
    Keywords:  lysosomal diseases; mouse models of disease; neuroinflammation; synuclein; tau
    DOI:  https://doi.org/10.3390/biomedicines9050446
  28. Int J Mol Sci. 2021 Apr 22. pii: 4371. [Epub ahead of print]22(9):
    Connectivity Map Analysis of a Single-Cell RNA-Sequencing -Derived Transcriptional Signature of mTOR Signaling.
    Naim Al Mahi, Erik Y Zhang, Susan Sherman, Jane J Yu, Mario Medvedovic.
      In the connectivity map (CMap) approach to drug repositioning and development, transcriptional signature of disease is constructed by differential gene expression analysis between the diseased tissue or cells and the control. The negative correlation between the transcriptional disease signature and the transcriptional signature of the drug, or a bioactive compound, is assumed to indicate its ability to "reverse" the disease process. A major limitation of traditional CMaP analysis is the use of signatures derived from bulk disease tissues. Since the key driver pathways are most likely dysregulated in only a subset of cells, the "averaged" transcriptional signatures resulting from bulk analysis lack the resolution to effectively identify effective therapeutic agents. The use of single-cell RNA-seq (scRNA-seq) transcriptomic assay facilitates construction of disease signatures that are specific to individual cell types, but methods for using scRNA-seq data in the context of CMaP analysis are lacking. Lymphangioleiomyomatosis (LAM) mutations in TSC1 or TSC2 genes result in the activation of the mTOR complex 1 (mTORC1). The mTORC1 inhibitor Sirolimus is the only FDA-approved drug to treat LAM. Novel therapies for LAM are urgently needed as the disease recurs with discontinuation of the treatment and some patients are insensitive to the drug. We developed methods for constructing disease transcriptional signatures and CMaP analysis using scRNA-seq profiling and applied them in the analysis of scRNA-seq data of lung tissue from naïve and sirolimus-treated LAM patients. New methods successfully implicated mTORC1 inhibitors, including Sirolimus, as capable of reverting the LAM transcriptional signatures. The CMaP analysis mimicking standard bulk-tissue approach failed to detect any connection between the LAM signature and mTORC1 signaling. This indicates that the precise signature derived from scRNA-seq data using our methods is the crucial difference between the success and the failure to identify effective therapeutic treatments in CMaP analysis.
    Keywords:  LINCS; connectivity analysis; lymphangioleiomyomatosis; mTOR; single-cell
    DOI:  https://doi.org/10.3390/ijms22094371
  29. Front Cell Dev Biol. 2021 ;9 647300
    An ω-3, but Not an ω-6 Polyunsaturated Fatty Acid Decreases Membrane Dipole Potential and Stimulates Endo-Lysosomal Escape of Penetratin.
    Florina Zakany, Mate Szabo, Gyula Batta, Levente Kárpáti, István M Mándity, Péter Fülöp, Zoltan Varga, Gyorgy Panyi, Peter Nagy, Tamas Kovacs.
      Although the largely positive intramembrane dipole potential (DP) may substantially influence the function of transmembrane proteins, its investigation is deeply hampered by the lack of measurement techniques suitable for high-throughput examination of living cells. Here, we describe a novel emission ratiometric flow cytometry method based on F66, a 3-hydroxiflavon derivative, and demonstrate that 6-ketocholestanol, cholesterol and 7-dehydrocholesterol, saturated stearic acid (SA) and ω-6 γ-linolenic acid (GLA) increase, while ω-3 α-linolenic acid (ALA) decreases the DP. These changes do not correlate with alterations in cell viability or membrane fluidity. Pretreatment with ALA counteracts, while SA or GLA enhances cholesterol-induced DP elevations. Furthermore, ALA (but not SA or GLA) increases endo-lysosomal escape of penetratin, a cell-penetrating peptide. In summary, we have developed a novel method to measure DP in large quantities of individual living cells and propose ALA as a physiological DP lowering agent facilitating cytoplasmic entry of penetratin.
    Keywords:  cholesterol; flow cytometry; membrane dipole potential; penetratin; polyunsaturated fatty acids
    DOI:  https://doi.org/10.3389/fcell.2021.647300
  30. J Bone Miner Res. 2021 Apr 26.
    Phosphate restriction impairs mTORC1 signaling leading to increased bone marrow adipose tissue and decreased bone in growing mice.
    Frank C Ko, Margaret M Kobelski, Wanlin Zhang, Gina M Grenga, Janaina S Martins, Marie B Demay.
      Bone Marrow Stromal Cells (BMSCs) are multipotent cells that differentiate into cells of the osteogenic and adipogenic lineage. A striking inverse relationship between Bone Marrow Adipose Tissue (BMAT) and bone volume is seen in several conditions, suggesting that differentiation of BMSCs into bone marrow adipocytes diverts cells from the osteogenic lineage, thereby compromising the structural and mechanical properties of bone. Phosphate restriction of growing mice acutely decreases bone formation, blocks osteoblast differentiation and increases BMAT. Studies performed to evaluate the cellular and molecular basis for the effects of acute phosphate restriction demonstrate that it acutely increases AMPK phosphorylation and inhibits mTORC1 signaling in osteoblasts. This is accompanied by decreased expression of Wnt10b in BMSCs. Phosphate restriction also promotes expression of the key adipogenic transcription factors, PPARγ and CEBPα, in CXCL12 Abundant Reticular (CAR) cells, which represent undifferentiated BMSCs and are the main source of BMAT and osteoblasts in the adult murine skeleton. Consistent with this, lineage tracing studies reveal that the BMAT observed in phosphate-restricted mice is of CAR cell origin. To determine whether circumventing the decrease in mTORC1 signaling in maturing osteoblasts attenuates the osteoblast and BMAT phenotype, phosphate restricted mice with OSX-CreERT2 -mediated haploinsufficiency of the mTORC1 inhibitor, TSC2 were generated. TSC2 haploinsufficiency in preosteoblasts/osteoblasts normalized bone volume and osteoblast number in phosphate restricted mice and attenuated the increase in BMAT observed. Thus, acute phosphate restriction leads to decreased bone and increases BMAT by impairing mTORC1 signaling in osterix-expressing cells.
    Keywords:  Bone QCT/μCT; Bone histomorphometry; Genetic animal models; mTORC1; osteoblast
    DOI:  https://doi.org/10.1002/jbmr.4312
  31. J Biol Chem. 2021 Apr 27. pii: S0021-9258(21)00412-9. [Epub ahead of print] 100626
    A comprehensive analysis of RAS-effector interactions reveals interaction hotspots and new binding partners.
    Soheila Rezaei Adariani, Neda S Kazemein Jasemi, Farhad Bazgir, Christoph Wittich, Ehsan Amin, Claus A M Seidel, Radovan Dvorsky, Mohammad R Ahmadian.
      RAS effectors specifically interact with GTP-bound RAS proteins to link extracellular signals to downstream signaling pathways. These interactions rely on two types of domains, called RAS binding (RB) and RAS association (RA) domains, which share common structural characteristics. Although the molecular nature of RAS-effector interactions is well-studied for some proteins, most of the RA/RB domain containing proteins remain largely uncharacterized. Here, we searched through human proteome databases, extracting 41 RA domains in 39 proteins and 16 RB domains in 14 proteins, each of which can specifically select at least one of the 25 members in the RAS family. We next comprehensively investigated the sequence-structure-function relationship between different representatives of the RAS family, including HRAS, RRAS, RALA, RAP1B, RAP2A, RHEB1, and RIT1, with all members of RA domain family proteins (RASSFs) and the RB domain-containing CRAF. The binding affinity for RAS-effector interactions, determined using fluorescence polarization, broadly ranged between high (0.3 μM) and very low (500 μM) affinities, raising interesting questions about the consequence of these variable binding affinities in the regulation of signaling events. Sequence and structural alignments pointed to two interaction hotspots in the RA/RB domains, consisting of an average of 19 RAS-binding residues. Moreover, we found novel interactions between RRAS1, RIT1, and RALA and RASSF7, RASSF9, and RASSF1, respectively, which were systematically explored in sequence-structure-property relationship analysis, and validated by mutational analysis. These data provide a set of distinct functional properties and putative biological roles that should now be investigated in the cellular context.
    Keywords:  Effectors; GTPase; NORE-1; RA domain; RAF kinase; RAS; RAS association domain; RAS binding domain; RASSF; RASSF1; RASSF5; RB domain; protein interactions
    DOI:  https://doi.org/10.1016/j.jbc.2021.100626
  32. Apoptosis. 2021 Apr 27.
    Rictor, an essential component of mTOR complex 2, undergoes caspase-mediated cleavage during apoptosis induced by multiple stimuli.
    Liqun Zhao, Lei Zhu, You-Take Oh, Guoqing Qian, Zhen Chen, Shi-Yong Sun.
      Caspase-mediated cleavage of proteins ensures the irreversible commitment of cells to undergo apoptosis, and is thus a hallmark of apoptosis. Rapamycin-insensitive companion of mTOR (rictor) functions primarily as a core and essential component of mTOR complex 2 (mTORC2) to critically regulate cellular homeostasis. However, its role in the regulation of apoptosis is largely unknown. In the current study, we found that rictor was cleaved to generate two small fragments at ~ 50 kD and ~ 130 kD in cells undergoing apoptosis upon treatment with different stimuli such as the death ligand, TRAIL, and the small molecule, AZD9291. This cleavage was abolished when caspases were inhibited and could be reproduced when directly incubating rictor protein and caspase-3 in vitro. Furthermore, the cleavage site of caspase-3 on rictor was mapped at D1244 (VGVD). These findings together robustly demonstrate that rictor is a substrate of caspase-3 and undergoes cleavage during apoptosis. These results add new information for understanding the biology of rictor in the regulation of cell survival and growth.
    Keywords:  Apoptosis; EGFR-TKIs; Rictor; TRAIL
    DOI:  https://doi.org/10.1007/s10495-021-01676-y
  33. Front Cell Dev Biol. 2021 ;9 650186
    A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets.
    Vineet Choudhary, Roger Schneiter.
      Lipid droplets (LDs) constitute compartments dedicated to the storage of metabolic energy in the form of neutral lipids. LDs originate from the endoplasmic reticulum (ER) with which they maintain close contact throughout their life cycle. These ER-LD junctions facilitate the exchange of both proteins and lipids between these two compartments. In recent years, proteins that are important for the proper formation of LDs and localize to ER-LD junctions have been identified. This junction is unique as it is generally believed to invoke a transition from the ER bilayer membrane to a lipid monolayer that delineates LDs. Proper formation of this junction requires the ordered assembly of proteins and lipids at specialized ER subdomains. Without such a well-ordered assembly of LD biogenesis factors, neutral lipids are synthesized throughout the ER membrane, resulting in the formation of aberrant LDs. Such ectopically formed LDs impact ER and lipid homeostasis, resulting in different types of lipid storage diseases. In response to starvation, the ER-LD junction recruits factors that tether the vacuole to these junctions to facilitate LD degradation. In addition, LDs maintain close contacts with peroxisomes and mitochondria for metabolic channeling of the released fatty acids toward beta-oxidation. In this review, we discuss the function of different components that ensure proper functioning of LD contact sites, their role in lipogenesis and lipolysis, and their relation to lipid storage diseases.
    Keywords:  ER subdomains; lipid droplet; lipid storage disorders; lipodystrophy; membrane contact site; membrane trafficking; organelle biogenesis; seipin
    DOI:  https://doi.org/10.3389/fcell.2021.650186
  34. Sci Adv. 2021 Apr;pii: eabf3873. [Epub ahead of print]7(18):
    Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes.
    Daniela Ivanova, Katharine L Dobson, Akshada Gajbhiye, Elizabeth C Davenport, Daniela Hacker, Sila K Ultanir, Matthias Trost, Michael A Cousin.
      Synaptic vesicle (SV) release probability (Pr), determines the steady state and plastic control of neurotransmitter release. However, how diversity in SV composition arises and regulates the Pr of individual SVs is not understood. We found that modulation of the copy number of the noncanonical vesicular SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), vesicle-associated membrane protein 4 (VAMP4), on SVs is key for regulating Pr. Mechanistically, this is underpinned by its reduced ability to form an efficient SNARE complex with canonical plasma membrane SNAREs. VAMP4 has unusually high synaptic turnover and is selectively sorted to endolysosomes during activity-dependent bulk endocytosis. Disruption of endolysosomal trafficking and function markedly increased the abundance of VAMP4 in the SV pool and inhibited SV fusion. Together, our results unravel a new mechanism for generating SV heterogeneity and control of Pr through coupling of SV recycling to a major clearing system that regulates protein homeostasis.
    DOI:  https://doi.org/10.1126/sciadv.abf3873
  35. Nature. 2021 Apr 28.
    PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism.
    Aileen A Ren, Daniel A Snellings, Yourong S Su, Courtney C Hong, Marco Castro, Alan T Tang, Matthew R Detter, Nicholas Hobson, Romuald Girard, Sharbel Romanos, Rhonda Lightle, Thomas Moore, Robert Shenkar, Christian Benavides, M Makenzie Beaman, Helge Mueller-Fielitz, Mei Chen, Patricia Mericko, Jisheng Yang, Derek C Sung, Michael T Lawton, Michael Ruppert, Markus Schwaninger, Jakob Körbelin, Michael Potente, Issam A Awad, Douglas A Marchuk, Mark L Kahn.
      Vascular malformations are considered monogenic disorders that result in dysregulated vessel growth. Cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex required to dampen MEKK3 activity1-4. Environmental factors explain differences in CCM natural history between individuals5, but why single CCMs often exhibit sudden, rapid growth culminating in stroke or seizure is unknown. Here we demonstrate that CCM growth requires increased PI3K-mTOR signalling and loss of CCM function. We identify PIK3CA gain of function (GOF) and CCM loss of function (LOF) somatic mutations in the same cells in a majority of human CCMs. Using mouse models, we show that CCM growth requires both PI3K GOF and CCM LOF in endothelial cells, and that both CCM LOF and increased expression of the transcription factor KLF4, a downstream MEKK3 effector, augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor Rapamycin effectively blocks CCM formation in mouse models. We establish a three-hit mechanism analogous to cancer in which aggressive vascular malformations arise through the loss of vascular "suppressor genes" that constrain vessel growth and gain of a vascular "oncogene" that stimulates excess vessel growth. These findings suggest that aggressive CCMs may be treated using clinically approved mTORC1 inhibitors.
    DOI:  https://doi.org/10.1038/s41586-021-03562-8
  36. Nat Rev Mol Cell Biol. 2021 Apr 28.
    Lysosome transport interrupted.
    Paulina Strzyz.
      
    DOI:  https://doi.org/10.1038/s41580-021-00376-4
  37. J Immunol. 2021 Apr 30. pii: ji2001215. [Epub ahead of print]
    mTORC1 Inhibition in Metastatic Breast Cancer Patients Negatively Affects Peripheral NK Cell Maturation and Number.
    Laurie Besson, Benoite Mery, Magali Morelle, Yamila Rocca, Pierre Etienne Heudel, Benoit You, Thomas Bachelot, Isabelle Ray-Coquard, Marine Villard, Emily Charrier, François Parant, Sébastien Viel, Gwenaële Garin, Romaine Mayet, David Perol, Thierry Walzer, Olivier Tredan, Antoine Marçais.
      NK cells are cytotoxic lymphocytes displaying strong antimetastatic activity. Mouse models and in vitro studies suggest a prominent role of the mechanistic target of rapamycin (mTOR) kinase in the control of NK cell homeostasis and antitumor functions. However, mTOR inhibitors are used as chemotherapies in several cancer settings. The impact of such treatments on patients' NK cells is unknown. We thus performed immunophenotyping of circulating NK cells from metastatic breast cancer patients treated with the mTOR inhibitor everolimus over a three-month period. Everolimus treatment resulted in inhibition of mTORC1 activity in peripheral NK cells, whereas mTORC2 activity was preserved. NK cell homeostasis was profoundly altered with a contraction of the NK cell pool and an overall decrease in their maturation. Phenotype and function of the remaining NK cell population was less affected. This is, to our knowledge, the first in vivo characterization of the role of mTOR in human NK cells.
    DOI:  https://doi.org/10.4049/jimmunol.2001215
  38. FEBS J. 2021 Apr 25.
    Brain-specific functions of the endocytic machinery.
    Santiago Camblor-Perujo, Natalia L Kononenko.
      Endocytosis is an essential cellular process required for multiple physiological functions, including communication with the extracellular environment, nutrient uptake, and signaling by the cell-surface receptors. In a broad sense, endocytosis is accomplished through either constitutive or ligand-induced invagination of the plasma membrane, which results in the formation of the plasma membrane-retrieved endocytic vesicles, which can either be sent for degradation to the lysosomes or recycled back to the PM. This additional function of endocytosis in membrane retrieval has been adopted by excitable cells, such as neurons, for membrane equilibrium maintenance at synapses. The last two decades were especially productive with respect to the identification of brain-specific functions of the endocytic machinery, which additionally include but not limited to regulation of neuronal differentiation and migration, maintenance of neuron morphology and synaptic plasticity, and prevention of neurotoxic aggregates spreading. In this review, we highlight the current knowledge of brain-specific functions of endocytic machinery with a specific focus on three brain cell types, neuronal progenitor cells, neurons, and glial cells.
    Keywords:  endocytosis; glial cells; neurodegeneration; neurodevelopment; neuronal progenitor cells; neurons
    DOI:  https://doi.org/10.1111/febs.15897
  39. Front Pediatr. 2021 ;9 631473
    The Clinical Significance and Potential Role of Cathepsin S in IgA Nephropathy.
    Jingying Zhao, Yongchang Yang, Yubin Wu.
      Objective: Cathepsin S (CTSS) is an important lysosomal cysteine protease. This study aimed at investigating the clinical significance of CTSS and underlying mechanism in immunoglobulin A nephropathy (IgAN). Methods: This study recruited 25 children with IgAN and age-matched controls and their serum CTSS levels were measured by enzyme-linked immunosorbent assay (ELISA). Following induction of IgAN in rats, their kidney CTSS expression, IgA accumulation and serum CTSS were characterized by immunohistochemistry, immunofluorescence, and ELISA. The impact of IgA1 aggregates on the proliferation of human mesangial cells (HMCs) was determined by Cell Counting Kit-8 and Western blot analysis of Ki67. Results: Compared to the non-IgAN controls, significantly up-regulated CTSS expression was detected in the renal tissues, particularly in the glomerular mesangium and tubular epithelial cells of IgAN patients, accompanied by higher levels of serum CTSS (P < 0.05), which were correlated with the levels of 24-h-urine proteins and microalbumin and urine erythrocytes and grades of IgAN Lee's classification in children with IgAN (P < 0.01 for all). Following induction of IgAN, we detected inducible IgA accumulation and increased levels of CTSS expression in the glomerular mesangium and glomerular damages in rats, which were mitigated by LY3000328, a CTSS-specific inhibitor. Treatment with LY3000328 significantly mitigated the Ki67 expression in the kidney of IgAN rats (P < 0.01) and significantly minimized the IgA1 aggregate-stimulated proliferation of HMCs and their Ki67 expression in vitro (P < 0.01). Conclusions: CTSS promoted the proliferation of glomerular mesangial cells, contributing to the pathogenesis of IgAN and may be a new therapeutic target for intervention of aberrant mesangial cell proliferation during the process of IgAN.
    Keywords:  CTSS; CTSS inhibitor 2; IgA nephropathy; Mesangial Cell Proliferation; Pediatrics
    DOI:  https://doi.org/10.3389/fped.2021.631473
  40. Int J Mol Sci. 2021 Apr 26. pii: 4516. [Epub ahead of print]22(9):
    In Silico Analysis of the Molecular-Level Impact of SMPD1 Variants on Niemann-Pick Disease Severity.
    François Ancien, Fabrizio Pucci, Marianne Rooman.
      Sphingomyelin phosphodiesterase (SMPD1) is a key enzyme in the sphingolipid metabolism. Genetic SMPD1 variants have been related to the Niemann-Pick lysosomal storage disorder, which has different degrees of phenotypic severity ranging from severe symptomatology involving the central nervous system (type A) to milder ones (type B). They have also been linked to neurodegenerative disorders such as Parkinson and Alzheimer. In this paper, we leveraged structural, evolutionary and stability information on SMPD1 to predict and analyze the impact of variants at the molecular level. We developed the SMPD1-ZooM algorithm, which is able to predict with good accuracy whether variants cause Niemann-Pick disease and its phenotypic severity; the predictor is freely available for download. We performed a large-scale analysis of all possible SMPD1 variants, which led us to identify protein regions that are either robust or fragile with respect to amino acid variations, and show the importance of aromatic-involving interactions in SMPD1 function and stability. Our study also revealed a good correlation between SMPD1-ZooM scores and in vitro loss of SMPD1 activity. The understanding of the molecular effects of SMPD1 variants is of crucial importance to improve genetic screening of SMPD1-related disorders and to develop personalized treatments that restore SMPD1 functionality.
    Keywords:  Niemann-Pick disease; Parkinson disease; disease severity prediction; genetic variants; sphingomyelin phosphodiesterase
    DOI:  https://doi.org/10.3390/ijms22094516
  41. Cells. 2021 Apr 08. pii: 849. [Epub ahead of print]10(4):
    Increased Alveolar Heparan Sulphate and Reduced Pulmonary Surfactant Amount and Function in the Mucopolysaccharidosis IIIA Mouse.
    Tamara L Paget, Emma J Parkinson-Lawrence, Paul J Trim, Chiara Autilio, Madhuriben H Panchal, Grielof Koster, Mercedes Echaide, Marten F Snel, Anthony D Postle, Janna L Morrison, Jésus Pérez-Gil, Sandra Orgeig.
      Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA.
    Keywords:  MPS IIIA; Sanfilippo syndrome; bronchoalveolar lavage; captive bubble surfactometry; heparan sulphate; lipids; pulmonary surfactant; respiratory dysfunction; surface activity; surfactant proteins
    DOI:  https://doi.org/10.3390/cells10040849
  42. Elife. 2021 04 26. pii: e66904. [Epub ahead of print]10
    Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology.
    Markus Terrey, Scott I Adamson, Jeffrey H Chuang, Susan L Ackerman.
      Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2. Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.
    Keywords:  cerebellum; chromosomes; gene expression; mouse; n-Tr20; neurogenesis; ribosome; translation
    DOI:  https://doi.org/10.7554/eLife.66904
  43. Biomolecules. 2021 Apr 20. pii: 611. [Epub ahead of print]11(4):
    Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development.
    Giulia Massaro, Amy F Geard, Wenfei Liu, Oliver Coombe-Tennant, Simon N Waddington, Julien Baruteau, Paul Gissen, Ahad A Rahim.
      Rare monogenic disorders such as lysosomal diseases have been at the forefront in the development of novel treatments where therapeutic options are either limited or unavailable. The increasing number of successful pre-clinical and clinical studies in the last decade demonstrates that gene therapy represents a feasible option to address the unmet medical need of these patients. This article provides a comprehensive overview of the current state of the field, reviewing the most used viral gene delivery vectors in the context of lysosomal storage disorders, a selection of relevant pre-clinical studies and ongoing clinical trials within recent years.
    Keywords:  gene therapy; lysosomal diseases; viral vectors
    DOI:  https://doi.org/10.3390/biom11040611
  44. Proc Natl Acad Sci U S A. 2021 May 04. pii: e2001681118. [Epub ahead of print]118(18):
    Genetic removal of p70 S6K1 corrects coding sequence length-dependent alterations in mRNA translation in fragile X syndrome mice.
    Sameer Aryal, Francesco Longo, Eric Klann.
      Loss of the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP is widely thought to repress protein synthesis, but its translational targets and modes of control remain in dispute. We previously showed that genetic removal of p70 S6 kinase 1 (S6K1) corrects altered protein synthesis as well as synaptic and behavioral phenotypes in FXS mice. In this study, we examined the gene specificity of altered messenger RNA (mRNA) translation in FXS and the mechanism of rescue with genetic reduction of S6K1 by carrying out ribosome profiling and RNA sequencing on cortical lysates from wild-type, FXS, S6K1 knockout, and double knockout mice. We observed reduced ribosome footprint (RF) abundance in the majority of differentially translated genes in the cortices of FXS mice. We used molecular assays to discover evidence that the reduction in RF abundance reflects an increased rate of ribosome translocation, which is captured as a decrease in the number of translating ribosomes at steady state and is normalized by inhibition of S6K1. We also found that genetic removal of S6K1 prevented a positive-to-negative gradation of alterations in translation efficiencies (RF/mRNA) with coding sequence length across mRNAs in FXS mouse cortices. Our findings reveal the identities of dysregulated mRNAs and a molecular mechanism by which reduction of S6K1 prevents altered translation in FXS.
    Keywords:  autism; fragile X syndrome; mRNA translation; protein synthesis; translation elongation
    DOI:  https://doi.org/10.1073/pnas.2001681118
  45. Int J Mol Sci. 2021 Apr 23. pii: 4434. [Epub ahead of print]22(9):
    Fabry Disease and the Heart: A Comprehensive Review.
    Olga Azevedo, Filipa Cordeiro, Miguel Fernandes Gago, Gabriel Miltenberger-Miltenyi, Catarina Ferreira, Nuno Sousa, Damião Cunha.
      Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations of the GLA gene that result in a deficiency of the enzymatic activity of α-galactosidase A and consequent accumulation of glycosphingolipids in body fluids and lysosomes of the cells throughout the body. GB3 accumulation occurs in virtually all cardiac cells (cardiomyocytes, conduction system cells, fibroblasts, and endothelial and smooth muscle vascular cells), ultimately leading to ventricular hypertrophy and fibrosis, heart failure, valve disease, angina, dysrhythmias, cardiac conduction abnormalities, and sudden death. Despite available therapies and supportive treatment, cardiac involvement carries a major prognostic impact, representing the main cause of death in FD. In the last years, knowledge has substantially evolved on the pathophysiological mechanisms leading to cardiac damage, the natural history of cardiac manifestations, the late-onset phenotypes with predominant cardiac involvement, the early markers of cardiac damage, the role of multimodality cardiac imaging on the diagnosis, management and follow-up of Fabry patients, and the cardiac efficacy of available therapies. Herein, we provide a comprehensive and integrated review on the cardiac involvement of FD, at the pathophysiological, anatomopathological, laboratory, imaging, and clinical levels, as well as on the diagnosis and management of cardiac manifestations, their supportive treatment, and the cardiac efficacy of specific therapies, such as enzyme replacement therapy and migalastat.
    Keywords:  Fabry disease; cardiomyopathy; enzyme replacement therapy; heart; migalastat
    DOI:  https://doi.org/10.3390/ijms22094434
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