bims-lysosi 2021-05-09 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–05–09
thirty-two papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Lysosome biogenesis: Regulation and functions.
The Contribution of Lysosomes to DNA Replication.
Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease.
Identification of lysosome-targeting drugs with anti-inflammatory activity as potential invasion inhibitors of treatment resistant HER2 positive cancers.
Cyclosporine A blocks autophagic flux in tubular epithelial cells by impairing TFEB-mediated lysosomal function.
TOR Signaling in Caenorhabditis elegans Development, Metabolism, and Aging.
Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis.
The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes.
Targeting SLC1A5 and SLC3A2/SLC7A5 as a Potential Strategy to Strengthen Anti-Tumor Immunity in the Tumor Microenvironment.
Implication of a lysosomal antigen in the pathogenesis of lupus erythematosus.
Three-dimensional tissue-engineered human skeletal muscle model of Pompe disease.
Activation of TOR signaling by diverse nitrogen signals in plants.
The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma.
Sit4 and PP2A Dephosphorylate Nitrogen Catabolite Repression-Sensitive Gln3 When TorC1 Is Up- as Well as Downregulated.
Umbilical mesenchymal stem cell-derived extracellular vesicles as enzyme delivery vehicle to treat Morquio A fibroblasts.
MicroRNA-34a activation in tuberous sclerosis complex during early brain development may lead to impaired corticogenesis.
mTOR signaling regulates gastric epithelial progenitor homeostasis and gastric tumorigenesis via MEK1-ERKs and BMP-Smad1 pathways.
An ABCG Transporter Functions in Rab Localization and Lysosome-Related Organelle Biogenesis in Caenorhabditis elegans.
Manifestations of Age on Autophagy, Mitophagy and Lysosomes in Skeletal Muscle.
TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae.
Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering.
RAG-ulating mTORC1 with amino acids.
Increasing mTORC1 Pathway Activity or Methionine Supplementation during Pregnancy Reverses the Negative Effect of Maternal Malnutrition on the Developing Kidney.
Sphingolipid lysosomal storage diseases: from bench to bedside.
pH induced conformational alteration in human peroxiredoxin 6 might be responsible for its resistance against lysosomal pH or high temperature.
A Stress-Responsive Signaling Network Regulating Pseudohyphal Growth and Ribonucleoprotein Granule Abundance in Saccharomyces cerevisiae.
Rapamycin Promotes ROS-Mediated Cell Death via Functional Inhibition of xCT Expression in Melanoma Under γ-Irradiation.
Conditional knockout of Tsc1 in RORγt-expressing cells induces brain damage and early death in mice.
Genotype-phenotype studies in a large cohort of Brazilian patients with Hunter syndrome.
Distinct fission signatures predict mitochondrial degradation or biogenesis.
Discovery of Highly Specific Catalytic-Site-Targeting Fluorescent Probes for Detecting Lysosomal PDE10A in Living Cells.
DNA-Based pH Nanosensor with Adjustable FRET Responses to Track Lysosomes and pH Fluctuations.

J Cell Biol. 2021 Jun 07. pii: e202102001. [Epub ahead of print]220(6):

Lysosome biogenesis: Regulation and functions.

Chonglin Yang, Xiaochen Wang.

Lysosomes are degradation centers and signaling hubs in cells and play important roles in cellular homeostasis, development, and aging. Changes in lysosome function are essential to support cellular adaptation to multiple signals and stimuli. Therefore, lysosome biogenesis and activity are regulated by a wide variety of intra- and extracellular cues. Here, we summarize current knowledge of the regulatory mechanisms of lysosome biogenesis, including synthesis of lysosomal proteins and their delivery via the endosome-lysosome pathway, reformation of lysosomes from degradative vesicles, and transcriptional regulation of lysosomal genes. We survey the regulation of lysosome biogenesis in response to nutrient and nonnutrient signals, the cell cycle, stem cell quiescence, and cell fate determination. Finally, we discuss lysosome biogenesis and functions in the context of organismal development and aging.

DOI: https://doi.org/10.1083/jcb.202102001
Cells. 2021 Apr 30. pii: 1068. [Epub ahead of print]10(5):

The Contribution of Lysosomes to DNA Replication.

Joanna Maria Merchut-Maya, Apolinar Maya-Mendoza.

  Lysosomes, acidic, membrane-bound organelles, are not only the core of the cellular recycling machinery, but they also serve as signaling hubs regulating various metabolic pathways. Lysosomes maintain energy homeostasis and provide pivotal substrates for anabolic processes, such as DNA replication. Every time the cell divides, its genome needs to be correctly duplicated; therefore, DNA replication requires rigorous regulation. Challenges that negatively affect DNA synthesis, such as nucleotide imbalance, result in replication stress with severe consequences for genome integrity. The lysosomal complex mTORC1 is directly involved in the synthesis of purines and pyrimidines to support DNA replication. Numerous drugs have been shown to target lysosomal function, opening an attractive avenue for new treatment strategies against various pathologies, including cancer. In this review, we focus on the interplay between lysosomal function and DNA replication through nucleic acid degradation and nucleotide biosynthesis and how these could be exploited for therapeutic purposes.

Keywords:  DNA synthesis; autophagy; cancer; lysosomes; mTORC1

DOI:  https://doi.org/10.3390/cells10051068
EMBO Mol Med. 2021 May 07. 13(5): e13376

Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease.

Kalliopi Sofou, Kolja Meier, Leslie E Sanderson, Debora Kaminski, Laia Montoliu-Gaya, Emma Samuelsson, Maria Blomqvist, Lotta Agholme, Jutta Gärtner, Chris Mühlhausen, Niklas Darin, Tahsin Stefan Barakat, Lars Schlotawa, Tjakko van Ham, Jorge Asin Cayuela, Fredrik H Sterky.

  Lysosomal storage diseases, including mucopolysaccharidoses, result from genetic defects that impair lysosomal catabolism. Here, we describe two patients from two independent families presenting with progressive psychomotor regression, delayed myelination, brain atrophy, neutropenia, skeletal abnormalities, and mucopolysaccharidosis-like dysmorphic features. Both patients were homozygous for the same intronic variant in VPS16, a gene encoding a subunit of the HOPS and CORVET complexes. The variant impaired normal mRNA splicing and led to an ~85% reduction in VPS16 protein levels in patient-derived fibroblasts. Levels of other HOPS/CORVET subunits, including VPS33A, were similarly reduced, but restored upon re-expression of VPS16. Patient-derived fibroblasts showed defects in the uptake and endosomal trafficking of transferrin as well as accumulation of autophagosomes and lysosomal compartments. Re-expression of VPS16 rescued the cellular phenotypes. Zebrafish with disrupted vps16 expression showed impaired development, reduced myelination, and a similar accumulation of lysosomes and autophagosomes in the brain, particularly in glia cells. This disorder resembles previously reported patients with mutations in VPS33A, thus expanding the family of mucopolysaccharidosis-like diseases that result from mutations in HOPS/CORVET subunits.

Keywords:  MPS; autophagy; endosome; lysosomal storage disease; myelination

DOI:  https://doi.org/10.15252/emmm.202013376
Cell Oncol (Dordr). 2021 May 03.

Identification of lysosome-targeting drugs with anti-inflammatory activity as potential invasion inhibitors of treatment resistant HER2 positive cancers.

Malene Bredahl Hansen, Maria Postol, Siri Tvingsholm, Inger Ødum Nielsen, Tiina Naumanen Dietrich, Pietri Puustinen, Kenji Maeda, Christoffel Dinant, Robert Strauss, David Egan, Marja Jäättelä, Tuula Kallunki.

   PURPOSE: Most HER2 positive invasive cancers are either intrinsic non-responsive or develop resistance when treated with 1st line HER2 targeting drugs. Both 1st and 2nd line treatments of HER2 positive cancers are aimed at targeting the HER2 receptor directly, thereby strongly limiting the treatment options of HER2/ErbB2 inhibition resistant invasive cancers.
METHODS: We used phenotypic high throughput microscopy screening to identify efficient inhibitors of ErbB2-induced invasion using 1st line HER2 inhibitor trastuzumab- and pertuzumab-resistant, p95-ErbB2 expressing breast cancer cells in conjunction with the Prestwick Chemical Library®. The screening entailed a drug's ability to inhibit ErbB2-induced, invasion-promoting positioning of lysosomes at the cellular periphery, a phenotype that defines their invasiveness. In addition, we used high throughput microscopy and biochemical assays to assess the effects of the drugs on lysosomal membrane permeabilization (LMP) and autophagy, two features connected to cancer treatment. Using 2nd line HER2 inhibitor lapatinib resistant 3-dimensional model systems, we assessed the effects of the drugs on ErbB2 positive breast cancer spheroids and developed a high-throughput invasion assay for HER2 positive ovarian cancer organoids for further evaluation.
RESULTS: We identified Auranofin, Colchicine, Monensin, Niclosamide, Podophyllotoxin, Quinacrine and Thiostrepton as efficient inhibitors of invasive growth of 2nd line HER2 inhibitor lapatinib resistant breast cancer spheroids and ovarian cancer organoids. We classified these drugs into four groups based on their ability to target lysosomes by inducing autophagy and/or LMP, i.e., drugs inducing early LMP, early autophagy with late LMP, late LMP, or neither.
CONCLUSIONS: Our results indicate that targetable lysosome-engaging cellular pathways downstream of ErbB2 contribute to invasion. They support lysosomal trafficking as an attractive target for therapy aiming at preventing the spreading of cancer cells. Since these drugs additionally possess anti-inflammatory activities, they could serve as multipurpose drugs simultaneously targeting infection/inflammation and cancer spreading.

Keywords:  Anti‐inflammatory activity; Drug multipurposing; HER2/ErbB2; Invasive growth; Lapatinib; Lysosome targeting drug; Tumor organoid; Tumor spheroid

DOI:  https://doi.org/10.1007/s13402-021-00603-2
J Cell Mol Med. 2021 May 04.

Cyclosporine A blocks autophagic flux in tubular epithelial cells by impairing TFEB-mediated lysosomal function.

Zhi-Hang Li, Ning An, Xi-Jie Huang, Chen Yang, Hong-Luan Wu, Xiao-Cui Chen, Qing-Jun Pan, Hua-Feng Liu.

  Cyclosporine A (CsA) is an immunosuppressor widely used for the prevention of acute rejection during solid organ transplantation. However, severe nephrotoxicity has substantially limited its long-term usage. Recently, an impaired autophagy pathway was suggested to be involved in the pathogenesis of chronic CsA nephrotoxicity. However, the underlying mechanisms of CsA-induced autophagy blockade in tubular cells remain unclear. In the present study, we observed that CsA suppressed the activation and expression of transcription factor EB (TFEB) by increasing the activation of mTOR, in turn promoting lysosomal dysfunction and autophagy flux blockade in tubular epithelial cells (TECs) in vivo and in vitro. Restoration of TFEB activation by Torin1-mediated mTOR inhibition significantly improved lysosomal function and rescued autophagy pathway activity, suppressing TEC injury. In summary, targeting TFEB-mediated autophagy flux represents a potential therapeutic strategy for CsA-induced nephrotoxicity.

Keywords:  TFEB; autophagy; cyclosporine A; lysosome; mTOR; tubular epithelial cells

DOI:  https://doi.org/10.1111/jcmm.16593
Genetics. 2019 Oct 01. 213(2): 329-360

TOR Signaling in Caenorhabditis elegans Development, Metabolism, and Aging.

T Keith Blackwell, Aileen K Sewell, Ziyun Wu, Min Han.

  The Target of Rapamycin (TOR or mTOR) is a serine/threonine kinase that regulates growth, development, and behaviors by modulating protein synthesis, autophagy, and multiple other cellular processes in response to changes in nutrients and other cues. Over recent years, TOR has been studied intensively in mammalian cell culture and genetic systems because of its importance in growth, metabolism, cancer, and aging. Through its advantages for unbiased, and high-throughput, genetic and in vivo studies, Caenorhabditis elegans has made major contributions to our understanding of TOR biology. Genetic analyses in the worm have revealed unexpected aspects of TOR functions and regulation, and have the potential to further expand our understanding of how growth and metabolic regulation influence development. In the aging field, C. elegans has played a leading role in revealing the promise of TOR inhibition as a strategy for extending life span, and identifying mechanisms that function upstream and downstream of TOR to influence aging. Here, we review the state of the TOR field in C. elegans, and focus on what we have learned about its functions in development, metabolism, and aging. We discuss knowledge gaps, including the potential pitfalls in translating findings back and forth across organisms, but also describe how TOR is important for C. elegans biology, and how C. elegans work has developed paradigms of great importance for the broader TOR field.

Keywords:   Caenorhabditis elegans development; DAF-15; NPRL-2; NPRL-3; Nprl2; Nprl3; RAGA-1; RSKS-1; RagA; RagC; Raptor; Rheb; Rheb-1; Rictor; S6 kinase; TOR; TORC1; TORC2; WormBook; aging; growth regulation; metabolism; nutrient signaling; sphingolipid

DOI:  https://doi.org/10.1534/genetics.119.302504
PLoS One. 2021 ;16(5): e0251184

Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis.

Jonathan R Florian, Samuel J DeMatte, Devon M Sweeder, Richard W Ordway, Fumiko Kawasaki.

The ESCRT pathway is evolutionarily conserved across eukaryotes and plays key roles in a variety of membrane remodeling processes. A new Drosophila mutant recovered in our forward genetic screens for synaptic transmission mutants mapped to the vps24 gene encoding a subunit of the ESCRT-III complex. Molecular characterization indicated a loss of VPS24 function, however the mutant is viable and thus loss of VPS24 may be studied in a developed multicellular organism. The mutant exhibits deficits in locomotion and lifespan and, notably, these phenotypes are rescued by neuronal expression of wild-type VPS24. At the cellular level, neuronal and muscle cells exhibit marked expansion of a ubiquitin-positive lysosomal compartment, as well as accumulation of autophagic intermediates, and these phenotypes are rescued cell-autonomously. Moreover, VPS24 expression in glia suppressed the mutant phenotype in muscle, indicating a cell-nonautonomous function for VPS24 in protective intercellular signaling. Ultrastructural analysis of neurons and muscle indicated marked accumulation of the lysosomal compartment in the vps24 mutant. In the neuronal cell body, this included characteristic lysosomal structures associated with an expansive membrane compartment with a striking tubular network morphology. These findings further define the in vivo roles of VPS24 and the ESCRT pathway in lysosome homeostasis and their potential contributions to neurodegenerative diseases characterized by defective ESCRT or lysosome function.

DOI: https://doi.org/10.1371/journal.pone.0251184
Cell Discov. 2021 May 04. 7(1): 31

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes.

Yabin Zhang, Hao Sun, Rongjuan Pei, Binli Mao, Zhenyu Zhao, Huihui Li, Yong Lin, Kefeng Lu.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 pandemic. How SARS-CoV-2 regulates cellular responses to escape clearance by host cells is unknown. Autophagy is an intracellular lysosomal degradation pathway for the clearance of various cargoes, including viruses. Here, we systematically screened 28 viral proteins of SARS-CoV-2 and identified that ORF3a strongly inhibited autophagic flux by blocking the fusion of autophagosomes with lysosomes. ORF3a colocalized with lysosomes and interacted with VPS39, a component of the homotypic fusion and protein sorting (HOPS) complex. The ORF3a-VPS39 interaction prohibited the binding of HOPS with RAB7, which prevented the assembly of fusion machinery, leading to the accumulation of unfused autophagosomes. These results indicated the potential mechanism by which SARS-CoV-2 escapes degradation; that is, the virus interferes with autophagosome-lysosome fusion. Furthermore, our findings will facilitate strategies targeting autophagy for conferring potential protection against the spread of SARS-CoV-2.

DOI: https://doi.org/10.1038/s41421-021-00268-z
Front Immunol. 2021 ;12 624324

Targeting SLC1A5 and SLC3A2/SLC7A5 as a Potential Strategy to Strengthen Anti-Tumor Immunity in the Tumor Microenvironment.

Marianna Nachef, Alaa Kassim Ali, Saeedah Musaed Almutairi, Seung-Hwan Lee.

  Cancer cells are metabolically vigorous and are superior in the uptake of nutrients and in the release of the tumor microenvironment (TME)-specific metabolites. They create an acidic, hypoxic, and nutrient-depleted TME that makes it difficult for the cytotoxic immune cells to adapt to the metabolically hostile environment. Since a robust metabolism in immune cells is required for optimal anti-tumor effector functions, the challenges caused by the TME result in severe defects in the invasion and destruction of the established tumors. There have been many recent developments in NK and T cell-mediated immunotherapy, such as engineering them to express chimeric antigen receptors (CARs) to enhance tumor-recognition and infiltration. However, to defeat the tumor and overcome the limitations of the TME, it is essential to fortify these novel therapies by improving the metabolism of the immune cells. One potential strategy to enhance the metabolic fitness of immune cells is to upregulate the expression of nutrient transporters, specifically glucose and amino acid transporters. In particular, the amino acid transporters SLC1A5 and SLC7A5 as well as the ancillary subunit SLC3A2, which are required for efficient uptake of glutamine and leucine respectively, could strengthen the metabolic capabilities and effector functions of tumor-directed CAR-NK and T cells. In addition to enabling the influx and efflux of essential amino acids through the plasma membrane and within subcellular compartments such as the lysosome and the mitochondria, accumulating evidence has demonstrated that the amino acid transporters participate in sensing amino acid levels and thereby activate mTORC1, a master metabolic regulator that promotes cell metabolism, and induce the expression of c-Myc, a transcription factor essential for cell growth and proliferation. In this review, we discuss the regulatory pathways of these amino acid transporters and how we can take advantage of these processes to strengthen immunotherapy against cancer.

Keywords:  SLC1A5; SLC3A2; SLC7A5; anti-tumor immunity; immunometabolism; natural killer cells; nutrient transporters; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2021.624324
J Autoimmun. 2021 Apr 28. pii: S0896-8411(21)00041-X. [Epub ahead of print]120 102633

Implication of a lysosomal antigen in the pathogenesis of lupus erythematosus.

Maud Wilhelm, Srinivasa Reddy Bonam, Nicolas Schall, Mykolas Bendorius, Anne-Sophie Korganow, Catherine Lumbroso, Sylviane Muller.

  Naturally-occurring autoantibodies to certain components of autophagy processes have been described in a few autoimmune diseases, but their fine specificity, their relationships with clinical phenotypes, and their potential pathogenic functions remain elusive. Here, we explored IgG autoantibodies reacting with a panel of cytoplasmic endosomal/lysosomal antigens and individual heat-shock proteins, all of which share links to autophagy. Sera from autoimmune patients and from MRL/lpr and NZB/W lupus-prone mice reacted with the C-terminal residues of lysosome-associated membrane glycoprotein (LAMP)2A. No cross-reaction was observed with LAMP2B or LAMP2C variants, with dsDNA or mononucleosomes, or with heat-shock protein A8. Moreover, administering chromatography-purified LAMP2A autoantibodies to MRL/lpr mice accelerated mortality. Furthermore, flow cytometry revealed elevated cell-surface expression of LAMP2A on MRL/lpr B cells. These findings reveal the involvement of a new class of autoantibodies targeting the C-terminus of LAMP2A, a receptor for cytosolic proteins targeted for degradation via chaperone-mediated autophagy. These autoantibodies could affect the autophagy process, which is abnormally upregulated in lupus. The data presented support a novel connection between autophagy dysregulation, autoimmune processes and pathophysiology in lupus.

Keywords:  Autoantibodies; Autophagy; LAMP2A; Lupus; Lysosomes

DOI:  https://doi.org/10.1016/j.jaut.2021.102633
Commun Biol. 2021 May 05. 4(1): 524

Three-dimensional tissue-engineered human skeletal muscle model of Pompe disease.

Jason Wang, Chris J Zhou, Alastair Khodabukus, Sabrina Tran, Sang-Oh Han, Aaron L Carlson, Lauran Madden, Priya S Kishnani, Dwight D Koeberl, Nenad Bursac.

In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA-/- mice and wild-type controls to establish a Pompe disease-specific transcriptional signature and reveal novel disease pathways. The mouse-derived signature is enriched in the transcriptomic profile of IOPD vs. healthy myobundles and partially reversed by in vitro rhGAA treatment, further confirming the utility of the human myobundle model for studies of Pompe disease and therapy.

DOI: https://doi.org/10.1038/s42003-021-02059-4
Dev Cell. 2021 May 03. pii: S1534-5807(21)00352-X. [Epub ahead of print]56(9): 1213-1214

Activation of TOR signaling by diverse nitrogen signals in plants.

Frej Tulin, Zhenzhen Zhang, Zhi-Yong Wang.

Growth control in eukaryotes depends on the TOR kinase, which integrates energy and nutrient signals. In this issue of Developmental Cell, Liu et al. demonstrate that, in plants, inorganic nitrogen and amino acids activate TOR via the GTPase ROP2 to promote cell proliferation and leaf growth in the shoot.

DOI: https://doi.org/10.1016/j.devcel.2021.04.014
Autophagy. 2021 May 07. 1-13

The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma.

Iolanda Ferro, Jacopo Gavini, Stefano Gallo, Lisamaria Bracher, Marc Landolfo, Daniel Candinas, Deborah M Stroka, Norbert Polacek.

  The small non-coding VTRNA1-1 (vault RNA 1-1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of VTRNA1-1 in regulating in vitro and in vivo tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of VTRNA1-1 in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of VTRNA1-1 potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of VTRNA1-1 reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation in vitro and in vivo. These findings reveal a so far unknown role of VTRNA1-1 in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance.

Keywords:  Chemoresistance; lysosome; non-coding RNA; tumorigenesis; vault RNA; vtRNA1-1

DOI:  https://doi.org/10.1080/15548627.2021.1922983
Genetics. 2019 Aug 01. 212(4): 1205-1225

Sit4 and PP2A Dephosphorylate Nitrogen Catabolite Repression-Sensitive Gln3 When TorC1 Is Up- as Well as Downregulated.

Jennifer J Tate, Elizabeth A Tolley, Terrance G Cooper.

  Saccharomyces cerevisiae lives in boom and bust nutritional environments. Sophisticated regulatory systems have evolved to rapidly cope with these changes while preserving intracellular homeostasis. Target of Rapamycin Complex 1 (TorC1), is a serine/threonine kinase complex and a principle nitrogen-responsive regulator. TorC1 is activated by excess nitrogen and downregulated by limiting nitrogen. Two of TorC1's many downstream targets are Gln3 and Gat1-GATA-family transcription activators-whose localization and function are Nitrogen Catabolite Repression- (NCR-) sensitive. In nitrogen replete environments, TorC1 is activated, thereby inhibiting the PTap42-Sit4 and PTap42-PP2A (Pph21/Pph22-Tpd3, Pph21,22-Rts1/Cdc55) phosphatase complexes. Gln3 is phosphorylated, sequestered in the cytoplasm and NCR-sensitive transcription repressed. In nitrogen-limiting conditions, TorC1 is downregulated and PTap42-Sit4 and PTap42-PP2A are active. They dephosphorylate Gln3, which dissociates from Ure2, relocates to the nucleus, and activates transcription. A paradoxical observation, however, led us to suspect that Gln3 control was more complex than appreciated, i.e., Sit4 dephosphorylates Gln3 more in excess than in limiting nitrogen conditions. This paradox motivated us to reinvestigate the roles of these phosphatases in Gln3 regulation. We discovered that: (i) Sit4 and PP2A actively function both in conditions where TorC1 is activated as well as down-regulated; (ii) nuclear Gln3 is more highly phosphorylated than when it is sequestered in the cytoplasm; (iii) in nitrogen-replete conditions, Gln3 relocates from the nucleus to the cytoplasm, where it is dephosphorylated by Sit4 and PP2A; and (iv) in nitrogen excess and limiting conditions, Sit4, PP2A, and Ure2 are all required to maintain cytoplasmic Gln3 in its dephosphorylated form.

Keywords:  Gln3; PP2A; Sit4; TorC1; Ure2; nitrogen catabolite repression; rapamycin

DOI:  https://doi.org/10.1534/genetics.119.302371
Stem Cell Res Ther. 2021 May 06. 12(1): 276

Umbilical mesenchymal stem cell-derived extracellular vesicles as enzyme delivery vehicle to treat Morquio A fibroblasts.

Michael Flanagan, Isha Pathak, Qi Gan, Linda Winter, Ryan Emnet, Salem Akel, Adriana M Montaño.

   BACKGROUND: Mucopolysaccharidosis IVA (Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which results in the accumulation of the glycosaminoglycans (GAGs), keratan sulfate, and chondroitin-6-sulfate in the lysosomes of all tissues causing systemic dysfunction. Current treatments include enzyme replacement therapy (ERT) which can treat only certain aspects of the disease such as endurance-related biological endpoints. A key challenge in ERT is ineffective enzyme uptake in avascular tissues, which makes the treatment of the corneal, cartilage, and heart valvular tissue difficult. The aim of this study was to culture human umbilical mesenchymal stem cells (UMSC), demonstrate presence of GALNS enzyme activity within the extracellular vesicles (EVs) derived from these UMSC, and study how these secreted EVs are taken up by GALNS-deficient cells and used by the deficient cell's lysosomes.
METHODS: We obtained and cultured UMSC from the umbilical cord tissue from anonymous donors from the Saint Louis Cord Blood Bank. We characterized UMSC cell surface markers to confirm phenotype by cell sorting analyses. In addition, we confirmed that UMSC secrete GALNS enzyme creating conditioned media for co-culture experiments with GALNS deficient cells. Lastly, we isolated EVs derived from UMSC by ultracentrifugation to confirm source of GALNS enzyme.
RESULTS: Co-culture and confocal microscopy experiments indicated that the lysosomal content from UMSC migrated to deficient cells as evidenced by the peak signal intensity occurring at 15 min. EVs released by UMSC were characterized indicating that the EVs contained the active GALNS enzyme. Uptake of GALNS within EVs by deficient fibroblasts was not affected by mannose-6-phosphate (M6P) inhibition, suggesting that EV uptake by these fibroblasts is gradual and might be mediated by a different means than the M6P receptor.
CONCLUSIONS: UMSC can deliver EVs containing functional GALNS enzyme to deficient cells. This enzyme delivery method, which was unaffected by M6P inhibition, can function as a novel technique for reducing GAG accumulation in cells in avascular tissues, thereby providing a potential treatment option for Morquio A syndrome.

Keywords:  Extracellular vesicles; Morquio A; Mucopolysaccharidosis IVA; Umbilical mesenchymal stem cell

DOI:  https://doi.org/10.1186/s13287-021-02355-0
Neuropathol Appl Neurobiol. 2021 May 03.

MicroRNA-34a activation in tuberous sclerosis complex during early brain development may lead to impaired corticogenesis.

A Korotkov, N S Sim, M J Luinenburg, J J Anink, J van Scheppingen, T S Zimmer, A Bongaarts, D W M Broekaart, C Mijnsbergen, F E Jansen, W Van Hecke, W G M Spliet, P C van Rijen, M Feucht, J A Hainfellner, P Krsek, J Zamecnik, P B Crino, K Kotulska, L Lagae, A C Jansen, D J Kwiatkowski, S Jozwiak, P Curatolo, A Mühlebner, J H Lee, J D Mills, E A van Vliet, E Aronica.

   AIMS: Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA-34a (miR-34a) among the most up-regulated miRs in tubers. Here, we characterized miR-34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR-34a.
METHODS: We analysed the expression of miR-34a in resected cortical tubers (n = 37) compared to autopsy-derived control tissue (n = 27). The effect of miR-34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from 3 patients with TSC and in SH-SY5Y cells following miR-34a transfection.
RESULTS: The peak of miR-34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. MiR-34a was also strongly expressed in fetal TSC cortex. Overexpression of miR-34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR-34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1.
CONCLUSIONS: MiR-34a is most highly overexpressed in tubers during fetal and early postnatal brain development. MiR-34a can negatively regulate mTORC1, however, it may also contribute to abnormal corticogenesis in TSC.

Keywords:  TSC; mechanistic target of rapamycin; miRNA; migration; neurodevelopmental disorder

DOI:  https://doi.org/10.1111/nan.12717
Cell Rep. 2021 May 04. pii: S2211-1247(21)00400-9. [Epub ahead of print]35(5): 109069

mTOR signaling regulates gastric epithelial progenitor homeostasis and gastric tumorigenesis via MEK1-ERKs and BMP-Smad1 pathways.

Ke Li, Hongguang Wu, Ao Wang, Jean Charron, Yuji Mishina, Samy L Habib, Huijuan Liu, Baojie Li.

  mTOR, the sensor of nutrients and growth factors, has important roles in tissue homeostasis and tumorigenesis. However, how mTOR controls gastric epithelial cell turnover and gastric cancer development, a leading malignancy, remains poorly understood. Here, we provide genetic evidence that mTOR activation promotes proliferation and inhibits differentiation of Lgr5+ gastric epithelial progenitors (GEPs) in gastric homeostasis and tumorigenesis. mTOR signaling increases MEK1 and Smad1 expression and enhances activation of MEK1-ERKs and BMP-Smad1 pathways, respectively, in GEPs and gastric tumors. Mek1 deletion or inhibition rescues hyperproliferation, whereas Bmpr1a ablation or inhibition rescues differentiation defects of Tsc1-/- GEPs. Tsc1 deficiency in Lgr5+ GEPs accelerates gastric tumor initiation and development, which require MEK1-ERKs for hyperplasia and BMP-Smad1 for differentiation suppression. These findings reveal how mTOR signaling controls Lgr5+ GEP homeostasis and cancerization and suggest that ERKs and Smad1 signaling can be safely targeted to substitute mTOR inhibitors in gastric cancer therapy.

Keywords:  Lgr5; MEK1; Smad1; TSC; differentiation; gastric cancer; gastric epithelial progenitor; homeostasis; mTOR; tumorigenesis

DOI:  https://doi.org/10.1016/j.celrep.2021.109069
Genetics. 2020 Feb 01. 214(2): 419-445

An ABCG Transporter Functions in Rab Localization and Lysosome-Related Organelle Biogenesis in Caenorhabditis elegans.

Laura Voss, Olivia K Foster, Logan Harper, Caitlin Morris, Sierra Lavoy, James N Brandt, Kimberly Peloza, Simran Handa, Amanda Maxfield, Marie Harp, Brian King, Victoria Eichten, Fiona M Rambo, Greg J Hermann.

  ABC transporters couple ATP hydrolysis to the transport of substrates across cellular membranes. This protein superfamily has diverse activities resulting from differences in their cargo and subcellular localization. Our work investigates the role of the ABCG family member WHT-2 in the biogenesis of gut granules, a Caenorhabditis elegans lysosome-related organelle. In addition to being required for the accumulation of birefringent material within gut granules, WHT-2 is necessary for the localization of gut granule proteins when trafficking pathways to this organelle are partially disrupted. The role of WHT-2 in gut granule protein targeting is likely linked to its function in Rab GTPase localization. We show that WHT-2 promotes the gut granule association of the Rab32 family member GLO-1 and the endolysosomal RAB-7, identifying a novel function for an ABC transporter. WHT-2 localizes to gut granules where it could play a direct role in controlling Rab localization. Loss of CCZ-1 and GLO-3, which likely function as a guanine nucleotide exchange factor (GEF) for GLO-1, lead to similar disruption of GLO-1 localization. We show that CCZ-1, like GLO-3, is localized to gut granules. WHT-2 does not direct the gut granule association of the GLO-1 GEF and our results point to WHT-2 functioning differently than GLO-3 and CCZ-1. Point mutations in WHT-2 that inhibit its transport activity, but not its subcellular localization, lead to the loss of GLO-1 from gut granules, while other WHT-2 activities are not completely disrupted, suggesting that WHT-2 functions in organelle biogenesis through transport-dependent and transport-independent activities.

Keywords:   Caenorhabditis elegans ; ABC transporter; Rab GTPase; lysosome-related organelle; membrane trafficking

DOI:  https://doi.org/10.1534/genetics.119.302900
Cells. 2021 Apr 29. pii: 1054. [Epub ahead of print]10(5):

Manifestations of Age on Autophagy, Mitophagy and Lysosomes in Skeletal Muscle.

Matthew Triolo, David A Hood.

  Sarcopenia is the loss of both muscle mass and function with age. Although the molecular underpinnings of sarcopenia are not fully understood, numerous pathways are implicated, including autophagy, in which defective cargo is selectively identified and degraded at the lysosome. The specific tagging and degradation of mitochondria is termed mitophagy, a process important for the maintenance of an organelle pool that functions efficiently in energy production and with relatively low reactive oxygen species production. Emerging data, yet insufficient, have implicated various steps in this pathway as potential contributors to the aging muscle atrophy phenotype. Included in this is the lysosome, the end-stage organelle possessing a host of proteolytic and degradative enzymes, and a function devoted to the hydrolysis and breakdown of defective molecular complexes and organelles. This review provides a summary of our current understanding of how the autophagy-lysosome system is regulated in aging muscle, highlighting specific areas where knowledge gaps exist. Characterization of the autophagy pathway with a particular focus on the lysosome will undoubtedly pave the way for the development of novel therapeutic strategies to combat age-related muscle loss.

Keywords:  aging; autophagy; lysosomes; mitophagy; sarcopenia; skeletal muscle

DOI:  https://doi.org/10.3390/cells10051054
BMC Biol. 2021 May 06. 19(1): 95

TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae.

Mohammad Alfatah, Jin Huei Wong, Vidhya Gomathi Krishnan, Yong Cheow Lee, Quan Feng Sin, Corinna Jie Hui Goh, Kiat Whye Kong, Wei Ting Lee, Jacqueline Lewis, Shawn Hoon, Prakash Arumugam.

   BACKGROUND: Target of Rapamycin Complex 1 (TORC1) is a highly conserved eukaryotic protein complex that couples the presence of growth factors and nutrients in the environment with cellular proliferation. TORC1 is primarily implicated in linking amino acid levels with cellular growth in yeast and mammals. Although glucose deprivation has been shown to cause TORC1 inactivation in yeast, the precise role of TORC1 in glucose signaling and the underlying mechanisms remain unclear.
RESULTS: We demonstrate that the presence of glucose in the growth medium is both necessary and sufficient for TORC1 activation. TORC1 activity increases upon addition of glucose to yeast cells growing in a non-fermentable carbon source. Conversely, shifting yeast cells from glucose to a non-fermentable carbon source reduces TORC1 activity. Analysis of transcriptomic data revealed that glucose and TORC1 co-regulate about 27% (1668/6004) of yeast genes. We demonstrate that TORC1 orchestrates the expression of glucose-responsive genes mainly via the Tap42-Sit4-Rrd1/2 pathway. To confirm TORC1's function in glucose signaling, we tested its role in spore germination, a glucose-dependent developmental state transition in yeast. TORC1 regulates the glucose-responsive genes during spore germination and inhibition of TORC1 blocks spore germination.
CONCLUSIONS: Our studies indicate that a regulatory loop that involves activation of TORC1 by glucose and regulation of glucose-responsive genes by TORC1, mediates nutritional control of growth and development in yeast.

Keywords:  Spore germination; TORC1; Tap42/Sit4/Rrd1-2 module; Transcriptional response to glucose

DOI:  https://doi.org/10.1186/s12915-021-01030-3
Elife. 2021 May 04. pii: e67578. [Epub ahead of print]10

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering.

Hongki Song, Thomas L Torng, Amy S Orr, Axel T Brunger, William T Wickner.

  Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/aSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

Keywords:  S. cerevisiae; biochemistry; chemical biology

DOI:  https://doi.org/10.7554/eLife.67578
Nat Rev Mol Cell Biol. 2021 May 04.

RAG-ulating mTORC1 with amino acids.

Jenna L Jewell.

DOI: https://doi.org/10.1038/s41580-021-00378-2
J Am Soc Nephrol. 2021 May 06. pii: ASN.2020091321. [Epub ahead of print]

Increasing mTORC1 Pathway Activity or Methionine Supplementation during Pregnancy Reverses the Negative Effect of Maternal Malnutrition on the Developing Kidney.

Yaniv Makayes, Elad Resnick, Liad Hinden, Elina Aizenshtein, Tomer Shlomi, Raphael Kopan, Morris Nechama, Oded Volovelsky.

   BACKGROUND: Low nephron number at birth is associated with a high risk of CKD in adulthood because nephrogenesis is completed in utero. Poor intrauterine environment impairs nephron endowment via an undefined molecular mechanism. A calorie-restricted diet (CRD) mouse model examined the effect of malnutrition during pregnancy on nephron progenitor cells (NPCs).
METHODS: Daily caloric intake was reduced by 30% during pregnancy. mRNA expression, the cell cycle, and metabolic activity were evaluated in sorted Six2 NPCs. The results were validated using transgenic mice, oral nutrient supplementation, and organ cultures.
RESULTS: Maternal CRD is associated with low nephron number in offspring, compromising kidney function at an older age. RNA-seq identified cell cycle regulators and the mTORC1 pathway, among other pathways, that maternal malnutrition in NPCs modifies. Metabolomics analysis of NPCs singled out the methionine pathway as crucial for NPC proliferation and maintenance. Methionine deprivation reduced NPC proliferation and lowered NPC number per tip in embryonic kidney cultures, with rescue from methionine metabolite supplementation. Importantly, in vivo, the negative effect of caloric restriction on nephrogenesis was prevented by adding methionine to the otherwise restricted diet during pregnancy or by removing one Tsc1 allele in NPCs.
CONCLUSIONS: These findings show that mTORC1 signaling and methionine metabolism are central to the cellular and metabolic effects of malnutrition during pregnancy on NPCs, contributing to nephrogenesis and later, to kidney health in adulthood.

Keywords:  intrauterine environment; kidney development; mTOR pathway; malnutrition; methionine; nephron progenitor cells; stem cell

DOI:  https://doi.org/10.1681/ASN.2020091321
Lipids Health Dis. 2021 May 03. 20(1): 44

Sphingolipid lysosomal storage diseases: from bench to bedside.

Muna Abed Rabbo, Yara Khodour, Laurie S Kaguni, Johnny Stiban.

  Johann Ludwig Wilhelm Thudicum described sphingolipids (SLs) in the late nineteenth century, but it was only in the past fifty years that SL research surged in importance and applicability. Currently, sphingolipids and their metabolism are hotly debated topics in various biochemical fields. Similar to other macromolecular reactions, SL metabolism has important implications in health and disease in most cells. A plethora of SL-related genetic ailments has been described. Defects in SL catabolism can cause the accumulation of SLs, leading to many types of lysosomal storage diseases (LSDs) collectively called sphingolipidoses. These diseases mainly impact the neuronal and immune systems, but other systems can be affected as well. This review aims to present a comprehensive, up-to-date picture of the rapidly growing field of sphingolipid LSDs, their etiology, pathology, and potential therapeutic strategies. We first describe LSDs biochemically and briefly discuss their catabolism, followed by general aspects of the major diseases such as Gaucher, Krabbe, Fabry, and Farber among others. We conclude with an overview of the available and potential future therapies for many of the diseases. We strive to present the most important and recent findings from basic research and clinical applications, and to provide a valuable source for understanding these disorders.

Keywords:  Fabry; Gaucher; Krabbe; gangliosidosis; inborn errors of metabolism; lysosomal storage diseases; neurological diseases; sphingolipidoses; sphingolipids

DOI:  https://doi.org/10.1186/s12944-021-01466-0
Sci Rep. 2021 May 06. 11(1): 9657

pH induced conformational alteration in human peroxiredoxin 6 might be responsible for its resistance against lysosomal pH or high temperature.

Rimpy Kaur Chowhan, Sunaina Hotumalani, Hamidur Rahaman, Laishram Rajendrakumar Singh.

Peroxiredoxin 6 (Prdx6), the ubiquitously expressed enzyme belonging to the family of peroxidases, namely, peroxiredoxins, exhibits a unique feature of functional compartmentalization within cells. Whereas, the enzyme localized in cytosol shows glutathione peroxidase activity, its lysosomal counterpart performs calcium independent phospholipase A2 (aiPLA2) activity. Like any true moonlighting protein, these two activities of Prdx6 are mutually exclusive of each other as a function of the pH of the cellular compartments. Differential substrate preference at different pH (i.e. peroxidised phospholipids at neutral pH and reduced phospholipids at acidic pH) is considered to be the reason for this behavior. To gain insight into the pH-induced structural-functional interplay we have systematically evaluated conformational variations, thermodynamic stability of the protein and quaternary state of the conformers at both pH 7.0 and 4.0. Our findings suggest that change in pH allows alterations in native states of Prdx6 at pH 7.0 and 4.0 such that the changes make the protein resistant to thermal denaturation at low pH.

DOI: https://doi.org/10.1038/s41598-021-89093-8
Genetics. 2019 Oct 01. 213(2): 705-720

A Stress-Responsive Signaling Network Regulating Pseudohyphal Growth and Ribonucleoprotein Granule Abundance in Saccharomyces cerevisiae.

Nebibe Mutlu, Daniel T Sheidy, Angela Hsu, Han Seol Jeong, Katherine J Wozniak, Anuj Kumar.

  The budding yeast Saccharomyces cerevisiae undergoes a stress-responsive transition to a pseudohyphal growth form in which cells elongate and remain connected in multicellular filaments. Pseudohyphal growth is regulated through conserved signaling networks that control cell growth and the response to glucose or nitrogen limitation in metazoans. These networks are incompletely understood, and our studies identify the TORC1- and PKA-regulated kinase Ksp1p as a key stress-responsive signaling effector in the yeast pseudohyphal growth response. The kinase-defective ksp1-K47D allele results in decreased pseudohyphal morphology at the cellular and colony level, indicating that Ksp1p kinase signaling is required for pseudohyphal filamentation. To determine the functional consequences of Ksp1p signaling, we implemented transcriptional profiling and quantitative phosphoproteomic analysis of ksp1-K47D on a global scale. Ksp1p kinase signaling maintains wild-type transcript levels of many pathways for amino acid synthesis and metabolism, relevant for the regulation of translation under conditions of nutrient stress. Proteins in stress-responsive ribonucleoprotein granules are regulated post-translationally by Ksp1p, and the Ksp1p-dependent phosphorylation sites S176 in eIF4G/Tif4631p and S436 in Pbp1p are required for wild-type levels of pseudohyphal growth and Protein Kinase A pathway activity. Pbp1p and Tif4631p localize in stress granules, and the ksp1 null mutant shows elevated abundance of Pbp1p puncta relative to wild-type. Collectively, the Ksp1p kinase signaling network integrates polarized pseudohyphal morphogenesis and translational regulation through the stress-responsive transcriptional control of pathways for amino acid metabolism and post-translational modification of translation factors affecting stress granule abundance.

Keywords:  functional genomics; proteomics; pseudohyphal growth; yeast

DOI:  https://doi.org/10.1534/genetics.119.302538
Front Oncol. 2021 ;11 665420

Rapamycin Promotes ROS-Mediated Cell Death via Functional Inhibition of xCT Expression in Melanoma Under γ-Irradiation.

Yunseo Woo, Hyo-Ji Lee, Jeongyeon Kim, Seung Goo Kang, Sungjin Moon, Jeong A Han, Young Mee Jung, Yu-Jin Jung.

  Although many cancer patients are administered radiotherapy for their treatment, the interaction between tumor cells and macrophages in the tumor microenvironment attenuates the curative effects of radiotherapy. The enhanced activation of mTOR signaling in the tumors promotes tumor radioresistance. In this study, the effects of rapamycin on the interaction between tumor cells and macrophages were investigated. Rapamycin and 3BDO were used to regulate the mTOR pathway. In vitro, tumor cells cocultured with macrophages in the presence of each drug under normoxic or hypoxic conditions were irradiated with γ-rays. In vivo, mice were irradiated with γ-radiation after injection with DMSO, rapamycin and 3BDO into tumoral regions. Rapamycin reduced the secretion of IL-4 in tumor cells as well as YM1 in macrophages. Mouse recombinant YM1 decreased the enhanced level of ROS and the colocalized proportion of both xCT and EEA1 in irradiated tumor cells. Human recombinant YKL39 also induced results similar to those of YM1. Moreover, the colocalized proportion of both xCT and LC3 in tumor tissues was elevated by the injection of rapamycin into tumoral regions. Overall, the suppression of mTOR signaling in the tumor microenvironment might be useful for the improvement of tumor radioresistance.

Keywords:  KEAP1; NRF2; YM1; mTOR; macrophage; melanoma; radiation

DOI:  https://doi.org/10.3389/fonc.2021.665420
J Neuroinflammation. 2021 May 06. 18(1): 107

Conditional knockout of Tsc1 in RORγt-expressing cells induces brain damage and early death in mice.

Yafei Deng, Qinglan Yang, Yao Yang, Yana Li, Hongyan Peng, Shuting Wu, Shuju Zhang, Baige Yao, Shuhui Li, Yuan Gao, Xiaohui Li, Liping Li, Youcai Deng.

   BACKGROUND: Tuberous sclerosis complex 1 (Tsc1) is known to regulate the development and function of various cell types, and RORγt is a critical transcription factor in the immune system. However, whether Tsc1 participates in regulating RORγt-expressing cells remains unknown.
METHODS: We generated a mouse model in which Tsc1 was conditionally deleted from RORγt-expressing cells (Tsc1RORγt) to study the role of RORγt-expressing cells with Tsc1 deficiency in brain homeostasis.
RESULTS: Type 3 innate lymphoid cells (ILC3s) in Tsc1RORγt mice displayed normal development and function, and the mice showed normal Th17 cell differentiation. However, Tsc1RORγt mice exhibited spontaneous tonic-clonic seizures and died between 4 and 6 weeks after birth. At the age of 4 weeks, mice in which Tsc1 was specifically knocked out in RORγt-expressing cells had cortical neuron defects and hippocampal structural abnormalities. Notably, over-activation of neurons and astrogliosis were observed in the cortex and hippocampus of Tsc1RORγt mice. Moreover, expression of the γ-amino butyric acid (GABA) receptor in the brains of Tsc1RORγt mice was decreased, and GABA supplementation prolonged the lifespan of the mice to some extent. Further experiments revealed the presence of a group of rare RORγt-expressing cells with high metabolic activity in the mouse brain.
CONCLUSIONS: Our study verifies the critical role of previously unnoticed RORγt-expressing cells in the brain and demonstrates that the Tsc1 signaling pathway in RORγt-expressing cells is important for maintaining brain homeostasis.

Keywords:  GABA; RORγt-expressing cells; Seizure; Tsc1

DOI:  https://doi.org/10.1186/s12974-021-02153-8
Am J Med Genet C Semin Med Genet. 2021 May 07.

Genotype-phenotype studies in a large cohort of Brazilian patients with Hunter syndrome.

Juliana Alves Josahkian, Ana Carolina Brusius-Facchin, Alice Brinckmann Oliveira Netto, Sandra Leistner-Segal, Diana Rojas Málaga, Maira Graeff Burin, Kristiane Michelin-Tirelli, Franciele Barbosa Trapp, Augusto César Cardoso-Dos-Santos, Erlane Marques Ribeiro, Chong Ae Kim, Ana Cecília Menezes de Siqueira, Mara Lucia Santos, Daniel Almeida do Valle, Raquel Tavares Boy da Silva, Dafne Dain Gandelman Horovitz, Paula Frassinetti Vasconcelos de Medeiros, Carolina Fischinger Moura de Souza, Liane de Rosso Giuliani, Diego Santana Chaves Geraldo Miguel, Luiz Carlos Santana-da-Silva, Marcial Francis Galera, Roberto Giugliani.

  Mucopolysaccharidosis type II (MPS II) is an X-linked inherited disease caused by pathogenic variants in the IDS gene, leading to deficiency of the lysosomal enzyme iduronate-2-sulfatase and consequent widespread storage of glycosaminoglycans, leading to several clinical consequences, with progressive manifestations which most times includes cognitive decline. MPS II has wide allelic and clinical heterogeneity and a complex genotype-phenotype correlation. We evaluated data from 501 Brazilian patients diagnosed with MPS II from 1982 to 2020. We genotyped 280 of these patients (55.9%), which were assigned to 206 different families. Point mutations were present in 70% of our patients, being missense variants the most frequent. We correlated the IDS pathogenic variants identified with the phenotype (neuronophatic or non-neuronopathic). Except for two half-brothers, there was no discordance in the genotype-phenotype correlation among family members, nor among MPS II patients from different families with the same single base-pair substitution variant. Mothers were carriers in 82.0% of the cases. This comprehensive study of the molecular profile of the MPS II cases in Brazil sheds light on the genotype-phenotype correlation and helps the better understanding of the disease and the prediction of its clinical course, enabling the provision of a more refined genetic counseling to the affected families.

Keywords:  Hunter syndrome; IDS gene; iduronate-2-sulfatase; lysosomal storage diseases; mucopolysaccharidosis type II

DOI:  https://doi.org/10.1002/ajmg.c.31915
Nature. 2021 May 05.

Distinct fission signatures predict mitochondrial degradation or biogenesis.

Tatjana Kleele, Timo Rey, Julius Winter, Sofia Zaganelli, Dora Mahecic, Hélène Perreten Lambert, Francesco Paolo Ruberto, Mohamed Nemir, Timothy Wai, Thierry Pedrazzini, Suliana Manley.

Mitochondrial fission is a highly regulated process that, when disrupted, can alter metabolism, proliferation and apoptosis1-3. Dysregulation has been linked to neurodegeneration3,4, cardiovascular disease3 and cancer5. Key components of the fission machinery include the endoplasmic reticulum6 and actin7, which initiate constriction before dynamin-related protein 1 (DRP1)8 binds to the outer mitochondrial membrane via adaptor proteins9-11, to drive scission12. In the mitochondrial life cycle, fission enables both biogenesis of new mitochondria and clearance of dysfunctional mitochondria through mitophagy1,13. Current models of fission regulation cannot explain how those dual fates are decided. However, uncovering fate determinants is challenging, as fission is unpredictable, and mitochondrial morphology is heterogeneous, with ultrastructural features that are below the diffraction limit. Here, we used live-cell structured illumination microscopy to capture mitochondrial dynamics. By analysing hundreds of fissions in African green monkey Cos-7 cells and mouse cardiomyocytes, we discovered two functionally and mechanistically distinct types of fission. Division at the periphery enables damaged material to be shed into smaller mitochondria destined for mitophagy, whereas division at the midzone leads to the proliferation of mitochondria. Both types are mediated by DRP1, but endoplasmic reticulum- and actin-mediated pre-constriction and the adaptor MFF govern only midzone fission. Peripheral fission is preceded by lysosomal contact and is regulated by the mitochondrial outer membrane protein FIS1. These distinct molecular mechanisms explain how cells independently regulate fission, leading to distinct mitochondrial fates.

DOI: https://doi.org/10.1038/s41586-021-03510-6
ACS Chem Biol. 2021 May 06.

Discovery of Highly Specific Catalytic-Site-Targeting Fluorescent Probes for Detecting Lysosomal PDE10A in Living Cells.

Yuqi Gao, Ju Huang, Qian Zhou, Runduo Liu, Sirui Zhang, Chen Zhang, Yi-You Huang, Zhe Li, Ling Huang, Deyan Wu, Yinuo Wu, Lehui Xiao, Lei Guo, Hai-Bin Luo.

A challenge for sensors targeting specific enzymes of interest in their native environment for direct imaging is that they rationally exploit a highly selective fluorescent probe with a high binding affinity to provide real-time detection. Immunohistochemical staining, proteomic analysis, or recent enzymatic fluorescent probes are not optimal for tracking specific enzymes directly in living cells. Herein, we introduce the concept of designing a highly effective fluorescent probe (BVQ1814) targeting phosphodiesterase 10A with a highly potent affinity and a >1000-fold subfamily selectivity by gaining insights into the three-dimensional structural information of the active site of the catalytic pocket. BVQ1814 showed an outstanding binding affinity for PDE10A in vitro and specifically detected PDE10A in living cells, indicating that most PDE10A was probably distributed in the lysosomes. We validated the PDE10A distribution in stable mCherry-PDE10A-overexpressing HepG2 cells. This probe delineated the profile of PDE10A in tissue sections and exhibited a remarkable therapeutic effect as a PDE10A inhibitor for treating pulmonary arterial hypertension. This concept will open up a new avenue for designing a highly effective fluorescent probe for tracking receptor proteins by taking full advantage of the structural information in the ligand-binding pocket of the target of interest.

DOI: https://doi.org/10.1021/acschembio.1c00018
Anal Chem. 2021 May 04.

DNA-Based pH Nanosensor with Adjustable FRET Responses to Track Lysosomes and pH Fluctuations.

Xinmin Yue, Yanqi Qiao, Dening Gu, Rui Qi, Hongjie Zhao, Yongmei Yin, Wei Zhao, Rimo Xi, Meng Meng.

Extensive attention has been recently focused on designing signal adjustable biosensors. However, there are limited approaches available in this field. In this work, to visually track lysosomes with high contrast, we used the i-motif structure as a pH-responsive unit and proposed a novel strategy to regulate the fluorescence resonance energy transfer (FRET) response of the pH sensor. By simply splitting the i-motif into two parts and modulating the split parameters, we can tune the pH transition midpoint (pHt) from 5.71 to 6.81 and the signal-to-noise ratio (S/N) from 1.94 to 18.11. To facilitate the lysosome tracking, we combined the i-motif split design with tetrahedral DNA (Td). The obtained pH nanosensor (pH-Td) displays appropriate pHt (6.12) to trace lysosomes with high S/N (10.3). Benefited from the improved stability, the superior cell uptake and lysosomal location of pH-Td, the visualization of the distribution of lysosomes, the lysosome-mitochondria interaction, and the pH changes of lysosomes in response to different stimuli were successfully achieved in NIH 3T3 cells. We believe that the design concept of controlling the split sequence distance will provide a novel insight into the design of i-motif-based nanosensors and even inspire the construction of smart DNA nanodevices for sensing, disease diagnosis, and controllable drug delivery.

DOI: https://doi.org/10.1021/acs.analchem.1c00436