bims-lysosi 2020-08-09 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2020–08–09
forty papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1.
Mislocalization of TORC1 to Lysosomes Caused by KIF11 Inhibition Leads to Aberrant TORC1 Activity.
Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy.
Cryo-EM structures of intact V-ATPase from bovine brain.
Synthesis and biological effect of lysosome-targeting fluorescent anion transporters with enhanced anionophoric activity.
Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens.
LZ-106, a potent lysosomotropic agent, causing TFEB-dependent cytoplasmic vacuolization.
Lysosome-targeted ratiometric fluorescent sensor for monitoring pH in living cells based on one-pot-synthesized carbon dots.
Pompe disease: pathogenesis, molecular genetics and diagnosis.
Experimental studies of mitochondrial and lysosomal function in in vitro and in vivo models relevant to Parkinson's disease genetic risk.
ATM loss disrupts the autophagy-lysosomal pathway.
Loss of Tmem106b exacerbates FTLD pathologies and causes motor deficits in progranulin-deficient mice.
Endo-Lysosomal Cation Channels and Infectious Diseases.
Lysosomal overloading and necrotizing enterocolitis.
Endosomal-lysosomal dysfunction in metabolic diseases and Alzheimer's disease.
A Case of Lysosomal Acid Lipase Deficiency Confirmed by Response to Sebelipase Alfa Therapy.
Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi β-Galactosidase.
Substrate reduction therapy using Genz-667161 reduces levels of pathogenic components in a mouse model of neuronopathic forms of Gaucher disease.
Treatment of mucopolysaccharidosis type II (Hunter syndrome): a Delphi derived practice resource of the American College of Medical Genetics and Genomics (ACMG).
Danon Disease-Associated LAMP-2 Deficiency Drives Metabolic Signature Indicative of Mitochondrial Aging and Fibrosis in Cardiac Tissue and hiPSC-Derived Cardiomyocytes.
Hearing Loss in Mucopolysaccharidoses: Current Knowledge and Future Directions.
Prediction of Improved Therapeutics for Fabry Disease Patients Generated by Mutagenesis of the á-Galactosidase A Active Site, Dimer Interface, and Glycosylation Region.
Upregulating β-hexosaminidase activity in rodents prevents α-synuclein lipid associations and protects dopaminergic neurons from α-synuclein-mediated neurotoxicity.
Transmissible Endosomal Intoxication: A Balance between Exosomes and Lysosomes at the Basis of Intercellular Amyloid Propagation.
Mucopolysaccharidosis Type I: A Review of the Natural History and Molecular Pathology.
Tuberous sclerosis 1 (Tsc1) mediated mTORC1 activation promotes glycolysis in tubular epithelial cells in kidney fibrosis.
TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.
Pathological Consequences of Hepatic mTORC1 Dysregulation.
Mitochondria-Associated ER Membranes - The Origin Site of Autophagy.
The Neurodevelopmental Pathogenesis of Tuberous Sclerosis Complex (TSC).
Genetics of tuberous sclerosis complex: an update.
Structural Insights into TOR Signaling.
p53 haploinsufficiency and increased mTOR signaling define a subset of aggressive hepatocellular carcinoma.
Cyclopamine and Rapamycin Synergistically Inhibit mTOR Signalling in Mouse Hepatocytes, Revealing an Interaction of Hedgehog and mTor Signalling in the Liver.
Selective inhibition of mTORC1 in tumor vessels increases antitumor immunity.
LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.
Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.
Emerging roles of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate as regulators of multiple steps in autophagy.
Endosomal membrane tension regulates ESCRT-III-dependent intra-lumenal vesicle formation.
SGK1 is a signalling hub that controls protein synthesis and proliferation in endothelial cells.

Elife. 2020 Aug 04. pii: e59555. [Epub ahead of print]9

Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1.

Marina Schrecker, Julia Korobenko, Richard K Hite.

  The chloride-proton exchanger CLC-7 plays critical roles in lysosomal homeostasis and bone regeneration and its mutation can lead to osteopetrosis, lysosomal storage disease and neurological disorders. In lysosomes and the ruffled border of osteoclasts, CLC-7 requires a b-subunit, OSTM1, for stability and activity. Here we present electron cryomicroscopy structures of CLC-7 in occluded states by itself and in complex with OSTM1, determined at resolutions up to 2.8 Å. In the complex, the luminal surface of CLC-7 is entirely covered by a dimer of the heavily glycosylated and disulfide-bonded OSTM1, which serves to protect CLC-7 from the degradative environment of the lysosomal lumen. OSTM1 binding does not induce large-scale rearrangements of CLC-7, but does have minor effects the conformation of the ion-conduction pathway, potentially contributing to its regulatory role. These studies provide insights into the role of OSTM1 and serve as a foundation for understanding the mechanisms of CLC-7 regulation.

Keywords:  human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.59555
Mol Cells. 2020 Aug 07.

Mislocalization of TORC1 to Lysosomes Caused by KIF11 Inhibition Leads to Aberrant TORC1 Activity.

Yoon-Gu Jang, Yujin Choi, Kyoungho Jun, Jongkyeong Chung.

  While the growth factors like insulin initiate a signaling cascade to induce conformational changes in the mechanistic target of rapamycin complex 1 (mTORC1), amino acids cause the complex to localize to the site of activation, the lysosome. The precise mechanism of how mTORC1 moves in and out of the lysosome is yet to be elucidated in detail. Here we report that microtubules and the motor protein KIF11 are required for the proper dissociation of mTORC1 from the lysosome upon amino acid scarcity. When microtubules are disrupted or KIF11 is knocked down, we observe that mTORC1 localizes to the lysosome even in the amino acid-starved situation where it should be dispersed in the cytosol, causing an elevated mTORC1 activity. Moreover, in the mechanistic perspective, we discover that mTORC1 interacts with KIF11 on the motor domain of KIF11, enabling the complex to move out of the lysosome along microtubules. Our results suggest not only a novel way of the regulation regarding amino acid availability for mTORC1, but also a new role of KIF11 and microtubules in mTOR signaling.

Keywords:  Drosophila; KIF11; lysosome; mTORC1; microtubule

DOI:  https://doi.org/10.14348/molcells.2020.0089
Cell Death Differ. 2020 Aug 06.

Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy.

Huijie Guo, Mei Pu, Yusi Tai, Yuxiang Chen, Henglei Lu, Junwen Qiao, Guanghui Wang, Jing Chen, Xinming Qi, Ruimin Huang, Zhouteng Tao, Jin Ren.

Lysosome is a crucial organelle in charge of degrading proteins and damaged organelles to maintain cellular homeostasis. Transcription factor EB (TFEB) is the master transcription factor regulating lysosomal biogenesis and autophagy. Under external stimuli such as starvation, dephosphorylated TFEB transports into the nucleus to specifically recognize and bind to the coordinated lysosomal expression and regulation (CLEAR) elements at the promotors of autophagy and lysosomal biogenesis-related genes. The function of TFEB in the nucleus is fine regulated but the molecular mechanism is not fully elucidated. In this study, we discovered that miR-30b-5p, a small RNA which is known to regulate a series of genes through posttranscriptional regulation in the cytoplasm, was translocated into the nucleus, bound to the CLEAR elements, suppressed the transcription of TFEB-dependent downstream genes, and further inhibited the lysosomal biogenesis and the autophagic flux; meanwhile, knocking out the endogenous miR-30b-5p by CRISPR/Cas9 technique significantly increased the TFEB-mediated transactivation, resulting in the increased expression of autophagy and lysosomal biogenesis-related genes. Overexpressing miR-30b-5p in mice livers showed a decrease in lysosomal biogenesis and autophagy. These in vitro and in vivo data indicate that miR-30b-5p may inhibit the TFEB-dependent transactivation by binding to the CLEAR elements in the nucleus to regulate the lysosomal biogenesis and autophagy. This novel mechanism of nuclear miRNA regulating gene transcription is conducive to further elucidating the roles of miRNAs in the lysosomal physiological functions and helps to understand the pathogenesis of abnormal autophagy-related diseases.

DOI: https://doi.org/10.1038/s41418-020-0602-4
Nat Commun. 2020 Aug 06. 11(1): 3921

Cryo-EM structures of intact V-ATPase from bovine brain.

Rong Wang, Tao Long, Abdirahman Hassan, Jin Wang, Yingyuan Sun, Xiao-Song Xie, Xiaochun Li.

The vacuolar-type H+-ATPases (V-ATPase) hydrolyze ATP to pump protons across the plasma or intracellular membrane, secreting acids to the lumen or acidifying intracellular compartments. It has been implicated in tumor metastasis, renal tubular acidosis, and osteoporosis. Here, we report two cryo-EM structures of the intact V-ATPase from bovine brain with all the subunits including the subunit H, which is essential for ATPase activity. Two type-I transmembrane proteins, Ac45 and (pro)renin receptor, along with subunit c", constitute the core of the c-ring. Three different conformations of A/B heterodimers suggest a mechanism for ATP hydrolysis that triggers a rotation of subunits DF, inducing spinning of subunit d with respect to the entire c-ring. Moreover, many lipid molecules have been observed in the Vo domain to mediate the interactions between subunit c, c", (pro)renin receptor, and Ac45. These two structures reveal unique features of mammalian V-ATPase and suggest a mechanism of V1-Vo torque transmission.

DOI: https://doi.org/10.1038/s41467-020-17762-9
Bioorg Med Chem Lett. 2020 Aug 02. pii: S0960-894X(20)30572-2. [Epub ahead of print] 127461

Synthesis and biological effect of lysosome-targeting fluorescent anion transporters with enhanced anionophoric activity.

Xiao-Qiao Hong, Xiang-Yu He, Kin Yip Tam, Wen-Hua Chen.

  Two lysosome-targeting fluorescent anion transporters derived from coumarins, trifluoromethylated arylsquaramides and morpholines were synthesized, and their specificity and efficiency to target and alkalize lysosomes were investigated. They are able to target lysosomes specifically. Compared with the previous analogue without trifluoromethyl substituents, these two conjugates, in particular the one having a 3,5-bis(trifluoromethyl) substituent, exhibit significantly higher ability to facilitate the transport of chloride anions, alkalize lysosomes and reduce the activity of lysosomal Cathepsin B enzyme. The present finding suggests that improving the anionophoric activity of lysosome-targeting fluorescent anion transporters is favorable to the efficiency to alkalize lysosomes and deactivate lysosomal Cathepsin B enzyme.

Keywords:  Cathepsin B enzyme; Fluorescent anion transporter; Lysosomal alkalization; Lysosome targeting

DOI:  https://doi.org/10.1016/j.bmcl.2020.127461
Nat Cell Biol. 2020 Aug;22(8): 973-985

Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens.

Suresh Kumar, Ashish Jain, Seong Won Choi, Gustavo Peixoto Duarte da Silva, Lee Allers, Michal H Mudd, Ryan Scott Peters, Jan Haug Anonsen, Tor-Erik Rusten, Michael Lazarou, Vojo Deretic.

Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.

DOI: https://doi.org/10.1038/s41556-020-0549-1
Gene. 2020 Aug 02. pii: S0378-1119(20)30686-7. [Epub ahead of print] 145017

LZ-106, a potent lysosomotropic agent, causing TFEB-dependent cytoplasmic vacuolization.

Xiaoqian Zou, Fei Meng, Chengyu Fu, Jieying Zhou, Yi Zhang, Ruixuan Wang, Chengwan Zhang, Zhiyu Li, Qinglong Guo, Lin Yang.

  Cytoplasmic vacuolization usually occurs in cells treated with different agents and substances. We found that LZ-106, an analog of enoxacin, is a potent lysosomotropic agent, contributing to the formation of cytoplasmic vacuoles in cells. Studies of LZ-106-induced vacuolization in H460 cells showed acid environment inside these vacuoles. Further study demonstrated that markers in the late endosomes and lysosomes, like LAMP1 and RAB7, on the surface of the vacuoles, implying that these vacuoles might derive from endosomes and/or lysosomes. By studying the fluorescence intensity of LZ-106, we discovered that LZ-106 tended to locate in acid organelles, and Bafilomycin A1, a V-ATPase inhibitor, was able to suppress its acid organelles localization. Also, we noticed that LZ-106 could induce lysosome stress, involving pH increment and lysosomal membrane damage. Moreover, the expression levels of some lysosome-related proteins, like LAMP1, EEA1, and Cathepsin B, were also altered upon LZ-106 treatment. At last, we confirmed LZ-106 can activate TFEB, a key regulator of lysosomes. Knockdown of TFEB could also reverse LZ-106's effect on vacuolization in H460 cells. Taken together, due to LZ-106's lysosomotropic properties, it is able to accumulate in the acid organelles and induce lysosomal dysfunction in H460 cells, leading to TFEB activation and the following cytoplasmic vacuolization.

Keywords:  Cytoplasmic vacuoles; Lysosomotropic agent; TFEB; lysosome stress

DOI:  https://doi.org/10.1016/j.gene.2020.145017
Mikrochim Acta. 2020 Aug 01. 187(8): 478

Lysosome-targeted ratiometric fluorescent sensor for monitoring pH in living cells based on one-pot-synthesized carbon dots.

Shaomei Xu, Xu He, Yibing Huang, Xin Liu, Lihe Zhao, Xinghua Wang, Ying Sun, Pinyi Ma, Daqian Song.

  A hydrothermal method has been employed to synthesize a green and one-pot carbon dots-based sensor for ratiometric monitoring and imaging lysosomal pH in living cells. The carbon dots were directly functionalized by abundant amino groups during synthesis and exhibited dual emission bands at 439 and 550 nm under single-wavelength excitation of 380 nm without any additional modification. In addition to its small size, the established sensor had good biocompatibility. Owing to its abundant amino groups and good hydrophilicity, the sensor is able to target lysosome with high Pearson's colocalization coefficients (0.935 and 0.924) and responds to change of lysosomal pH in living cells. It also had excellent pH sensitivity and reversibility, and anti-interference capability, thus enabling sensing pH change in intracellular environment in real time, as demonstrated by successful monitoring of lysosomal pH changes during lysosomal alkalization, dexamethasone-induced stimulation, and stress in Michigan Cancer Foundation-7 cells (blue channel, excitation = 405 nm and emission = 419-459 nm bandpass; and yellow channel, excitation = 405 nm and emission = 530-570 nm bandpass). Graphical abstract.

Keywords:  Carbon dots; Intracellular pH probe; Living cell imaging; Lysosome; Ratiometric fluorescence sensor

DOI:  https://doi.org/10.1007/s00604-020-04462-w
Aging (Albany NY). 2020 Aug 03. 12

Pompe disease: pathogenesis, molecular genetics and diagnosis.

Simona Taverna, Giuseppe Cammarata, Paolo Colomba, Serafina Sciarrino, Carmela Zizzo, Daniele Francofonte, Marco Zora, Simone Scalia, Chiara Brando, Alessia Lo Curto, Emanuela Maria Marsana, Roberta Olivieri, Silvia Vitale, Giovanni Duro.

  Pompe disease (PD) is a rare autosomal recessive disorder caused by mutations in the GAA gene, localized on chromosome 17 and encoding for acid alpha-1,4-glucosidase (GAA). Currently, more than 560 mutations spread throughout GAA gene have been reported. GAA catalyzes the hydrolysis of α-1,4 and α-1,6-glucosidic bonds of glycogen and its deficiency leads to lysosomal storage of glycogen in several tissues, particularly in muscle. PD is a chronic and progressive pathology usually characterized by limb-girdle muscle weakness and respiratory failure. PD is classified as infantile and childhood/adult forms. PD patients exhibit a multisystemic manifestation that depends on age of onset.Early diagnosis is essential to prevent or reduce the irreversible organ damage associated with PD progression. Here, we make an overview of PD focusing on pathogenesis, clinical phenotypes, molecular genetics, diagnosis, therapies, autophagy and the role of miRNAs as potential biomarkers for PD.

Keywords:  GAA; Pompe disease; acid alpha-1,4-glucosidase; glycogen; lysosomal storage disorder

DOI:  https://doi.org/10.18632/aging.103794
Int Rev Neurobiol. 2020 ;pii: S0074-7742(20)30013-1. [Epub ahead of print]154 279-302

Experimental studies of mitochondrial and lysosomal function in in vitro and in vivo models relevant to Parkinson's disease genetic risk.

Ria Thomas, Penelope J Hallett, Ole Isacson.

  Several studies have identified the involvement of mitochondrial and lysosomal dysfunction in Parkinson's disease (PD) pathology. In this review we discuss recent work that has identified deficits in mitophagy, mitochondrial network formation, increased sensitivity to mitochondrial stressors and alterations in proteins regulating mitochondrial fission and fusion associated with patient-derived fibroblasts harboring mutations in LRRK2 gene and from sporadic PD patient cells. We further focus on alterations of lysosomal enzymes, in particular glucocerebrosidase activity, and resultant lipid dyshomeostasis in PD and aging, in human tissue and in vivo rodent models. Future studies aimed at understanding the convergence of mitochondrial and lysosomal pathways will be of essence for the identification of unique cellular defects in PD and for the development of new treatments.

Keywords:  GBA; LRRK2; Lysosomal dysfunction; Mitochondrial dysfunction; Parkinson's disease; Patient fibroblasts

DOI:  https://doi.org/10.1016/bs.irn.2020.02.004
Autophagy. 2020 Aug 05.

ATM loss disrupts the autophagy-lysosomal pathway.

Aifang Cheng, Kai-Hei Tse, Hei-Man Chow, Yunqiao Gan, Xuan Song, Fulin Ma, Yi Xuan Yvonne Qian, Weiyi She, Karl Herrup.

  ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm-/- neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm-/- lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm-/- neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T.

Keywords:  SLC2A4/GLUT4; ataxia-telangiectasia; autophagy; lysosome; neurodegeneration; protein degradation; trafficking

DOI:  https://doi.org/10.1080/15548627.2020.1805860
EMBO Rep. 2020 Aug 05. e50197

Loss of Tmem106b exacerbates FTLD pathologies and causes motor deficits in progranulin-deficient mice.

Xiaolai Zhou, Mieu Brooks, Peizhou Jiang, Shunsuke Koga, Aamir R Zuberi, Matthew C Baker, Tammee M Parsons, Monica Castanedes-Casey, Virginia Phillips, Ariston L Librero, Aishe Kurti, John D Fryer, Guojun Bu, Cathleen Lutz, Dennis W Dickson, Rosa Rademakers.

  Progranulin (PGRN) and transmembrane protein 106B (TMEM106B) are important lysosomal proteins implicated in frontotemporal lobar degeneration (FTLD) and other neurodegenerative disorders. Loss-of-function mutations in progranulin (GRN) are a common cause of FTLD, while TMEM106B variants have been shown to act as disease modifiers in FTLD. Overexpression of TMEM106B leads to lysosomal dysfunction, while loss of Tmem106b ameliorates lysosomal and FTLD-related pathologies in young Grn-/- mice, suggesting that lowering TMEM106B might be an attractive strategy for therapeutic treatment of FTLD-GRN. Here, we generate and characterize older Tmem106b-/- Grn-/- double knockout mice, which unexpectedly show severe motor deficits and spinal cord motor neuron and myelin loss, leading to paralysis and premature death at 11-12 months. Compared to Grn-/- , Tmem106b-/- Grn-/- mice have exacerbated FTLD-related pathologies, including microgliosis, astrogliosis, ubiquitin, and phospho-Tdp43 inclusions, as well as worsening of lysosomal and autophagic deficits. Our findings confirm a functional interaction between Tmem106b and Pgrn and underscore the need to rethink whether modulating TMEM106B levels is a viable therapeutic strategy.

Keywords:  Tdp-43; Tmem106b; frontotemporal lobar degeneration; lysosomes; progranulin

DOI:  https://doi.org/10.15252/embr.202050197
Rev Physiol Biochem Pharmacol. 2020 Aug 04.

Endo-Lysosomal Cation Channels and Infectious Diseases.

Yu-Kai Chao, Sui-Yuan Chang, Christian Grimm.

  Among the infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi, the most prevalent ones today are malaria, tuberculosis, influenza, HIV/AIDS, Ebola, dengue fever, and methicillin-resistant Staphylococcus aureus (MRSA) infection, and most recently Covid-19 (SARS-CoV2). Others with a rather devastating history and high fatality rates such as plague, cholera, or typhus seem less threatening today but have not been eradicated, and with a declining efficacy of current antibiotics they ought to be watched carefully. Another emerging issue in this context is health-care associated infection. About 100,000 hospitalized patients in the USA ( www.cdc.gov ) and 33,000 in Europe ( https://www.ecdc.europa.eu ) die each year as a direct consequence of an infection caused by bacteria resistant to antibiotics. Among viral infections, influenza is responsible for about 3-5 million cases of severe illness, and about 250,000 to 500,000 deaths annually ( www.who.int ). About 37 million people are currently living with HIV infection and about one million die from it each year. Coronaviruses such as MERS-CoV, SARS-CoV, but in particular the recent outbreak of Covid-19 (caused by SARS-CoV2) have resulted in large numbers of infections worldwide with an estimated several hundred thousand deaths (anticipated fatality rate: <5%). With a comparatively low mortality rate dengue virus causes between 50 and 100 million infections every year, leading to 50,000 deaths. In contrast, Ebola virus is the causative agent for one of the deadliest viral diseases. The Ebola outbreak in West Africa in 2014 is considered the largest outbreak in history with more than 11,000 deaths. Many of the deadliest pathogens such as Ebola virus, influenza virus, mycobacterium tuberculosis, dengue virus, and cholera exploit the endo-lysosomal trafficking system of host cells for penetration into the cytosol and replication. Defects in endo-lysosomal maturation, trafficking, fusion, or pH homeostasis can efficiently reduce the cytotoxicity caused by these pathogens. Most of these functions critically depend on endo-lysosomal membrane proteins such as transporters and ion channels. In particular, cation channels such as the mucolipins (TRPMLs) or the two-pore channels (TPCs) are involved in all of these aspects of endo-lysosomal integrity. In this review we will discuss the correlations between pathogen toxicity and endo-lysosomal cation channel function, and their potential as drug targets for infectious disease therapy.

Keywords:  Endosome; Lysosome; TPC1; TPC2; TRPML1; TRPML2; TRPML3

DOI:  https://doi.org/10.1007/112_2020_31
Pediatr Surg Int. 2020 Aug 01.

Lysosomal overloading and necrotizing enterocolitis.

Masaya Yamoto, Mashriq Alganabi, Sinobol Chusilp, Dorothy Lee, Yuta Yazaki, Carol Lee, Bo Li, Agostino Pierro.

   PURPOSE: Intestinal absorption in premature infants occurs via direct epithelial cellular endocytosis and degradation by intracellular lysosomes. Autophagy is a mechanism by which cytoplasmic organelles contribute to lysosomal degradation. However, excessive autophagy can lead to cell death. The purpose of this study was to investigate whether autophagy and endocytosis are present in the small intestinal mucosa during experimental necrotizing enterocolitis (NEC).
METHODS: NEC was induced by gavage feeding of hyperosmolar formula, lipopolysaccharide and hypoxia between P5 and P9 (ethical approval 44032). Breastfed mice were used as control. Distal ileum was harvested on P5, P7 and P9 and analyzed for intestinal epithelial cellular morphology as well as autophagy/lysosomal activity, and cell death. Groups were compared using Student's t test.
RESULTS: During NEC, giant lysosomes were present in the intestinal villi, with some exceeding their degradation ability leading to their rupture. The NEC group had significantly increased inflammation and autophagy activity, decreased lysosome activity, and increased apoptosis compared to control.
CONCLUSIONS: NEC induction causes excessive autophagy and endocytosis leading to lysosomal overloading, lysosomal membrane permeabilization and rupture which results in cell death. These novel findings may help to clarify the pathogenesis of NEC. Reduction of lysosome overload and assisting in their degradation capability may reduce the burden of NEC.

Keywords:  Autophagy; Endocytosis; Lysosomal membrane permeabilization; Necrotizing enterocolitis

DOI:  https://doi.org/10.1007/s00383-020-04724-x
Int Rev Neurobiol. 2020 ;pii: S0074-7742(20)30021-0. [Epub ahead of print]154 303-324

Endosomal-lysosomal dysfunction in metabolic diseases and Alzheimer's disease.

Michael F Almeida, Ben A Bahr, Stephen T Kinsey.

  The endosomal-lysosomal pathways and related autophagic processes are responsible for proteostasis, involving complexes between lysosomes and autophagosomes. Lysosomes are a key component of homeostasis, involved in cell signaling, metabolism, and quality control, and they experience functional compromise in metabolic diseases, aging, and neurodegenerative diseases. Many genetic mutations and risk factor genes associated with proteinopathies, as well as with metabolic diseases like diabetes, negatively influence endocytic trafficking and autophagic clearance. In contrast, health-improving exercise induces autophagy-lysosomal degradation, perhaps promoting efficient digestion of injured organelles so that undamaged organelles ensure cellular healthiness. Reductions in lysosomal hydrolases are implicated in Alzheimer's, Parkinson's, and lysosomal storage diseases, as well as obesity-related pathology, and members of the cathepsin enzyme family are involved in clearing both Aβ42 and α-synuclein. Upregulation of cathepsin hydrolases improves synaptic and memory functions in models of dementia and in exercising humans, thus identifying lysosomal-related systems as vital for healthy cognitive aging.

Keywords:  Age-related neurodegenerative disorders; Autophagic-lysosomal pathway; Autophagy; Cathepsin B; Endocytic trafficking; Protein accumulation disorders; Proteinopathy; Quality control; Synaptic pathology; Trafficking

DOI:  https://doi.org/10.1016/bs.irn.2020.02.012
J Pediatr Gastroenterol Nutr. 2020 Jul 30.

A Case of Lysosomal Acid Lipase Deficiency Confirmed by Response to Sebelipase Alfa Therapy.

Joseph J Shen, Jessica L Davis, Xinying Hong, Fred H Laningham, Michael H Gelb, Grace E Kim.

Lysosomal acid lipase (LAL) deficiency, or cholesterol ester storage disease, is a disorder affecting the breakdown of cholesterol esters and triglycerides within lysosomes. Clinical findings include hepatomegaly, hepatic dysfunction, and dyslipidemia, with a wide range of phenotypic variability and age of onset (1-3). The available clinical and molecular information of the patient presented herein was consistent with a diagnosis of LAL deficiency, but her LAL activity assay repeatedly showed normal or borderline low results. Her response to enzyme replacement therapy (4,5) and demonstrable deficiency on a newer specific enzymatic assay (6) ultimately confirmed her diagnosis of LAL deficiency.

DOI: https://doi.org/10.1097/MPG.0000000000002870
Chem Pharm Bull (Tokyo). 2020 ;68(8): 753-761

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi β-Galactosidase.

Kazuki Miura, Chihiro Onodera, Motonari Takagi, Ryosuke Koyama, Takako Hirano, Toshiyuki Nishio, Wataru Hakamata.

  The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit β-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its β-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated β-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel β-galactosidase activity in the Golgi apparatus of human cells; however, the protein responsible for this activity could not be identified. Inhibitor-derived chemical probes can serve as powerful tools to identify the responsible protein. In this study, we first constructed a cell-based high-throughput screening (HTS) system for Golgi β-galactosidase inhibitors, and then screened inhibitors from two compound libraries using the HTS system, in vitro assay, and cytotoxicity assay. An isoflavone derivative was identified among the final Golgi β-galactosidase inhibitor compound hits. Molecular docking simulations were performed to redesign the isoflavone derivative into a more potent inhibitor, and six designed derivatives were then synthesized. One of the derivatives, ARM07, exhibited potent inhibitory activity against β-galactosidase, with an IC50 value of 14.8 µM and competitive inhibition with Ki value of 13.3 µM. Furthermore, the in vitro and cellular inhibitory activities of ARM07 exceeded those of deoxygalactonojirimycin. ARM07 may contribute to the development of affinity-based chemical probes to identify the protein responsible for the newly discovered Golgi β-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.

Keywords:  inhibitor screening; isoflavone; lysosomal storage disease; senescence-associated β-galactosidase; β-galactosidase

DOI:  https://doi.org/10.1248/cpb.c20-00194
J Neurochem. 2020 Aug 03.

Substrate reduction therapy using Genz-667161 reduces levels of pathogenic components in a mouse model of neuronopathic forms of Gaucher disease.

Shani Blumenreich, Chen Yaacobi, Ayelet Vardi, Or B Barav, Einat B Vitner, Hyejung Park, Bing Wang, Seng H Cheng, S Pablo Sardi, Anthony H Futerman.

  Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the blood-brain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B-epoxide (CBE), an irreversible inhibitor of acid beta-glucosidase (GCase), the enzyme defective in nGD, with or without co-injection with Genz-667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co-injection with Genz-667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz-667161 largely reversed the changes in genes and pathways that were differentially-expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain.

Keywords:  Gaucher disease; Substrate reduction therapy; glycosphingolipids; pathogenic pathways

DOI:  https://doi.org/10.1111/jnc.15136
Genet Med. 2020 Aug 03.

Treatment of mucopolysaccharidosis type II (Hunter syndrome): a Delphi derived practice resource of the American College of Medical Genetics and Genomics (ACMG).

ACMG Therapeutics Committee

  Mucopolysaccharidosis, type II (MPS II, MIM 309900) is a severe lysosomal storage disease with multisystem involvement. There is one product approved by the FDA, an enzyme replacement therapy, based on a phase III trial in older, attenuated MPS II individuals. Guidance on treatment of MPS II is lacking, not only in general, but for specific clinical situations. A previous systematic evidence-based review of treatment for MPS II demonstrated insufficient strength in all data analyzed to create a definitive practice guideline based solely on published evidence. The American College of Medical Genetics and Genomics (ACMG) Therapeutics Committee conducted a Delphi study to generate an MPS II clinical practice resource of the treatment for these individuals for the genetics community, based on the evidence-based review and subsequent literature. This report describes the process, including consensus development and areas where consensus could not be obtained due to lack of quality evidence. Recommendations from the Delphi process were generated, and areas were highlighted that need further study to help guide clinical care of these individuals.

Keywords:  consensus; evidence-based practice; practice guideline; therapeutics

DOI:  https://doi.org/10.1038/s41436-020-0909-z
J Clin Med. 2020 Jul 31. pii: E2457. [Epub ahead of print]9(8):

Danon Disease-Associated LAMP-2 Deficiency Drives Metabolic Signature Indicative of Mitochondrial Aging and Fibrosis in Cardiac Tissue and hiPSC-Derived Cardiomyocytes.

Giorgia Del Favero, Alois Bonifacio, Teisha J Rowland, Shanshan Gao, Kunhua Song, Valter Sergo, Eric D Adler, Luisa Mestroni, Orfeo Sbaizero, Matthew R G Taylor.

  Danon disease is a severe X-linked disorder caused by deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Clinical manifestations are phenotypically diverse and consist of hypertrophic and dilated cardiomyopathies, skeletal myopathy, retinopathy, and intellectual dysfunction. Here, we investigated the metabolic landscape of Danon disease by applying a multi-omics approach and combined structural and functional readouts provided by Raman and atomic force microscopy. Using these tools, Danon patient-derived cardiac tissue, primary fibroblasts, and human induced pluripotent stem cells differentiated into cardiomyocytes (hiPSC-CMs) were analyzed. Metabolic profiling indicated LAMP-2 deficiency promoted a switch toward glycolysis accompanied by rerouting of tryptophan metabolism. Cardiomyocytes' energetic balance and NAD+/NADH ratio appeared to be maintained despite mitochondrial aging. In turn, metabolic adaption was accompanied by a senescence-associated signature. Similarly, Danon fibroblasts appeared more stress prone and less biomechanically compliant. Overall, shaping of both morphology and metabolism contributed to the loss of cardiac biomechanical competence that characterizes the clinical progression of Danon disease.

Keywords:  Danon disease; LAMP-2 deficiency; cardiac fibrosis; cell biomechanics; mitochondrial aging phenotype; multi-omics profiling

DOI:  https://doi.org/10.3390/jcm9082457
Diagnostics (Basel). 2020 Aug 04. pii: E554. [Epub ahead of print]10(8):

Hearing Loss in Mucopolysaccharidoses: Current Knowledge and Future Directions.

Jeremy Wolfberg, Keerthana Chintalapati, Shunji Tomatsu, Kyoko Nagao.

  Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency of one of the enzymes involved in the degradation of glycosaminoglycans. Hearing loss is a common clinical presentation in MPS. This paper reviews the literature on hearing loss for each of the seven recognized subtypes of MPS. Hearing loss was found to be common in MPS I, II, III, IVA, VI, and VII, and absent from MPS IVB and MPS IX. MPS VI presents primarily with conductive hearing loss, while the other subtypes (MPS I, MPS II, MPS III, MPS IVA, and MPS VII) can present with any type of hearing loss (conductive, sensorineural, or mixed hearing loss). The sensorineural component develops as the disease progresses, but there is no consensus on the etiology of the sensorineural component. Enzyme replacement therapy (ERT) is the most common therapy utilized for MPS, but the effects of ERT on hearing function have been inconclusive. This review highlights a need for more comprehensive and multidisciplinary research on hearing function that includes behavioral testing, objective testing, and temporal bone imaging. This information would allow for better understanding of the progression and etiology of hearing loss. Owing to the prevalence of hearing loss in MPS, early diagnosis of hearing loss and annual comprehensive audiological evaluations are recommended.

Keywords:  hearing loss; inner ear; middle ear; otitis media

DOI:  https://doi.org/10.3390/diagnostics10080554
Protein Expr Purif. 2020 Jul 29. pii: S1046-5928(20)30301-6. [Epub ahead of print] 105710

Prediction of Improved Therapeutics for Fabry Disease Patients Generated by Mutagenesis of the á-Galactosidase A Active Site, Dimer Interface, and Glycosylation Region.

Erin S Stokes, M Lane Gilchrist, David H Calhoun.

  Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A that induces the accumulation of the substrate globotriaosylceramide. Currently approved enzyme replacement therapy using recombinant human α-galactosidase A improves patient symptoms but a majority of patients experience adverse events due to the multiple infusions required for full therapeutic efficacy. Our approach is to use medicinal chemistry and phylogenic comparisons to introduce mutations into the human enzyme to increase catalytic activity and/or stability to generate an improved therapeutic enzyme that may require fewer infusions. We designed mutations at three regions of the human α-galactosidase A: the active site, the dimer interface, and a site for glycosylation. The M208E mutation, adjacent to the Y207 active site residue, increased enzyme activity 3.01-fold. This mutation introduced a charged Glu residue that is adjacent to the Y207 active site residue and close to a site of N-glycosylation.. The W277C mutation, designed to promote dimer stability, introduced a strong thiol-aromatic interaction (Cys-Phe) at the dimer interface and increased activity 2.31-fold. The W277C and M208E mutations modify the structure of the enzyme into forms with enhanced thermal stability 3.7- and 3.9-fold, respectively and positive cooperativity resulting in increased Hill coefficient from 1.0 to 4.60 and 3.47, respectively. Enhanced thermal stability and positive cooperativity predict improved in vivo activity and superior therapeutic properties. Our results demonstrate the value of in vitro mutagenesis for α-galactosidase A and support future perspectives to validate these results in Fabry disease patients.

Keywords:  Enzyme replacement therapy; Improved Therapeutics for Fabry Disease Patients; In Vitro Mutagenesis; Medicinal chemistry; Phylogenetic comparisons; α-Galactosidase A

DOI:  https://doi.org/10.1016/j.pep.2020.105710
Acta Neuropathol Commun. 2020 Aug 06. 8(1): 127

Upregulating β-hexosaminidase activity in rodents prevents α-synuclein lipid associations and protects dopaminergic neurons from α-synuclein-mediated neurotoxicity.

Oeystein R Brekk, Joanna A Korecka, Cecile C Crapart, Mylene Huebecker, Zachary K MacBain, Sara Ann Rosenthal, Miguel Sena-Esteves, David A Priestman, Frances M Platt, Ole Isacson, Penelope J Hallett.

  Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of β-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson's disease (PD). α-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and HEX knockout mice, and HEX activity is reduced in the substantia nigra in PD. In this study, we biochemically demonstrate that HEX deficiency in mice causes formation of high-molecular weight (HMW) aSYN and ubiquitin in the brain. As expected from HEX enzymatic function requirements, overexpression in vivo of HEXA and B combined, but not either of the subunits expressed alone, increased HEX activity as evidenced by histochemical assays. Biochemically, such HEX gene expression resulted in increased conversion of GM2 to its breakdown product GM3. In a neurodegenerative model of overexpression of aSYN in rats, increasing HEX activity by AAV6 gene transfer in the substantia nigra reduced aSYN embedding in lipid compartments and rescued dopaminergic neurons from degeneration. Overall, these data are consistent with a paradigm shift where lipid abnormalities are central to or preceding protein changes typically associated with PD.

Keywords:  Lipid binding; Neuroprotection; Parkinson’s disease; Sandhoff disease; α-Synuclein; β-Hexosaminidase

DOI:  https://doi.org/10.1186/s40478-020-01004-6
Biomedicines. 2020 Aug 04. pii: E272. [Epub ahead of print]8(8):

Transmissible Endosomal Intoxication: A Balance between Exosomes and Lysosomes at the Basis of Intercellular Amyloid Propagation.

Anaïs Bécot, Charlotte Volgers, Guillaume van Niel.

  In Alzheimer's disease (AD), endolysosomal dysfunctions are amongst the earliest cellular features to appear. Each organelle of the endolysosomal system, from the multivesicular body (MVB) to the lysosome, contributes to the homeostasis of amyloid precursor protein (APP) cleavage products including β-amyloid (Aβ) peptides. Hence, this review will attempt to disentangle how changes in the endolysosomal system cumulate to the generation of toxic amyloid species and hamper their degradation. We highlight that the formation of MVBs and the generation of amyloid species are closely linked and describe how the molecular machineries acting at MVBs determine the generation and sorting of APP cleavage products towards their degradation or release in association with exosomes. In particular, we will focus on AD-related distortions of the endolysomal system that divert it from its degradative function to favour the release of exosomes and associated amyloid species. We propose here that such an imbalance transposed at the brain scale poses a novel concept of transmissible endosomal intoxication (TEI). This TEI would initiate a self-perpetuating transmission of endosomal dysfunction between cells that would support the propagation of amyloid species in neurodegenerative diseases.

Keywords:  Alzheimer’s disease; Aβ peptide; C99; amyloid propagation; amyloidogenesis; autophagy; endosomal sorting; exosomes; extracellular vesicles; lysosomes; proteostasis network; transmissible endosomal intoxication

DOI:  https://doi.org/10.3390/biomedicines8080272
Cells. 2020 Aug 05. pii: E1838. [Epub ahead of print]9(8):

Mucopolysaccharidosis Type I: A Review of the Natural History and Molecular Pathology.

Christiane S Hampe, Julie B Eisengart, Troy C Lund, Paul J Orchard, Monika Swietlicka, Jacob Wesley, R Scott McIvor.

  Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive inherited disease, caused by deficiency of the enzyme α-L-iduronidase, resulting in accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in organs and tissues. If untreated, patients with the severe phenotype die within the first decade of life. Early diagnosis is crucial to prevent the development of fatal disease manifestations, prominently cardiac and respiratory disease, as well as cognitive impairment. However, the initial symptoms are nonspecific and impede early diagnosis. This review discusses common phenotypic manifestations in the order in which they develop. Similarities and differences in the three animal models for MPS I are highlighted. Earliest symptoms, which present during the first 6 months of life, include hernias, coarse facial features, recurrent rhinitis and/or upper airway obstructions in the absence of infection, and thoracolumbar kyphosis. During the next 6 months, loss of hearing, corneal clouding, and further musculoskeletal dysplasias develop. Finally, late manifestations including lower airway obstructions and cognitive decline emerge. Cardiac symptoms are common in MPS I and can develop in infancy. The underlying pathogenesis is in the intra- and extracellular accumulation of partially degraded GAGs and infiltration of cells with enlarged lysosomes causing tissue expansion and bone deformities. These interfere with the proper arrangement of collagen fibrils, disrupt nerve fibers, and cause devastating secondary pathophysiological cascades including inflammation, oxidative stress, and other disruptions to intracellular and extracellular homeostasis. A greater understanding of the natural history of MPS I will allow early diagnosis and timely management of the disease facilitating better treatment outcomes.

Keywords:  animal models; mucopolysaccharidosis type I; α-L-iduronidase

DOI:  https://doi.org/10.3390/cells9081838
Kidney Int. 2020 Apr 27. pii: S0085-2538(20)30428-2. [Epub ahead of print]

Tuberous sclerosis 1 (Tsc1) mediated mTORC1 activation promotes glycolysis in tubular epithelial cells in kidney fibrosis.

Hongdi Cao, Jing Luo, Yu Zhang, Xiaoming Mao, Ping Wen, Hao Ding, Jing Xu, Qi Sun, Weichun He, Chunsun Dai, Ke Zen, Yang Zhou, Junwei Yang, Lei Jiang.

  Energy reprogramming to glycolysis is closely associated with the development of chronic kidney disease. As an important negative regulatory factor of the mammalian target of rapamycin complex 1 (mTORC1) signal, tuberous sclerosis complex 1 (Tsc1) is also a key regulatory point of glycolysis. Here, we investigated whether Tsc1 could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells. We induced mTORC1 signal activation in tubular epithelial cells in kidneys with fibrosis via unilateral ureteral occlusion. This resulted in increased tubular epithelial cell proliferation and glycolytic enzyme upregulation. Prior incubation with rapamycin inhibited mTORC1 activation and abolished the enhanced glycolysis and tubular epithelial cell proliferation. Furthermore, knockdown of Tsc1 expression promoted glycolysis in the rat kidney epithelial cell line NRK-52E. Specific deletion of Tsc1 in the proximal tubules of mice resulted in enlarged kidneys characterized by a high proportion of proliferative tubular epithelial cells, dilated tubules with cyst formation, and a large area of interstitial fibrosis in conjunction with elevated glycolysis. Treatment of the mice with the glycolysis inhibitor 2-deoxyglucose notably ameliorated tubular epithelial cell proliferation, cystogenesis, and kidney fibrosis. Thus, our findings suggest that Tsc1-associated mTORC1 signaling mediates the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells.

Keywords:  glycolysis; mTOR signaling; renal fibrosis; tubular epithelial cells

DOI:  https://doi.org/10.1016/j.kint.2020.03.035
JCI Insight. 2020 Aug 06. pii: 142898. [Epub ahead of print]5(15):

TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.

Matthew R Lynch, Mei T Tran, Kenneth M Ralto, Zsuzsanna K Zsengeller, Vinod Raman, Swati S Bhasin, Nuo Sun, Xiuying Chen, Daniel Brown, Ilsa I Rovira, Kensei Taguchi, Craig R Brooks, Isaac E Stillman, Manoj K Bhasin, Toren Finkel, Samir M Parikh.

DOI: https://doi.org/10.1172/jci.insight.142898
Genes (Basel). 2020 Aug 05. pii: E896. [Epub ahead of print]11(8):

Pathological Consequences of Hepatic mTORC1 Dysregulation.

Chun-Seok Cho, Allison Ho Kowalsky, Jun Hee Lee.

  The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of metabolism that integrates environmental inputs, including nutrients, growth factors, and stress signals. mTORC1 activation upregulates anabolism of diverse macromolecules, such as proteins, lipids, and nucleic acids, while downregulating autolysosomal catabolism. mTORC1 dysregulation is often found in various diseases, including cancer, cardiovascular and neurodegenerative diseases, as well as metabolic syndromes involving obesity and type II diabetes. As an essential metabolic organ, the liver requires proper regulation of mTORC1 for maintaining homeostasis and preventing pathologies. For instance, aberrant hyper- or hypoactivation of mTORC1 disrupts hepatocellular homeostasis and damages the structural and functional integrity of the tissue, leading to prominent liver injury and the development of hepatocellular carcinogenesis. Proper regulation of mTORC1 during liver diseases may be beneficial for restoring liver function and ameliorating the detrimental consequences of liver failure.

Keywords:  liver; mTORC1; metabolism

DOI:  https://doi.org/10.3390/genes11080896
Front Cell Dev Biol. 2020 ;8 595

Mitochondria-Associated ER Membranes - The Origin Site of Autophagy.

Ming Yang, Chenrui Li, Shikun Yang, Ying Xiao, Xiaofen Xiong, Wei Chen, Hao Zhao, Qin Zhang, Yachun Han, Lin Sun.

  Autophagy is a process of intracellular self-recycling and degradation that plays an important role in maintaining cell homeostasis. However, the molecular mechanism of autophagy remains to be further studied. Mitochondria-associated endoplasmic reticulum membranes (MAMs) are the region of the ER that mediate communication between the ER and mitochondria. MAMs have been demonstrated to be involved in autophagy, Ca2+ transport and lipid metabolism. Here, we discuss the composition and function of MAMs, more specifically, to emphasize the role of MAMs in regulating autophagy. Finally, some key information that may be useful for future research is summarized.

Keywords:  autophagy; endoplasmic reticulum; mitochondria; mitochondria-associated endoplasmic reticulum membranes (MAMs); mitophagy

DOI:  https://doi.org/10.3389/fcell.2020.00595
Front Neuroanat. 2020 ;14 39

The Neurodevelopmental Pathogenesis of Tuberous Sclerosis Complex (TSC).

David M Feliciano.

  Tuberous sclerosis complex (TSC) is a model disorder for understanding brain development because the genes that cause TSC are known, many downstream molecular pathways have been identified, and the resulting perturbations of cellular events are established. TSC, therefore, provides an intellectual framework to understand the molecular and biochemical pathways that orchestrate normal brain development. The TSC1 and TSC2 genes encode Hamartin and Tuberin which form a GTPase activating protein (GAP) complex. Inactivating mutations in TSC genes (TSC1/TSC2) cause sustained Ras homologue enriched in brain (RHEB) activation of the mammalian isoform of the target of rapamycin complex 1 (mTORC1). TOR is a protein kinase that regulates cell size in many organisms throughout nature. mTORC1 inhibits catabolic processes including autophagy and activates anabolic processes including mRNA translation. mTORC1 regulation is achieved through two main upstream mechanisms. The first mechanism is regulation by growth factor signaling. The second mechanism is regulation by amino acids. Gene mutations that cause too much or too little mTORC1 activity lead to a spectrum of neuroanatomical changes ranging from altered brain size (micro and macrocephaly) to cortical malformations to Type I neoplasias. Because somatic mutations often underlie these changes, the timing, and location of mutation results in focal brain malformations. These mutations, therefore, provide gain-of-function and loss-of-function changes that are a powerful tool to assess the events that have gone awry during development and to determine their functional physiological consequences. Knowledge about the TSC-mTORC1 pathway has allowed scientists to predict which upstream and downstream mutations should cause commensurate neuroanatomical changes. Indeed, many of these predictions have now been clinically validated. A description of clinical imaging and histochemical findings is provided in relation to laboratory models of TSC that will allow the reader to appreciate how human pathology can provide an understanding of the fundamental mechanisms of development.

Keywords:  SEGA; TSC1; TSC2; Tuber; mTOR; subependymal giant cell astrocytoma

DOI:  https://doi.org/10.3389/fnana.2020.00039
Childs Nerv Syst. 2020 Aug 06.

Genetics of tuberous sclerosis complex: an update.

Daphna Marom.

   PURPOSE: To review the current genetic aspects of tuberous sclerosis complex.
METHODS: Review of the literature.
RESULTS: Tuberous sclerosis complex (TSC), a long known childhood-onset monogenic disorder, characterized by hamartoma formation affecting mainly the brain, heart, kidney, lung, and skin, is associated with a high morbidity burden and risk of a reduced life span. The identification of TSC1 and TSC2, as tumor suppressor genes causative of the disorder, led to the elucidation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway and its pivotal role in the pathogenesis of hamartoma formation. This knowledge was translated into standard clinical practice with the discovery of rapamycin, and additional analogues, as inhibitors of mTORC1.
CONCLUSION: Next-generation sequencing was proven to be fundamental to drive research of tumorigenesis in TSC, hopefully leading to new therapeutic options in the future.

Keywords:  Hamartoma; TSC1; TSC2; mTOR pathway

DOI:  https://doi.org/10.1007/s00381-020-04726-z
Genes (Basel). 2020 Aug 04. pii: E885. [Epub ahead of print]11(8):

Structural Insights into TOR Signaling.

Lucas Tafur, Jennifer Kefauver, Robbie Loewith.

  The Target of Rapamycin (TOR) is a highly conserved serine/threonine protein kinase that performs essential roles in the control of cellular growth and metabolism. TOR acts in two distinct multiprotein complexes, TORC1 and TORC2 (mTORC1 and mTORC2 in humans), which maintain different aspects of cellular homeostasis and orchestrate the cellular responses to diverse environmental challenges. Interest in understanding TOR signaling is further motivated by observations that link aberrant TOR signaling to a variety of diseases, ranging from epilepsy to cancer. In the last few years, driven in large part by recent advances in cryo-electron microscopy, there has been an explosion of available structures of (m)TORC1 and its regulators, as well as several (m)TORC2 structures, derived from both yeast and mammals. In this review, we highlight and summarize the main findings from these reports and discuss both the fascinating and unexpected molecular biology revealed and how this knowledge will potentially contribute to new therapeutic strategies to manipulate signaling through these clinically relevant pathways.

Keywords:  cell growth homeostasis; structural biology; target of rapamycin

DOI:  https://doi.org/10.3390/genes11080885
J Hepatol. 2020 Jul 29. pii: S0168-8278(20)30489-X. [Epub ahead of print]

p53 haploinsufficiency and increased mTOR signaling define a subset of aggressive hepatocellular carcinoma.

Yuan-Deng Luo, Lei Fang, Hong-Qiang Yu, Jie Zhang, Xiao-Tong Lin, Xiao-Yu Liu, Di Wu, Gui-Xi Li, Deng Huang, Yu-Jun Zhang, Shu Chen, Yan Jiang, Ling Shuai, Yu He, Lei-Da Zhang, Ping Bie, Chuan-Ming Xie.

   BACKGROUND AND AIMS: p53 mutations occur frequently in human hepatocellular carcinoma (HCC). Activation of the mammalian target of rapamycin (mTOR) pathway is also associated with HCC. However, it is still unknown whether these changes together initiate HCC and can be targeted as a potential therapeutic strategy.
METHODS: We generated mouse models in which mTOR was hyperactivated by loss of tuberous sclerosis complex 1 (Tsc1) with or without p53 haplodeficiency. Primary cells were isolated from mouse livers. Oncogenic signaling was assessed in vitro and in vivo, with or without targeted inhibition of a single molecule or multiple molecules. Transcriptional profiling was used to identify biomarkers predictive of HCC. Human HCC materials were used to corroborate the findings from mouse models.
RESULTS: p53 haploinsufficiency facilitates mTOR signaling via the Pten/PI3k/Akt axis, promoting HCC tumorigenesis and lung metastasis. Inhibition of PI3K/Akt reduced mTOR activity, which effectively enhanced the anticancer effort of an mTOR inhibitor. Abcc4 was found to be responsible for p53 haploinsufficiency- and Tsc1 loss-driven HCC tumorigenesis. Moreover, in clinical HCC samples, Abcc4 specifically identified an aggressive subtype. The mTOR inhibitor rapamycin significantly reduced hepatocarcinogenesis triggered by Tsc1 loss and p53 haploinsufficiency in vivo, as well as the biomarker Abcc4.
CONCLUSIONS: Our data advance the current understanding of the activation of the Pten/PI3K/Akt/mTOR axis and its downstream target Abcc4 in hepatocarcinogenesis driven by p53 reduction and Tsc1 loss. Targeting mTOR, an unexpected vulnerability in p53 (haplo)deficiency HCC, can be exploited therapeutically to treat Abcc-4-positive HCC patients.

Keywords:  Abcc4; PI3K/Akt; Pten; Tsc1/mTOR; hepatocellular carcinoma; p53; poor survival; rapamycin; sapanisertib

DOI:  https://doi.org/10.1016/j.jhep.2020.07.036
Cells. 2020 Jul 31. pii: E1817. [Epub ahead of print]9(8):

Cyclopamine and Rapamycin Synergistically Inhibit mTOR Signalling in Mouse Hepatocytes, Revealing an Interaction of Hedgehog and mTor Signalling in the Liver.

Luise Spormann, Christiane Rennert, Erik Kolbe, Fritzi Ott, Carolin Lossius, Robert Lehmann, Rolf Gebhardt, Thomas Berg, Madlen Matz-Soja.

  In the liver, energy homeostasis is mainly regulated by mechanistic target of rapamycin (mTOR) signalling, which influences relevant metabolic pathways, including lipid metabolism. However, the Hedgehog (Hh) pathway is one of the newly identified drivers of hepatic lipid metabolism. Although the link between mTOR and Hh signalling was previously demonstrated in cancer development and progression, knowledge of their molecular crosstalk in healthy liver is lacking. To close this information gap, we used a transgenic mouse model, which allows hepatocyte-specific deletion of the Hh pathway, and in vitro studies to reveal interactions between Hh and mTOR signalling. The study was conducted in male and female mice to investigate sexual differences in the crosstalk of these signalling pathways. Our results reveal that the conditional Hh knockout reduces mitochondrial adenosine triphosphate (ATP) production in primary hepatocytes from female mice and inhibits autophagy in hepatocytes from both sexes. Furthermore, in vitro studies show a synergistic effect of cyclopamine and rapamycin on the inhibition of mTor signalling and oxidative respiration in primary hepatocytes from male and female C57BL/6N mice. Overall, our results demonstrate that the impairment of Hh signalling influences mTOR signalling and therefore represses oxidative phosphorylation and autophagy.

Keywords:  cyclopamine; hedgehog; hepatocytes; liver; mTOR; mTORC2; rapamycin

DOI:  https://doi.org/10.3390/cells9081817
JCI Insight. 2020 Aug 06. pii: 139237. [Epub ahead of print]5(15):

Selective inhibition of mTORC1 in tumor vessels increases antitumor immunity.

Shan Wang, Ariel Raybuck, Eileen Shiuan, Sung Hoon Cho, Qingfei Wang, Dana M Brantley-Sieders, Deanna Edwards, Margaret M Allaman, James Nathan, Keith T Wilson, David DeNardo, Siyuan Zhang, Rebecca Cook, Mark Boothby, Jin Chen.

  A tumor blood vessel is a key regulator of tissue perfusion, immune cell trafficking, cancer metastasis, and therapeutic responsiveness. mTORC1 is a signaling node downstream of multiple angiogenic factors in the endothelium. However, mTORC1 inhibitors have limited efficacy in most solid tumors, in part due to inhibition of immune function at high doses used in oncology patients and compensatory PI3K signaling triggered by mTORC1 inhibition in tumor cells. Here we show that low-dose RAD001/everolimus, an mTORC1 inhibitor, selectively targets mTORC1 signaling in endothelial cells (ECs) without affecting tumor cells or immune cells, resulting in tumor vessel normalization and increased antitumor immunity. Notably, this phenotype was recapitulated upon targeted inducible gene ablation of the mTORC1 component Raptor in tumor ECs (RaptorECKO). Tumors grown in RaptorECKO mice displayed a robust increase in tumor-infiltrating lymphocytes due to GM-CSF-mediated activation of CD103+ dendritic cells and displayed decreased tumor growth and metastasis. GM-CSF neutralization restored tumor growth and metastasis, as did T cell depletion. Importantly, analyses of human tumor data sets support our animal studies. Collectively, these findings demonstrate that endothelial mTORC1 is an actionable target for tumor vessel normalization, which could be leveraged to enhance antitumor immune therapies.

Keywords:  Cancer immunotherapy; Immunology; Oncology; endothelial cells

DOI:  https://doi.org/10.1172/jci.insight.139237
EMBO Rep. 2020 Aug 07. e50103

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Wonyoung Jeong, Soyoung Kim, Ukjin Lee, Zhendong A Zhong, Mikhail Savitsky, Hyeryun Kwon, Jiyoung Kim, Taebok Lee, Jin Won Cho, Bart O Williams, Vladimir L Katanaev, Eek-Hoon Jho.

  Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

Keywords:   YAP ; Hippo signaling; LRP6; O-GlcNAcylation; Starvation

DOI:  https://doi.org/10.15252/embr.202050103
Elife. 2020 Aug 03. pii: e58246. [Epub ahead of print]9

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

Vasyl Ivashov, Johannes Zimmer, Sinead Schwabl, Jennifer Kahlhofer, Sabine Weys, Ronald Gstir, Thomas Jakschitz, Leopold Kremser, Günther K Bonn, Herbert Lindner, Lukas A Huber, Sebastien Leon, Oliver Schmidt, David Teis.

  How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

Keywords:  S. cerevisiae; cell biology

DOI:  https://doi.org/10.7554/eLife.58246
J Biochem. 2020 Aug 03. pii: mvaa089. [Epub ahead of print]

Emerging roles of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate as regulators of multiple steps in autophagy.

Takashi Baba, Tamas Balla.

  Inositol phospholipids are low-abundance regulatory lipids that orchestrate diverse cellular functions in eukaryotic organisms. Recent studies have uncovered involvement of the lipids in multiple steps in autophagy. The late endosome-lysosome compartment plays critical roles in cellular nutrient sensing and in the control of both the initiation of autophagy and the late stage of eventual degradation of cytosolic materials destined for elimination. It is particularly notable that inositol lipids are involved in almost all steps of the autophagic process. In this review, we summarize how inositol lipids regulate and contribute to autophagy through the endomembrane compartments, primarily focusing on PI4P and PI(4,5)P2.

Keywords:  Autophagy and Late endosome/Lysosome; Phosphoinositide

DOI:  https://doi.org/10.1093/jb/mvaa089
Nat Cell Biol. 2020 Aug;22(8): 947-959

Endosomal membrane tension regulates ESCRT-III-dependent intra-lumenal vesicle formation.

Vincent Mercier, Jorge Larios, Guillaume Molinard, Antoine Goujon, Stefan Matile, Jean Gruenberg, Aurélien Roux.

The plasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether the membrane tension of organelles regulates their functions, notably intracellular traffic. The endosomal sorting complexes required for transport (ESCRT)-III complex is the major membrane remodelling complex that drives intra-lumenal-vesicle (ILV) formation on endosomal membranes. Here we used a fluorescent membrane-tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when the membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro and correlates with increased ILV formation in ESCRT-III-decorated endosomes in vivo. Finally, we find that the endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and endosome trafficking, leading us to conclude that membrane tension can control organelle functions.

DOI: https://doi.org/10.1038/s41556-020-0546-4
FEBS Lett. 2020 Aug 07.

SGK1 is a signalling hub that controls protein synthesis and proliferation in endothelial cells.

Ferran Medina-Jover, Núria Gendrau-Sanclemente, Francesc Viñals.

  BMP9 is a cytokine involved in the maturation phase of the angiogenic process that signals through its serine/threonine receptor ALK1 and its coreceptor endoglin. In this paper, we explain how BMP9 directs the regulation of endothelial cell proliferation blockage while in turn stimulating protein synthesis. To achieve this, BMP9 promotes SGK1 synthesis and activation through mTORC2 in order to stimulate the mTORC1/S6K/S6 axis. Moreover, BMP9 blocks proliferation also through SGK1 by reducing the activity of the MEK/ERK signalling pathway. Inhibition of SGK1 activity is sufficient to prevent BMP9-mediated inhibition of ERK, leading to an increase in endothelial cell proliferation. Overall, our findings reveal that SGK1 is a key player during angiogenesis, mediating the pro-quiescent and maturation effects of BMP9/ALK1.

Keywords:  ALK1; BMP9; ERK; HHT; SGK1; angiogenesis; endothelial cells; protein synthesis

DOI:  https://doi.org/10.1002/1873-3468.13901