bims-lysosi 2021-08-22 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒08‒22
forty-two papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).
Protein aggregation and autophagy dysfunction: new lessons from mucopolysaccharidoses.
Cell-type selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies.
Disease modeling for Mucopolysaccharidosis type IIIB using patient derived induced pluripotent stem cells.
Hepatic lysosomal acid lipase drives the autophagy-lysosomal response and alleviates cholesterol metabolic disorder in ApoE deficient mice.
Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry.
Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked Thiel-Behnke corneal dystrophy.
Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.
Efficacy of a Novel Bi-Steric mTORC1 Inhibitor in Models of B-Cell Acute Lymphoblastic Leukemia.
Lysosome Depletion-Triggered Autophagy Impairment in Progressive Kidney Injury.
AMPK: a key regulator of energy stress and calcium-induced autophagy.
Selective Neutral pH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal pH Conditions.
Improved systemic AAV gene therapy with a neurotrophic capsid in Niemann-Pick disease type C1 mice.
Pex30-like proteins function as adaptors at distinct ER membrane contact sites.
MPS VI associated ocular phenotypes in an MPS VI murine model and the therapeutic effects of odiparcil treatment.
TFEB, a master regulator of autophagy and biogenesis, unexpectedly promotes apoptosis in response to the cyclopentenone prostaglandin 15d-PGJ2.
Tuberous sclerosis complex (TSC) inactivation increases neuronal network activity by enhancing Ca2+ influx via L-type Ca2+ channels.
A New Brain-Penetrant Glucosylceramide Synthase Inhibitor as Potential Therapeutics for Gaucher Disease.
mTOR Signaling Regulates Metabolic Function in Oligodendrocyte Precursor Cells and Promotes Efficient Brain Remyelination in the Cuprizone Model.
Partners with a killer: Metabolic signaling promotes inflammatory cell death.
Contribution of Energy Dysfunction to Impaired Protein Translation in Neurodegenerative Diseases.
Saturated very long chain fatty acid configures glycosphingolipid for lysosome homeostasis in long-lived C. elegans.
Rab GTPases in Parkinson's disease: a primer.
The role of membrane contact sites at the bacteria-host interface.
Lysosome-targeting chimeras evolve.
Nhp2 is a reader of H2AQ105me and part of a network integrating metabolism with rRNA synthesis.
Endolysosomal Ca2+ signaling in cardiovascular health and disease.
TFEB phosphorylation on Serine 211 is induced by autophagy in human synovial fibroblasts and by p62/SQSTM1 overexpression in HEK293 cells.
The microglial lysosomal system in Alzheimer's disease: Guardian against proteinopathy.
Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.
Lysosome purinergic receptor P2X4 regulates neoangiogenesis induced by microvesicles from sarcoma patients.
Cerebellar Long Noncoding RNA Expression Profile in a Niemann-Pick C Disease Mouse Model.
Expanding TOR Complex 2 Signaling: Emerging Regulators and New Connections.
Blockage of Extracellular Signal-Regulated Kinase Exerts an Antitumor Effect via Regulating Energy Metabolism and Enhances the Efficacy of Autophagy Inhibitors by Regulating Transcription Factor EB Nuclear Translocation in Osteosarcoma.
Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line.
Linking transport and translation of mRNAs with endosomes and mitochondria.
Bifunctional small molecules that mediate the degradation of extracellular proteins.
Dysfunction of RAB39B-Mediated Vesicular Trafficking in Lewy Body Diseases.
NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells.
Hypo-Expression of Tuberin Promotes Adenomyosis via the mTOR1-Autophagy Axis.
Ca2+ dysregulation in the pathogenesis of amyotrophic lateral sclerosis.
Intravenous delivery of adeno-associated viral gene therapy in feline GM1 gangliosidosis.

Biochem J. 2021 Aug 18. pii: BCJ20210415. [Epub ahead of print]

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).

Björn Kowalewski, Heike Lange, Sabrina Galle, Thomas Dierks, Torben Lübke, Markus Damme.

  The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wildtype and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate.

Keywords:  3-O sulfated; Arylsulfatase G; Heparan sulfate; Lysosomes; glycosaminoglycans

DOI:  https://doi.org/10.1042/BCJ20210415
Autophagy. 2021 Aug 18. 1-2

Protein aggregation and autophagy dysfunction: new lessons from mucopolysaccharidoses.

Antonio Monaco, Alessandro Fraldi.

  Mucopolysaccharidoses (MPS) are inherited metabolic diseases with strong neurological involvement. MPSs are caused by defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs), which consequently accumulate into the lysosomes as primary storage. Macroautophagy/autophagy impairment is well known to drive neurodegeneration in MPSs, however, mechanisms underlying such dysfunction are still poorly understood. Recently, by studying a mouse model for MPS-III (Sanfilippo syndrome) we have shown that the progressive aggregation of amyloid proteins in neuronal cell bodies occurs downstream of the GAG storage and, in turn, impairs the autophagy pathway by affecting lysosomal-dependent autophagosome clearance.

Keywords:  Autophagy; lysosomal storage diseases; lysosome; mucopolysaccharidoses; protein aggregation

DOI:  https://doi.org/10.1080/15548627.2021.1961076
Mol Ther. 2021 Aug 13. pii: S1525-0016(21)00414-7. [Epub ahead of print]

Cell-type selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies.

Andrew D Baik, Philip Calafati, Xiaoli Zhang, Nina A Aaron, Antonia Mehra, Sven Moller-Tank, Lawrence Miloscio, Maria Praggastis, Nicholas Giovannone, Cheryl Pan, Yajun Tang, Susannah Bridges, Alejo Mujica, Peter Barbounis, Jean Yanolatos, Nicholas Gale, Ning Li, Christos A Kyratsous, Christopher J Schoenherr, Andrew J Murphy, Aris N Economides, Katherine D Cygnar.

Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy.

DOI: https://doi.org/10.1016/j.ymthe.2021.08.020
Exp Cell Res. 2021 Aug 16. pii: S0014-4827(21)00338-4. [Epub ahead of print] 112785

Disease modeling for Mucopolysaccharidosis type IIIB using patient derived induced pluripotent stem cells.

Wei Huang, Yu-Shan Cheng, Shu Yang, Manju Swaroop, Miao Xu, Wenwei Huang, Wei Zheng.

  Mucopolysaccharidosis type IIIB (MPS IIIB) is a lysosomal disease caused by mutations in the NAGLU gene encoding α-N-acetylglucosaminidase (NAGLU) which degrades heparan sulfate in lysosomes. Deficiency in NAGLU results in lysosomal accumulation of glycosaminoglycans (GAGs) and neurological symptoms. Currently, there is no effective treatment or cure for this disease. In this study, induced pluripotent stem cell lines were established from two MPS IIIB patient fibroblast lines and differentiated into neural stem cells and neurons. MPS IIIB neural stem cells exhibited NAGLU deficiency accompanied with GAG accumulation, as well as lysosomal enlargement and secondary lipid accumulation. Treatments with recombinant NAGLU, δ-tocopherol, and 2-hydroxypropyl-b-cyclodextrin significantly reduced the disease phenotypes in these cells. These results indicate the MPS IIIB neural stem cells and neurons have the disease relevant phenotype and can be used as a cell-based disease model system for evaluation of drug efficacy and compound screening for drug development.

Keywords:  2-hydroxypropyl-b-cyclodextrin; Mucopolysaccharidosis type IIIB; Sanfilippo syndrome B; iPSC disease model; lysosomal storage disease; δ-tocopherol

DOI:  https://doi.org/10.1016/j.yexcr.2021.112785
Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Aug 17. pii: S1388-1981(21)00155-4. [Epub ahead of print] 159027

Hepatic lysosomal acid lipase drives the autophagy-lysosomal response and alleviates cholesterol metabolic disorder in ApoE deficient mice.

Feifei Li, Xiaojie Zhao, Hao Li, Yu Liu, Yu Zhang, Xiaopeng Huang, Jia Cao, Fen Du, Dongfang Wu, Hong Yu.

  Lysosomal acid lipase (LAL)-dependent lipolysis degrades cholesteryl ester (CE) and triglyceride in the lysosome. LAL deficiency in human and mice leads to hypercholesterolemia, hepatic CE deposition, and atherosclerosis. Despite its hepatocyte-specific deficiency leads to CE accumulation, the regulation of LAL in cholesterol metabolic disease remains elusive. For the in vitro study, the target gene Lipa was transfected with recombinant shRNA or lentiviral vector in Hepa1-6 cells. It was found that LAL silencing in cells affected lysosomal function by reducing LAL activity and proteolytic activity, and altered the expression of genes related to cholesterol metabolism and autophagy, leading to cholesterol accumulation; whereas LAL overexpression improved the above effects. To explore the impacts of hepatic LAL on cholesterol metabolic disease in vivo, apolipoprotein E deficient (ApoE-/-) mice were intravenously injected with lentivirus to achieve hepatic LAL overexpression and fed a Western diet for 16 weeks. The results showed that hepatic LAL overexpression significantly reduced plasma lipid levels, alleviated inflammation and oxidative status in plasma and liver, and attenuated hepatic steatosis and fibrosis in ApoE-/- mice. Mechanically, hepatic LAL promoted cholesterol transport and biliary excretion by increasing liver X receptor alpha (LXRα) and its downstream genes, and modulated the compliance of the autophagy-lysosomal pathway. Our data provide the original evidence of the validity of hepatic LAL in controlling cholesterol metabolism and liver homeostasis, suggesting that targeting hepatic LAL may provide a promising approach to rescue cholesterol metabolic disorders, such as hypercholesterolemia and liver disease.

Keywords:  Autophagic flux; Cholesterol metabolism; Hepatic steatosis; Lysosomal acid lipase; Lysosome

DOI:  https://doi.org/10.1016/j.bbalip.2021.159027
J Vis Exp. 2021 Aug 02.

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry.

Amine Belaid, Harilaos Filippakis.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically. In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

DOI: https://doi.org/10.3791/62793
Autophagy. 2021 Aug 17. 1-18

Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked Thiel-Behnke corneal dystrophy.

Liyuan Wang, Chuchu Zhao, Tao Zheng, Yi Zhang, Hanruo Liu, Xi Wang, Xianling Tang, Baowen Zhao, Ping Liu.

  Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD.

Keywords:  Autophagy; TBCD; TFEB; TGFBI; lysosome dysfunction; torin 1

DOI:  https://doi.org/10.1080/15548627.2021.1955469
EMBO Mol Med. 2021 Aug 19. e13742

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.

Chiara Soldati, Irene Lopez-Fabuel, Luca G Wanderlingh, Marina Garcia-Macia, Jlenia Monfregola, Alessandra Esposito, Gennaro Napolitano, Marta Guevara-Ferrer, Anna Scotto Rosato, Einar K Krogsaeter, Dominik Paquet, Christian M Grimm, Sandro Montefusco, Thomas Braulke, Stephan Storch, Sara E Mole, Maria A De Matteis, Andrea Ballabio, Julio L Sampaio, Tristan McKay, Ludger Johannes, Juan P Bolaños, Diego L Medina.

  Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.

Keywords:  CLN3; CLN7; TFEB; high content imaging screening; tamoxifen

DOI:  https://doi.org/10.15252/emmm.202013742
Front Oncol. 2021 ;11 673213

Efficacy of a Novel Bi-Steric mTORC1 Inhibitor in Models of B-Cell Acute Lymphoblastic Leukemia.

Bianca J Lee, Sharmila Mallya, Nuntana Dinglasan, Amos Fung, Tram Nguyen, Lee-Or Herzog, Joshua Thao, Edward G Lorenzana, David Wildes, Mallika Singh, Jacqueline A M Smith, David A Fruman.

  The mechanistic target of rapamycin (mTOR) is a kinase whose activity is elevated in hematological malignancies. mTOR-complex-1 (mTORC1) phosphorylates numerous substrates to promote cell proliferation and survival. Eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) are mTORC1 substrates with an integral role in oncogenic protein translation. Current pharmacological approaches to inhibit mTORC1 activity and 4E-BP phosphorylation have drawbacks. Recently we described a series of bi-steric compounds that are potent and selective inhibitors of mTORC1, inhibiting 4E-BP phosphorylation at lower concentrations than mTOR kinase inhibitors (TOR-KIs). Here we report the activity of the mTORC1-selective bi-steric inhibitor, RMC-4627, in BCR-ABL-driven models of B-cell acute lymphoblastic leukemia (B-ALL). RMC-4627 exhibited potent and selective inhibition of 4E-BP1 phosphorylation in B-ALL cell lines without inhibiting mTOR-complex-2 (mTORC2) activity. RMC-4627 suppressed cell cycle progression, reduced survival, and enhanced dasatinib cytotoxicity. Compared to a TOR-KI compound, RMC-4627 was more potent, and its effects on cell viability were sustained after washout in vitro. Notably, a once-weekly, well tolerated dose reduced leukemic burden in a B-ALL xenograft model and enhanced the activity of dasatinib. These preclinical studies suggest that intermittent dosing of a bi-steric mTORC1-selective inhibitor has therapeutic potential as a component of leukemia regimens, and further study is warranted.

Keywords:  4EBP1; Ph+ B-ALL; combination therapy; mTORC1; targeted (selective) treatment

DOI:  https://doi.org/10.3389/fonc.2021.673213
Kidney Dis (Basel). 2021 Jul;7(4): 254-267

Lysosome Depletion-Triggered Autophagy Impairment in Progressive Kidney Injury.

Xiao-Cui Chen, Zhi-Hang Li, Chen Yang, Ji-Xin Tang, Hui-Yao Lan, Hua-Feng Liu.

  Background: Macroautophagy (autophagy) is a cellular recycling process involving the destruction of damaged organelles and proteins in intracellular lysosomes for efficient nutrient reuse.Summary: Impairment of the autophagy-lysosome pathway is tightly associated with multiple kidney diseases, such as diabetic nephropathy, proteinuric kidney disease, acute kidney injury, crystalline nephropathy, and drug- and heavy metal-induced renal injury. The impairment in the process of autophagic clearance may induce injury in renal intrinsic cells by activating the inflammasome, inducing cell cycle arrest, and cell death. The lysosome depletion may be a key mechanism triggering this process. In this review, we discuss this pathway and summarize the protective mechanisms for restoration of lysosome function and autophagic flux via the endosomal sorting complex required for transport (ESCRT) machinery, lysophagy, and transcription factor EB-mediated lysosome biogenesis.
Key Message: Further exploring mechanisms of ESCRT, lysophagy, and lysosome biogenesis may provide novel therapy strategies for the management of kidney diseases.

Keywords:  Autophagy; Kidney disease; Lysosome; Podocyte; Tubular epithelial cell

DOI:  https://doi.org/10.1159/000515035
J Mol Med (Berl). 2021 Aug 16.

AMPK: a key regulator of energy stress and calcium-induced autophagy.

Rimpi Saikia, Jomon Joseph.

  Autophagy is a well-known cell-survival strategy orchestrated by a conserved set of proteins. It equips the cells with mechanisms to attain homeostasis during unfavorable conditions such as stress by breaking down the cellular components and reusing them for energy as well as for building new components required for survival. A basal level of autophagy is required for achieving homeostasis under normal conditions through regular turnover of macromolecules and organelles. Initiation of autophagy is regulated by two key components of the nutrient/energy sensor pathways; mammalian target of rapamycin 1 (mTORC1) and AMP-activated kinase (AMPK). Under energy-deprived conditions, AMPK is activated triggering autophagy, whereas, in nutrient-rich conditions, the growth-promoting kinase mTORC1 is activated inhibiting autophagy. Thus, the reciprocal regulation of autophagy by AMPK and mTORC1 defines a fundamental mechanism by which cells respond to nutrient availability. Interestingly, cytoplasmic calcium is also found to be an activator of AMPK and autophagy through a calmodulin/CaMKKβ pathway. However, the physiological significance of the regulation of autophagy by cytoplasmic calcium is currently unclear. This review focuses on the current understanding of the mechanism of autophagy and its regulation by AMPK.

Keywords:  AMPK; Autophagy; CaMKKβ; Calcium; Energy homeostasis; LKB1; mTORC1

DOI:  https://doi.org/10.1007/s00109-021-02125-8
ACS Chem Biol. 2021 Aug 20.

Selective Neutral pH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal pH Conditions.

Michael C Yoon, Angelo Solania, Zhenze Jiang, Mitchell P Christy, Sonia Podvin, Charles Mosier, Christopher B Lietz, Gen Ito, William H Gerwick, Dennis W Wolan, Gregory Hook, Anthony J O'Donoghue, Vivian Hook.

Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.

DOI: https://doi.org/10.1021/acschembio.1c00138
Life Sci Alliance. 2021 Oct;pii: e202101040. [Epub ahead of print]4(10):

Improved systemic AAV gene therapy with a neurotrophic capsid in Niemann-Pick disease type C1 mice.

Cristin D Davidson, Alana L Gibson, Tansy Gu, Laura L Baxter, Benjamin E Deverman, Keith Beadle, Arturo A Incao, Jorge L Rodriguez-Gil, Hideji Fujiwara, Xuntian Jiang, Randy J Chandler, Daniel S Ory, Viviana Gradinaru, Charles P Venditti, William J Pavan.

Niemann-Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1 m1N/m1N mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1 m1N/m1N mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1 m1N/m1N mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.

DOI: https://doi.org/10.26508/lsa.202101040
J Cell Biol. 2021 Oct 04. pii: e202103176. [Epub ahead of print]220(10):

Pex30-like proteins function as adaptors at distinct ER membrane contact sites.

Joana Veríssimo Ferreira, Pedro Carvalho.

Membrane lipids and proteins synthesized in the ER are used for de novo assembly of organelles, such as lipid droplets and peroxisomes. After assembly, the growth of these organelles is supported by ER-derived lipids transferred at membrane contact sites (MCSs). How ER sites for organelle biogenesis and lipid transfer are established and regulated is unclear. Here, we investigate how the ER membrane protein Pex30 and its family members Pex28, Pex29, Pex31, and Pex32 target and function at multiple MCSs. We show that different Pex30 complexes function at distinct ER domains and MCSs. Pex30 targets ER-peroxisome MCSs when bound to Pex28 and Pex32, organizes the nuclear-vacuolar junction when bound to Pex29, and promotes the biogenesis of lipid droplets independently of other family members. Importantly, the reticulon homology domain (RHD) mediates the assembly of the various Pex30 complexes. Given the role of RHD in membrane shaping, our findings offer a mechanistic link between MCS and regulation of membrane curvature.

DOI: https://doi.org/10.1083/jcb.202103176
Mol Genet Metab. 2021 Jul 25. pii: S1096-7192(21)00759-9. [Epub ahead of print]

MPS VI associated ocular phenotypes in an MPS VI murine model and the therapeutic effects of odiparcil treatment.

Eugeni Entchev, Sophie Antonelli, Virginie Mauro, Nicolas Cimbolini, Ingrid Jantzen, Aurelie Roussey, Jeanne-Marie Germain, Haoyue Zhang, Jean-Michel Luccarrini, Olivier Lacombe, Sarah P Young, Laurence Feraille, Mireille Tallandier.

  Maroteaux - Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is a lysosomal storage disease resulting from insufficient enzymatic activity for degradation of the specific glycosaminoglycans (GAG) chondroitin sulphate (CS) and dermatan sulphate (DS). Among the most pronounced MPS VI clinical manifestations caused by cellular accumulation of excess CS and DS are eye disorders, in particular those that affect the cornea. Ocular manifestations are not treated by the current standard of care, enzyme replacement therapy (ERT), leaving patients with a significant unmet need. Using in vitro and in vivo models, we previously demonstrated the potential of the β-D-xyloside, odiparcil, as an oral GAG clearance therapy for MPS VI. Here, we characterized the eye phenotypes in MPS VI arylsulfatase B deficient mice (Arsb-) and studied the effects of odiparcil treatment in early and established disease models. Severe levels of opacification and GAG accumulation were detected in the eyes of MPS VI Arsb- mice. Histological examination of MPS VI Arsb- eyes showed an aggregate of corneal phenotypes, including reduction in the corneal epithelium thickness and number of epithelial cell layers, and morphological malformations in the stroma. In addition, colloidal iron staining showed specifically GAG accumulation in the cornea. Orally administered odiparcil markedly reduced GAG accumulation in the eyes of MPS VI Arsb- mice in both disease models and restored the corneal morphology (epithelial layers and stromal structure). In the early disease model of MPS VI, odiparcil partially reduced corneal opacity area, but did not affect opacity area in the established model. Analysis of GAG types accumulating in the MPS VI Arsb- eyes demonstrated major contribution of DS and CS, with some increase in heparan sulphate (HS) as well and all were reduced with odiparcil treatment. Taken together, we further reveal the potential of odiparcil to be an effective therapy for eye phenotypes associated with MPS VI disease.

Keywords:  Cornea; Eye; MPS VI therapy; Maroteaux – Lamy syndrome; Mouse model; Mucopolysaccharidosis type VI

DOI:  https://doi.org/10.1016/j.ymgme.2021.07.008
Acta Pharmacol Sin. 2021 Aug 20.

TFEB, a master regulator of autophagy and biogenesis, unexpectedly promotes apoptosis in response to the cyclopentenone prostaglandin 15d-PGJ2.

Chuan-Bin Yang, Jia Liu, Benjamin Chun-Kit Tong, Zi-Ying Wang, Zhou Zhu, Cheng-Fu Su, Sravan Gopalkrishnashetty Sreenivasmurthy, Jia-Xi Wu, Ashok Iyaswamy, Senthilkumar Krishnamoorthi, Shi-Ying Huang, King-Ho Cheung, Ju-Xian Song, Jie-Qiong Tan, Jia-Hong Lu, Min Li.

  Transcriptional factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, is generally regarded as a pro-survival factor. Here, we identify that besides its effect on autophagy induction, TFEB exerts a pro-apoptotic effect in response to the cyclopentenone prostaglandin 15-deoxy-∆-12,14-prostaglandin J2 (15d-PGJ2). Specifically, 15d-PGJ2 promotes TFEB translocation from the cytoplasm into the nucleus to induce autophagy and lysosome biogenesis via reactive oxygen species (ROS) production rather than mTORC1 inactivation. Surprisingly, TFEB promotes rather than inhibits apoptosis in response to 15d-PGJ2. Mechanistically, ROS-mediated TFEB translocation into the nucleus transcriptionally upregulates the expression of ATF4, which is required for apoptosis elicited by 15d-PGJ2. Additionally, inhibition of TFEB activation by ROS scavenger N-acetyl cysteine or inhibition of protein synthesis by cycloheximide effectively compromises ATF4 upregulation and apoptosis in response to 15d-PGJ2. Collectively, these results indicate that ROS-induced TFEB activation exerts a novel role in promoting apoptosis besides its role in regulating autophagy in response to 15d-PGJ2. This work not only evidences how TFEB is activated by 15d-PGJ2, but also unveils a previously unexplored role of ROS-dependent activation of TFEB in modulating cell apoptosis in response to 15d-PGJ2.

Keywords:  15-deoxy-∆-12,14-prostaglandin J2 (15d-PGJ2); ATF4; ER stress; apoptosis; autophagy and lysosome biogenesis; transcriptional factor EB (TFEB)

DOI:  https://doi.org/10.1038/s41401-021-00711-7
J Neurosci. 2021 Aug 20. pii: JN-RM-1930-20. [Epub ahead of print]

Tuberous sclerosis complex (TSC) inactivation increases neuronal network activity by enhancing Ca2+ influx via L-type Ca2+ channels.

Chihiro Hisatsune, Tadayuki Shimada, Akitoshi Miyamoto, Amy Lee, Kanato Yamagata.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder characterized by hamartomas in various organs such as the brain, lungs, and kidneys. Epilepsy, along with autism and intellectual disability, is one of the neurological impairments associated with TSC that has an intimate relationship with developmental outcomes and quality of life. Sustained activation of the mammalian target of rapamycin (mTOR) via TSC1 or TSC2 mutations is known to be involved in the onset of epilepsy in TSC. However, the mechanism by which mTOR causes seizures remain unknown. In this study, we showed that, human induced pluripotent stem cell-derived TSC2-deficient (TSC2-/- ) neurons exhibited elevated neuronal activity with highly synchronized Ca2+ spikes. Notably, TSC2-/- neurons presented enhanced Ca2+ influx via L-type Ca2+ channels (LTCCs), which contributed to the abnormal neurite extension and sustained activation of cAMP response element binding protein (CREB), a critical mediator of synaptic plasticity. Expression of Cav1.3, a subtype of LTCCs, was increased in TSC2-/- neurons, but long-term rapamycin treatment suppressed this increase and reversed the altered neuronal activity and neurite extensions. Thus, we identified Cav1.3 LTCC as a critical downstream component of TSC-mTOR signaling that would trigger enhanced neuronal network activity of TSC2-/- neurons. We suggest that LTCCs could be potential novel targets for the treatment of epilepsy in TSC.Significant statementThere is a close relationship between elevated mTOR activity and epilepsy in tuberous sclerosis complex (TSC). However, the underlying mechanism by which mTOR causes epilepsy remains unknown. In this study, using human TSC2-/- neurons, we identified elevated Ca2+ influx via LTCCs as a critical downstream component of TSC-mTOR signaling and a potential cause of both elevated neuronal activity and neurite extension in TSC2-/- neurons. Our findings demonstrate a previously unrecognized connection between sustained mTOR activation and elevated Ca2+ signaling via LTCC in human TSC neurons, which could cause epilepsy in TSC.

DOI: https://doi.org/10.1523/JNEUROSCI.1930-20.2021
J Neurochem. 2021 Aug 16.

A New Brain-Penetrant Glucosylceramide Synthase Inhibitor as Potential Therapeutics for Gaucher Disease.

Takahiro Fujii, Yuta Tanaka, Hideyuki Oki, Sho Sato, Sachio Shibata, Takamitsu Maru, Yuta Tanaka, Maiko Tanaka, Tomohiro Onishi.

  Gaucher disease (GD), the most common lysosomal storage disorders, is caused by GBA gene mutations resulting in glycosphingolipids accumulations in various tissues, such as the brain. While suppressing glycosphingolipid accumulation is the central strategy for treating peripheral symptoms of GD, there is no effective treatment for the central nervous system symptoms. As glycosphingolipid biosynthesis starts from ceramide glycosylation by glucosylceramide synthase (GCS), inhibiting GCS in the brain is a promising strategy for neurological GD. Herein, we discovered T-036, a potent and brain-penetrant GCS inhibitor with a unique chemical structure and binding property. T-036 does not harbor an aliphatic amine moiety and has a non-competitive, inhibitory binding mode to the substrates, unlike other known inhibitors. T-036 exhibited sufficient exposure and a significant reduction of glucosylsphingolipids in the plasma and brain of the GD mouse model. Therefore, T-036 could be a promising lead molecule for treating central nervous system symptoms of GD.

Keywords:  Gaucher disease; Glucosylceramide synthase inhibitor; Glucosylsphingosine; Lysosomal storage disorder; drug discovery

DOI:  https://doi.org/10.1111/jnc.15492
J Neurosci. 2021 Aug 20. pii: JN-RM-1377-20. [Epub ahead of print]

mTOR Signaling Regulates Metabolic Function in Oligodendrocyte Precursor Cells and Promotes Efficient Brain Remyelination in the Cuprizone Model.

Marisa A Jeffries, Lauren E McLane, Luipa Khandker, Marie L Mather, Angelina V Evangelou, Divyangi Kantak, Jennifer N Bourne, Wendy B Macklin, Teresa L Wood.

In demyelinating diseases such as multiple sclerosis (MS), primary loss of myelin and subsequent neuronal degeneration throughout the central nervous system (CNS) impair patient functionality. While the importance of mechanistic target of rapamycin (mTOR) signaling during developmental myelination is known, no studies have yet directly examined the function of mTOR signaling specifically in the oligodendrocyte (OL) lineage during remyelination. Here, we conditionally deleted mTOR from adult oligodendrocyte precursor cells (OPCs) using Ng2-CreERT in male adult mice to test its function in new OLs responsible for remyelination. During early remyelination after cuprizone-induced demyelination, mice lacking mTOR in adult OPCs had unchanged OL numbers but thinner myelin. Myelin thickness recovered by late-stage repair, suggesting a delay in myelin production when mTOR is deleted from adult OPCs. Surprisingly, loss of mTOR in OPCs had no effect on efficiency of remyelination after lysophosphatidylcholine lesions in either the spinal cord or corpus callosum, suggesting that mTOR signaling functions specifically in a pathway dysregulated by cuprizone to promote remyelination efficiency. We further determined that cuprizone and inhibition of mTOR cooperatively compromise metabolic function in primary rat OLs undergoing differentiation. Taken together, our results support the conclusion that mTOR signaling in OPCs is required to overcome the metabolic dysfunction in the cuprizone-demyelinated adult brain.Significance Statement Impaired remyelination by oligodendrocytes contributes to the progressive pathology in multiple sclerosis, so it is critical to identify mechanisms of improving remyelination. The goal of this study was to examine mTOR signaling in remyelination. Here, we provide evidence that mTOR signaling promotes efficient remyelination of the brain after cuprizone-mediated demyelination but has no effect on remyelination after lysophosphatidylcholine demyelination in the spinal cord or brain. We also present novel data revealing that mTOR inhibition and cuprizone treatment additively affect the metabolic profile of differentiating oligodendrocytes, supporting a mechanism for the observed remyelination delay. These data suggest that altered metabolic function may underly failure of remyelination in MS lesions and that mTOR signaling may be of therapeutic potential for promoting remyelination.

DOI: https://doi.org/10.1523/JNEUROSCI.1377-20.2021
Cell. 2021 Aug 19. pii: S0092-8674(21)00939-9. [Epub ahead of print]184(17): 4374-4376

Partners with a killer: Metabolic signaling promotes inflammatory cell death.

Zhonghua Liu, Tsan Sam Xiao.

In this issue of Cell, Evavold et al. (2021) report that mTOR Complex 1 (mTORC1), a metabolic signaling complex, controls reactive oxygen species (ROS) production in mitochondria, which in turn promotes inflammatory cell death mediated by gasdermin D (GSDMD). This provides a new mechanistic connection between metabolic signaling and inflammatory cell death.

DOI: https://doi.org/10.1016/j.cell.2021.07.036
Front Cell Neurosci. 2021 ;15 668500

Contribution of Energy Dysfunction to Impaired Protein Translation in Neurodegenerative Diseases.

Yu-Ju Liu, Yijuang Chern.

  Impaired energy homeostasis and aberrant translational control have independently been implicated in the pathogenesis of neurodegenerative diseases. AMP kinase (AMPK), regulated by the ratio of cellular AMP and ATP, is a major gatekeeper for cellular energy homeostasis. Abnormal regulation of AMPK has been reported in several neurodegenerative diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Most importantly, AMPK activation is known to suppress the translational machinery by inhibiting the mechanistic target of rapamycin complex 1 (mTORC1), activating translational regulators, and phosphorylating nuclear transporter factors. In this review, we describe recent findings on the emerging role of protein translation impairment caused by energy dysregulation in neurodegenerative diseases.

Keywords:  AMP kinase; Alzheimer’s disease; amyotrophic lateral sclerosis; protein translation; stress granules

DOI:  https://doi.org/10.3389/fncel.2021.668500
Nat Commun. 2021 Aug 20. 12(1): 5073

Saturated very long chain fatty acid configures glycosphingolipid for lysosome homeostasis in long-lived C. elegans.

Feng Wang, Yuxi Dai, Xufeng Zhu, Qilong Chen, Huanhu Zhu, Ben Zhou, Haiqing Tang, Shanshan Pang.

The contents of numerous membrane lipids change upon ageing. However, it is unknown whether and how any of these changes are causally linked to lifespan regulation. Acyl chains contribute to the functional specificity of membrane lipids. In this study, working with C. elegans, we identified an acyl chain-specific sphingolipid, C22 glucosylceramide, as a longevity metabolite. Germline deficiency, a conserved lifespan-extending paradigm, induces somatic expression of the fatty acid elongase ELO-3, and behenic acid (22:0) generated by ELO-3 is incorporated into glucosylceramide for lifespan regulation. Mechanistically, C22 glucosylceramide is required for the membrane localization of clathrin, a protein that regulates membrane budding. The reduction in C22 glucosylceramide impairs the clathrin-dependent autophagic lysosome reformation, which subsequently leads to TOR activation and longevity suppression. These findings reveal a mechanistic link between membrane lipids and ageing and suggest a model of lifespan regulation by fatty acid-mediated membrane configuration.

DOI: https://doi.org/10.1038/s41467-021-25398-6
Essays Biochem. 2021 Aug 20. pii: EBC20210016. [Epub ahead of print]

Rab GTPases in Parkinson's disease: a primer.

Antonio Jesús Lara Ordóñez, Rachel Fasiczka, Yahaira Naaldijk, Sabine Hilfiker.

  Parkinson's disease is a prominent and debilitating movement disorder characterized by the death of vulnerable neurons which share a set of structural and physiological properties. Over the recent years, increasing evidence indicates that Rab GTPases can directly as well as indirectly contribute to the cellular alterations leading to PD. Rab GTPases are master regulators of intracellular membrane trafficking events, and alterations in certain membrane trafficking steps can be particularly disruptive to vulnerable neurons. Here, we describe current knowledge on the direct links between altered Rab protein function and PD pathomechanisms.

Keywords:  Parkinson´s disease; Rab GTPase; leucine rich repeat kinase; membrane trafficking; protein phosphorylation

DOI:  https://doi.org/10.1042/EBC20210016
Crit Rev Microbiol. 2021 Aug 17. 1-13

The role of membrane contact sites at the bacteria-host interface.

Chen Jiang, Xue Huang, Jia Yao, Lihua Yu, Fujing Wei, Aimin Yang.

  Membrane contact sites (MCSs) refer to the areas of close proximity between heterologous membranes. A growing body of evidence indicates that MCSs are involved in important cellular functions, such as cellular material transfer, organelle biogenesis, and cell growth. Importantly, the study of MCSs at the bacteria-host interface is an emerging popular research topic. Intracellular bacterial pathogens have evolved a variety of fascinating strategies to interfere with MCSs by injecting effectors into infected host cells. Bacteria-containing vacuoles establish direct physical contact with organelles within the host, ensuring vacuolar membrane integrity and energy supply from host organelles and protecting the vacuoles from the host endocytic pathway and lysosomal degradation. An increasing number of bacterial effectors from various bacterial pathogens hijack components of host MCSs to form the vacuole-organelle MCSs for material exchange. MCS-related events have been identified as new mechanisms of microbial pathogenesis to greatly improve bacterial survival and replication within host cells. In this review, we will discuss the recent advances in MCSs at the bacteria-host interface, focussing on the roles of MCSs mediated by bacterial effectors in microbial pathogenesis.

Keywords:  Membrane contact sites; bacteria-containing vacuoles; bacteria-host interface; bacterial effectors; microbial pathogenesis

DOI:  https://doi.org/10.1080/1040841X.2021.1961678
Nat Chem Biol. 2021 Aug 19.

Lysosome-targeting chimeras evolve.

Joshiawa Paulk.

DOI: https://doi.org/10.1038/s41589-021-00835-1
EMBO Rep. 2021 Aug 19. e52435

Nhp2 is a reader of H2AQ105me and part of a network integrating metabolism with rRNA synthesis.

Julia S P Mawer, Jennifer Massen, Christina Reichert, Niklas Grabenhorst, Constantine Mylonas, Peter Tessarz.

  Ribosome biogenesis is an essential cellular process that requires integration of extracellular cues, such as metabolic state, with intracellular signalling, transcriptional regulation and chromatin accessibility at the ribosomal DNA. Here, we demonstrate that the recently identified histone modification, methylation of H2AQ105 (H2AQ105me), is an integral part of a dynamic chromatin network at the rDNA locus. Its deposition depends on a functional mTor signalling pathway and acetylation of histone H3 at position K56, thus integrating metabolic and proliferative signals. Furthermore, we identify a first epigenetic reader of this modification, the ribonucleoprotein Nhp2, which specifically recognizes H2AQ105me. Based on functional and proteomic data, we suggest that Nhp2 functions as an adapter to bridge rDNA chromatin with components of the small subunit processome to efficiently coordinate transcription of rRNA with its post-transcriptional processing. We support this by showing that an H2AQ105A mutant has a mild defect in early processing of rRNA.

Keywords:  chromatin; epigenetic reader; rRNA; ribosome biogenesis; small subunit processome

DOI:  https://doi.org/10.15252/embr.202152435
Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00023-X. [Epub ahead of print]363 203-269

Endolysosomal Ca2+ signaling in cardiovascular health and disease.

Sharon Negri, Pawan Faris, Francesco Moccia.

  An increase in intracellular Ca2+ concentration ([Ca2+]i) regulates a plethora of functions in the cardiovascular (CV) system, including contraction in cardiomyocytes and vascular smooth muscle cells (VSMCs), and angiogenesis in vascular endothelial cells and endothelial colony forming cells. The sarco/endoplasmic reticulum (SR/ER) represents the largest endogenous Ca2+ store, which releases Ca2+ through ryanodine receptors (RyRs) and/or inositol-1,4,5-trisphosphate receptors (InsP3Rs) upon extracellular stimulation. The acidic vesicles of the endolysosomal (EL) compartment represent an additional endogenous Ca2+ store, which is targeted by several second messengers, including nicotinic acid adenine dinucleotide phosphate (NAADP) and phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], and may release intraluminal Ca2+ through multiple Ca2+ permeable channels, including two-pore channels 1 and 2 (TPC1-2) and Transient Receptor Potential Mucolipin 1 (TRPML1). Herein, we discuss the emerging, pathophysiological role of EL Ca2+ signaling in the CV system. We describe the role of cardiac TPCs in β-adrenoceptor stimulation, arrhythmia, hypertrophy, and ischemia-reperfusion injury. We then illustrate the role of EL Ca2+ signaling in VSMCs, where TPCs promote vasoconstriction and contribute to pulmonary artery hypertension and atherosclerosis, whereas TRPML1 sustains vasodilation and is also involved in atherosclerosis. Subsequently, we describe the mechanisms whereby endothelial TPCs promote vasodilation, contribute to neurovascular coupling in the brain and stimulate angiogenesis and vasculogenesis. Finally, we discuss about the possibility to target TPCs, which are likely to mediate CV cell infection by the Severe Acute Respiratory Disease-Coronavirus-2, with Food and Drug Administration-approved drugs to alleviate the detrimental effects of Coronavirus Disease-19 on the CV system.

Keywords:  Cardiomyocytes; Cardiovascular system; Endolysosomal Ca(2+) signaling; Endothelial cells; Endothelial colony forming cells; Nicotinic acid adenine dinucleotide phosphate; Phosphatidylinositol 3,5-bisphosphate; Transient receptor potential mucolipin 1; Two-pore channels 1 and 2; Vascular smooth muscle cells

DOI:  https://doi.org/10.1016/bs.ircmb.2021.03.001
Biochem J. 2021 Aug 18. pii: BCJ20210174. [Epub ahead of print]

TFEB phosphorylation on Serine 211 is induced by autophagy in human synovial fibroblasts and by p62/SQSTM1 overexpression in HEK293 cells.

Biserka Relic, Celine Deroyer, Olivier Malaise, Zelda Plener, Philippe Gillet, Dominique de Seny, Michel G Malaise.

  Autophagy receptor p62/SQSTM1 signals a complex network that links autophagy-lysosomal system to proteasome. Phosphorylation of p62 on Serine 349 (P-Ser349 p62) is involved in a cell protective, antioxidant pathway. We have shown previously that P-Ser349 p62 occurs and is rapidly degraded during human synovial fibroblasts autophagy. In this work we observed that fingolimod (FTY720), used as a medication for multiple sclerosis, induced coordinated expression of p62, P-Ser349 p62 and inhibitory TFEB form, phosphorylated on Serine 211 (P-Ser211 TFEB), in human synovial fibroblasts. These effects were mimicked and potentiated by proteasome inhibitor MG132. In addition, FTY720 induced autophagic flux, LC3B-II upregulation, Akt phosphorylation inhibition on Serine 473 but downregulated TFEB, suggesting stalled autophagy. FTY720 decreased cytoplasmic fraction contained TFEB but induced TFEB in nuclear fraction. FTY720-induced P-Ser211 TFEB was mainly found in membrane fraction. Autophagy and VPS34 kinase inhibitor, autophinib, further increased FTY720-induced P-Ser349 p62 but inhibited concomitant expression of P-Ser211 TFEB. These results suggested that P-Ser211 TFEB expression depends on autophagy. Overexpression of GFP tagged TFEB in HEK293 cells showed concomitant expression of its phosphorylated form on Serine 211, that was downregulated by autophinib. These results suggested that autophagy might be autoregulated through P-Ser211 TFEB as a negative feedback loop. Of interest, overexpression of p62, p62 phosphorylation mimetic (S349E) mutant and phosphorylation deficient mutant (S349A) in HEK293 cells markedly induced P-Ser211 TFEB. These results showed that p62 is involved in regulation of TFEB phosphorylation on Serine 211 but that this involvement does not depend on p62 phosphorylation on Serine 349.

Keywords:  TFEB; autophagy; p62/SQSTM1; phosphorylation/dephosphorylation

DOI:  https://doi.org/10.1042/BCJ20210174
Ageing Res Rev. 2021 Aug 12. pii: S1568-1637(21)00191-4. [Epub ahead of print]71 101444

The microglial lysosomal system in Alzheimer's disease: Guardian against proteinopathy.

Zoë P Van Acker, Anika Perdok, Marine Bretou, Wim Annaert.

  Microglia, the brain-resident immune cells, play an essential role in the upkeep of brain homeostasis. They actively adapt into specific activation states based on cues from the microenvironment. One of these encompasses the activated response microglia (ARMs) phenotype. It arises along a healthy aging process and in a range of neurodegenerative diseases, including Alzheimer's disease (AD). As the phenotype is characterized by an increased lipid metabolism, phagocytosis rate, lysosomal protease content and secretion of neuroprotective agents, it leaves to reason that the phenotype is adapted in an attempt to restore homeostasis. This is important to the conundrum of inflammatory processes. Inflammation per se may not be deleterious; it is only when microglial reactions become chronic or the microglial subtype is made dysfunctional by (multiple) risk proteins with single-nucleotide polymorphisms that microglial involvement becomes deleterious instead of beneficial. Interestingly, the ARMs up- and downregulate many late-onset AD-associated risk factor genes, the products of which are particularly active in the endolysosomal system. Hence, in this review, we focus on how the endolysosomal system is placed at the crossroad of inflammation and microglial capacity to keep pace with degradation.

Keywords:  Endolysosomal homeostasis; Late-onset Alzheimer’s disease; Membrane transport; Microglia; Phagocytosis

DOI:  https://doi.org/10.1016/j.arr.2021.101444
Circ Res. 2021 Aug 18.

Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.

Radhika Agarwal, Joao Paulo, Christopher N Toepfer, Jourdan K Ewoldt, Subramanian Sundaram, Anant Chopra, Qi Zhang, Joshua Gorham, Steven DePalma, Christopher S Chen, Steven Gygi, Christine E Seidman, Jonathan G Seidman.

Rationale: Dominant heterozygous variants in Filamin C (FLNC) cause diverse cardiomyopathies, though the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (WT), ablated expression (FLNC-/-) or haploinsufficiency (FLNC+/-) that causes DCM. We also studied a heterozygous in-frame deletion (FLNC+/∆7aa) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy (HCM). FLNC-/- hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. While sarcomere formation and function were unaffected in FLNC+/- and FLNC+/∆7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiologic function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms, and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC-related cardiomyopathies.

DOI: https://doi.org/10.1161/CIRCRESAHA.120.317076
Cell Death Dis. 2021 Aug 17. 12(9): 797

Lysosome purinergic receptor P2X4 regulates neoangiogenesis induced by microvesicles from sarcoma patients.

Wulf Palinski, Maria Monti, Rosa Camerlingo, Ilaria Iacobucci, Serena Bocella, Federica Pinto, Clara Iannuzzi, Gelsomina Mansueto, Sara Pignatiello, Flavio Fazioli, Michele Gallo, Laura Marra, Flora Cozzolino, Annarosaria De Chiara, Piero Pucci, Antonio Bilancio, Filomena de Nigris.

The tumor microenvironment modulates cancer growth. Extracellular vesicles (EVs) have been identified as key mediators of intercellular communication, but their role in tumor growth is largely unexplored. Here, we demonstrate that EVs from sarcoma patients promote neoangiogenesis via a purinergic X receptor 4 (P2XR4) -dependent mechanism in vitro and in vivo. Using a proteomic approach, we analyzed the protein content of plasma EVs and identified critical activated pathways in human umbilical vein endothelial cells (HUVECs) and human progenitor hematopoietic cells (CD34+). We then showed that vessel formation was due to rapid mitochondrial activation, intracellular Ca2+ mobilization, increased extracellular ATP, and trafficking of the lysosomal P2XR4 to the cell membrane, which is required for cell motility and formation of stable branching vascular networks. Cell membrane translocation of P2XR4 was induced by proteins and chemokines contained in EVs (e.g. Del-1 and SDF-1). Del-1 was found expressed in many EVs from sarcoma tumors and several tumor types. P2XR4 blockade reduced EVs-induced vessels in angioreactors, as well as intratumor vascularization in mouse xenografts. Together, these findings identify P2XR4 as a key mediator of EVs-induced tumor angiogenesis via a signaling mediated by mitochondria-lysosome-sensing response in endothelial cells, and indicate a novel target for therapeutic interventions.

DOI: https://doi.org/10.1038/s41419-021-04069-w
Mol Neurobiol. 2021 Aug 19.

Cerebellar Long Noncoding RNA Expression Profile in a Niemann-Pick C Disease Mouse Model.

Shiqian Han, Meng Ren, Tianyin Kuang, Mao Pang, Dongwei Guan, Yesong Liu, Yong Wang, Wengeng Zhang, Zhijia Ye.

  Niemann-Pick type C (NP-C) disease is a neurodegenerative lysosomal storage disorder primarily caused by mutations in NPC1. However, its pathogenesis remains poorly understood. While mounting evidence has demonstrated the involvement of long noncoding RNAs (lncRNAs) in the pathogenesis of neurodegenerative disorders, the lncRNA expression profile in NP-C has not been determined. Here, we used RNA-seq analysis to determine lncRNA and mRNA expression profiles of the cerebella of NPC1-/- mice. We found that 272 lncRNAs and 856 mRNAs were significantly dysregulated in NPC1-/- mice relative to controls (≥ 2.0-fold, p < 0.05). Quantitative real-time PCR (qRT-PCR) was utilized to validate the expression of selected lncRNAs and mRNAs. Next, a lncRNA-mRNA coexpression network was employed to examine the potential roles of the differentially expressed (DE) lncRNAs. Functional analysis revealed that mRNAs coexpressed with lncRNAs are mainly linked to immune system-related processes and neuroinflammation. Moreover, knockdown of the lncRNA H19 ameliorated changes in ROS levels and cell viability and suppressed the lipopolysaccharide (LPS)-induced inflammatory response in vitro. Our findings indicate that dysregulated lncRNA expression patterns are associated with NP-C pathogenesis and offer insight into the development of novel therapeutics based on lncRNAs.

Keywords:  Cerebellum; Coexpression network; LncRNA H19; Long noncoding RNAs; Niemann-Pick type C disease

DOI:  https://doi.org/10.1007/s12035-021-02526-3
Front Cell Dev Biol. 2021 ;9 713806

Expanding TOR Complex 2 Signaling: Emerging Regulators and New Connections.

Peng An, Wenyi Xu, Junjie Luo, Yongting Luo.

  Almost three decades after its seminal discovery, our understanding of the remarkable TOR pathway continues to expand. As a TOR complex, TORC2 lies at the nexus of many signaling pathways and directs a diverse array of fundamental processes such as cell survival, proliferation, and metabolism by integrating environmental and intracellular cues. The dysregulation of TORC2 activity disrupts cellular homeostasis and leads to many pathophysiological conditions. With continued efforts at mapping the signaling landscape, the pace of discovery in TORC2 regulation has been accelerated in recent years. Consequently, emerging evidence has expanded the repertoire of upstream regulators and has revealed unexpected diversity in the modes of TORC2 regulation. Multiple environmental cues and plasma membrane proteins that fine-tune TORC2 activity are unfolding. Furthermore, TORC2 signaling is intricately intertwined with other major signaling pathways. Therefore, feedback and crosstalk regulation also extensively modulate TORC2. In this context, we provide a comprehensive overview of revolutionary concepts regarding emerging regulators of TORC2 and discuss evidence of feedback and crosstalk regulation that shed new light on TORC2 biology.

Keywords:  TORC2; crosstalk; feedback; metabolites; pathogen; plasma membrane

DOI:  https://doi.org/10.3389/fcell.2021.713806
Front Cell Dev Biol. 2021 ;9 650846

Blockage of Extracellular Signal-Regulated Kinase Exerts an Antitumor Effect via Regulating Energy Metabolism and Enhances the Efficacy of Autophagy Inhibitors by Regulating Transcription Factor EB Nuclear Translocation in Osteosarcoma.

Man Zhang, Yang Bai, Chang Xu, Yiying Qi, Jiahong Meng, Wenkan Zhang, Hang Su, Weiqi Yan.

  Accumulating evidence suggests that extracellular signal-regulated kinase (ERK) is a valuable target molecule for cancer. However, antitumor drugs targeting ERK are still in their clinical phase and no FDA-approved medications exist. In this study, we identified an ERK inhibitor (ERKi; Vx-11e) with potential antitumor activities, which was reflected by the inhibition in the survival and proliferation of Osteosarcoma (OS) cells. Mechanistically, the ERKi regulated autophagic flux by promoting the translocation of transcription factor EB (TFEB) in OS cells, thereby increasing the dependence of OS cells on autophagy and sensitivity to treatment with autophagy inhibitors in OS. Besides, we also found that the ERKi could regulate mitochondrial apoptosis through the ROS/mitochondria pathway and aerobic glycolysis in OS, which also increases the dependence of OS cells on autophagy to clear metabolites to a certain extent. These results may provide a reference for the clinically improved efficacy of ERKis in combination with autophagy inhibitors in the treatment of OS and indicate its potential as a therapeutic agent.

Keywords:  ERK; autophagy; energy metabolism; osteosarcoma; transcription factor EB

DOI:  https://doi.org/10.3389/fcell.2021.650846
Front Microbiol. 2021 ;12 694489

Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line.

David Krones, Marcel Rühling, Katrin Anne Becker, Tobias C Kunz, Carolin Sehl, Kerstin Paprotka, Erich Gulbins, Martin Fraunholz.

  Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

Keywords:  Staphylococcus aureus; acid sphingomyelinase; lysosomal recruitment; sphingomyelinase release; staphylococcal alpha-toxin

DOI:  https://doi.org/10.3389/fmicb.2021.694489
EMBO Rep. 2021 Aug 17. e52445

Linking transport and translation of mRNAs with endosomes and mitochondria.

Kira Müntjes, Senthil Kumar Devan, Andreas S Reichert, Michael Feldbrügge.

  In eukaryotic cells, proteins are targeted to their final subcellular locations with precise timing. A key underlying mechanism is the active transport of cognate mRNAs, which in many systems can be linked intimately to membrane trafficking. A prominent example is the long-distance endosomal transport of mRNAs and their local translation. Here, we describe current highlights of fundamental mechanisms of the underlying transport process as well as of biological functions ranging from endosperm development in plants to fungal pathogenicity and neuronal processes. Translation of endosome-associated mRNAs often occurs at the cytoplasmic surface of endosomes, a process that is needed for membrane-assisted formation of heteromeric protein complexes and for accurate subcellular targeting of proteins. Importantly, endosome-coupled translation of mRNAs encoding mitochondrial proteins, for example, seems to be particularly important for efficient organelle import and for regulating subcellular mitochondrial activity. In essence, these findings reveal a new mechanism of loading newly synthesised proteins onto endocytic membranes enabling intimate crosstalk between organelles. The novel link between endosomes and mitochondria adds an inspiring new level of complexity to trafficking and organelle biology.

Keywords:  RNA transport; endosomes; local translation; microtubules; mitochondria; organelle

DOI:  https://doi.org/10.15252/embr.202152445
Nat Chem Biol. 2021 Aug 19.

Bifunctional small molecules that mediate the degradation of extracellular proteins.

David F Caianiello, Mengwen Zhang, Jason D Ray, Rebecca A Howell, Jake C Swartzel, Emily M J Branham, Egor Chirkin, Venkata R Sabbasani, Angela Z Gong, David M McDonald, Viswanathan Muthusamy, David A Spiegel.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

DOI: https://doi.org/10.1038/s41589-021-00851-1
Mov Disord. 2021 Aug;36(8): 1744-1758

Dysfunction of RAB39B-Mediated Vesicular Trafficking in Lewy Body Diseases.

David J Koss, Susanna Campesan, Flaviano Giorgini, Tiago F Outeiro.

  Intracellular vesicular trafficking is essential for neuronal development, function, and homeostasis and serves to process, direct, and sort proteins, lipids, and other cargo throughout the cell. This intricate system of membrane trafficking between different compartments is tightly orchestrated by Ras analog in brain (RAB) GTPases and their effectors. Of the 66 members of the RAB family in humans, many have been implicated in neurodegenerative diseases and impairment of their functions contributes to cellular stress, protein aggregation, and death. Critically, RAB39B loss-of-function mutations are known to be associated with X-linked intellectual disability and with rare early-onset Parkinson's disease. Moreover, recent studies have highlighted altered RAB39B expression in idiopathic cases of several Lewy body diseases (LBDs). This review contextualizes the role of RAB proteins in LBDs and highlights the consequences of RAB39B impairment in terms of endosomal trafficking, neurite outgrowth, synaptic maturation, autophagy, as well as alpha-synuclein homeostasis. Additionally, the potential for therapeutic intervention is examined via a discussion of the recent progress towards the development of specific RAB modulators. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords:  Lewy body diseases; RAB39B; alpha-synuclein; endocytosis; neurodegeneration

DOI:  https://doi.org/10.1002/mds.28605
iScience. 2021 Aug 20. 24(8): 102890

NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells.

Ryuki Shimada, Hiroko Koike, Takamasa Hirano, Yuzuru Kato, Yumiko Saga.

  During murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male-specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity through the repression of Rheb in a post-transcriptional manner.

Keywords:  biological sciences; developmental biology; molecular biology

DOI:  https://doi.org/10.1016/j.isci.2021.102890
Front Cell Dev Biol. 2021 ;9 710407

Hypo-Expression of Tuberin Promotes Adenomyosis via the mTOR1-Autophagy Axis.

Ni-Hao Gu, Guo-Jing Li, Bing-Xin Yang, Min You, Yu Lin, Feng Sun, Hong Xu.

  Adenomyosis (AM) is a disease in which endometrial tissue invades the myometrium and has a 10-60% prevalence in reproductive-aged women. TSC2 regulates autophagy via mTOR1 signalling in colorectal cancer and endometrial carcinoma. Dysregulation of autophagy is implicated in adenomyosis pathogenesis. However, whether TSC2 participates in adenomyosis via autophagy remains obscure. Here, we found that the expression of TSC2 in adenomyosis was significantly decreased than that in normal endometrium during the secretory phase. Moreover, TSC2 and autophagy marker expression was significantly lower in ectopic lesions than in eutopic samples. TSC2 downregulation inhibited autophagy through mTOR1 signalling pathway activation in endometrial cells, leading to excessive proliferation, migration, and EMT; TSC2 overexpression induced the opposite effects. Rapamycin treatment suppressed cell proliferation, migration and EMT in the absence of TSC2. In parallel, an autophagy-specific inhibitor (SAR-405) restored migration and EMT under rapamycin treatment in TSC2-knockdown Ishikawa cells. Finally, SAR-405 treatment promoted EMT and migration of overexpressing cells. Collectively, our results suggest that TSC2 controls endometrial epithelial cell migration and EMT by regulating mTOR1-autophagy axis activation and that hypo-expression of TSC2 in the endometrium might promote adenomyosis.

Keywords:  EMT; TSC2; adenomyosis; autophagy; endometrial cell; mTOR1; migration

DOI:  https://doi.org/10.3389/fcell.2021.710407
Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00019-8. [Epub ahead of print]363 21-47

Ca2+ dysregulation in the pathogenesis of amyotrophic lateral sclerosis.

Valentina Tedeschi, Tiziana Petrozziello, Agnese Secondo.

  Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease without appropriate cure. One of the main reasons for the lack of a proper pharmacotherapy in ALS is the narrow knowledge on the molecular causes of the disease. In this respect, the identification of dysfunctional pathways in ALS is now considered a critical medical need. Among the causative factors involved in ALS, Ca2+ dysregulation is one of the most important pathogenetic mechanisms of the disease. Of note, Ca2+ dysfunction may induce, directly or indirectly, motor neuron degeneration and loss. Interestingly, both familial (fALS) and sporadic ALS (sALS) share the progressive dysregulation of Ca2+ homeostasis as a common noxious mechanism. Mechanicistically, Ca2+ dysfunction involves both plasma membrane and intracellular mechanisms, including AMPA receptor (AMPAR)-mediated excitotoxicity, voltage-gated Ca2+ channels (VGCCs) and Ca2+ transporter dysregulation, endoplasmic reticulum (ER) Ca2+ deregulation, mitochondria-associated ER membranes (MAMs) dysfunction, lysosomal Ca2+ leak, etc. Here, a comprehensive analysis of the main pathways involved in the dysregulation of Ca2+ homeostasis has been reported with the aim to focus the attention on new putative druggable targets.

Keywords:  ALS; ALS preclinical models; Ca(2+) dysregulation; ER stress; Excitotoxicity; Organellar Ca(2+)

DOI:  https://doi.org/10.1016/bs.ircmb.2021.02.014
Brain. 2021 Aug 19. pii: awab309. [Epub ahead of print]

Intravenous delivery of adeno-associated viral gene therapy in feline GM1 gangliosidosis.

Amanda L Gross, Heather L Gray-Edwards, Cassie N Bebout, Nathan L Ta, Kayly Nielsen, Brandon L Brunson, Kalajan R Lopez Mercado, Devin E Osterhoudt, Ana Rita Batista, Stacy Maitland, Thomas N Seyfried, Miguel Sena-Esteves, Douglas R Martin.

  GM1 gangliosidosis is a fatal neurodegenerative disease caused by a deficiency of lysosomal β-galactosidase. In its most severe form, GM1 gangliosidosis causes death by 4 years of age, and no effective treatments exist. Previous work has shown that injection of the brain parenchyma with an adeno-associated viral vector provides pronounced therapeutic benefit in a feline GM1 model. To develop a less invasive treatment for the brain and increase systemic biodistribution, intravenous injection of AAV9 was evaluated. AAV9 expressing feline β-galactosidase was intravenously administered at 1.5x1013 vector genomes/kilogram body weight to six GM1 cats at approximately 1 month of age. The animals were divided into two cohorts: 1) a long-term group, which was followed to humane endpoint, and 2) a short-term group, which was analyzed 16-weeks post treatment. Clinical assessments included neurological exams, cerebrospinal fluid and urine biomarkers, and 7-Telsa magnetic resonance imaging and spectroscopy. Postmortem analysis included β-galactosidase and virus distribution, histological analysis, and ganglioside content. Untreated GM1 animals survived 8.0 ± 0.6 months while intravenous treatment increased survival to an average of 3.5 years (n = 2) with substantial improvements in quality of life and neurologic function. Neurological abnormalities, which in untreated animals progress to the inability to stand and debilitating neurological disease by 8 months of age, were mild in all treated animals. Cerebrospinal fluid biomarkers were normalized, indicating decreased central nervous system cell damage in the treated animals. Urinary glycosaminoglycans decreased to normal levels in the long-term cohort. Magnetic resonance imaging and spectroscopy showed partial preservation of the brain in treated animals, which was supported by postmortem histological evaluation. β-galactosidase activity was increased throughout the central nervous system, reaching carrier levels in much of the cerebrum and normal levels in the cerebellum, spinal cord and cerebrospinal fluid. Ganglioside accumulation was significantly reduced by treatment. Peripheral tissues such as heart, skeletal muscle, and sciatic nerve also had normal β-galactosidase activity in treated GM1 cats. GM1 histopathology was largely corrected with treatment. There was no evidence of tumorigenesis or toxicity. Restoration of β-galactosidase activity in the central nervous system and peripheral organs by intravenous gene therapy led to profound increases in lifespan and quality of life in GM1 cats. This data supports the promise of intravenous gene therapy as a safe, effective treatment for GM1 gangliosidosis.

Keywords:  GM1 gangliosidosis; adeno-associated virus; gene therapy; lysosomal storage disease; neurodegeneration

DOI:  https://doi.org/10.1093/brain/awab309