bims-lysosi 2020-07-05 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2020‒07‒05
twenty-two papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

The Lysosome at the Intersection of Cellular Growth and Destruction.
A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.
AMPK is activated during lysosomal damage via a galectin-ubiquitin signal transduction system.
Endocytic regulation of cellular ion homeostasis controls lysosome biogenesis.
A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.
The Unfolded Protein Response Regulates Hepatic Autophagy by sXBP1-mediated Activation of TFEB.
TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast.
AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions.
Amino acids bearing aromatic or heteroaromatic substituents as a new class of ligands for the lysosomal sialic acid transporter sialin (SLC17A5).
ULK1 and ULK2 are less redundant than previously thought: computational analysis uncovers distinct regulation and functions of these autophagy induction proteins.
Systematic quantitative analysis of ribosome inventory during nutrient stress.
Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes.
Metabolic Communication and Healthy Aging: Where Should We Focus Our Energy?
Enhancing the Activity of Glucocerebrosidase as a Treatment for Parkinson Disease.
Neuropathophysiology, Genetic Profile, and Clinical Manifestation of Mucolipidosis IV-A Review and Case Series.
Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.
Engineering monocyte/macrophage-specific glucocerebrosidase expression in human hematopoietic stem cells using genome editing.
Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.
miR-143 Regulates Lysosomal Enzyme Transport across the Blood-Brain Barrier and Transforms CNS Treatment for Mucopolysaccharidosis Type I.
Loss of CLN3, the gene mutated in juvenile neuronal ceroid lipofuscinosis, leads to metabolic impairment and autophagy induction in retina pigment epithelium.
A tailored Cln3Q352X mouse model for testing therapeutic interventions in CLN3 Batten disease.
ATP6V1B2-related epileptic encephalopathy.

Dev Cell. 2020 Jun 30. pii: S1534-5807(20)30460-3. [Epub ahead of print]

The Lysosome at the Intersection of Cellular Growth and Destruction.

Hijai R Shin, Roberto Zoncu.

  The lysosome is an essential catabolic organelle that consumes cellular biomass to regenerate basic building blocks that can fuel anabolic reactions. This simple view has evolved more recently to integrate novel functions of the lysosome as a key signaling center, which can steer the metabolic trajectory of cells in response to changes in nutrients, growth factors, and stress. Master protein kinases and transcription factors mediate the growth-promoting and catabolic activities of the lysosome and undergo a complex interplay that enables cellular adaptation to ever-changing metabolic conditions. Understanding how this coordination occurs will shed light on the fundamental logic of how the lysosome functions to control growth in the context of development, tissue homeostasis, and cancer.

Keywords:  TFEB; anabolism; autophagy; catabolism; lysosome; mTORC1; nutrient sensing

DOI:  https://doi.org/10.1016/j.devcel.2020.06.010
Nature. 2020 Jul 01.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

Gennaro Napolitano, Chiara Di Malta, Alessandra Esposito, Mariana E G de Araujo, Salvatore Pece, Giovanni Bertalot, Maria Matarese, Valerio Benedetti, Angela Zampelli, Taras Stasyk, Diletta Siciliano, Alessandro Venuta, Marcella Cesana, Claudia Vilardo, Edoardo Nusco, Jlenia Monfregola, Alessia Calcagnì, Pier Paolo Di Fiore, Lukas A Huber, Andrea Ballabio.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

DOI: https://doi.org/10.1038/s41586-020-2444-0
Autophagy. 2020 Jun 28.

AMPK is activated during lysosomal damage via a galectin-ubiquitin signal transduction system.

Jingyue Jia, Bhawana Bissa, Lukas Brecht, Lee Allers, Seong Won Choi, Yuexi Gu, Mark Zbinden, Mark R Burge, Graham Timmins, Kenneth Hallows, Christian Behrends, Vojo Deretic.

  Lysosomal damage activates AMPK, a regulator of macroautophagy/autophagy and metabolism, and elicits a strong ubiquitination response. Here we show that the cytosolic lectin LGALS9 detects lysosomal membrane breach by binding to lumenal glycoepitopes, and directs both the ubiquitination response and AMPK activation. Proteomic analyses have revealed increased LGALS9 association with lysosomes, and concomitant changes in LGALS9 interactions with its newly identified partners that control ubiquitination-deubiquitination processes. An LGALS9-inetractor, deubiquitinase USP9X, dissociates from damaged lysosomes upon recognition of lumenal glycans by LGALS9. USP9X's departure from lysosomes promotes K63 ubiquitination and stimulation of MAP3K7/TAK1, an upstream kinase and activator of AMPK hitherto orphaned for a precise physiological function. Ubiquitin-activated MAP3K7/TAK1 controls AMPK specifically during lysosomal injury, caused by a spectrum of membrane-damaging or -permeabilizing agents, including silica crystals, the intracellular pathogen Mycobacterium tuberculosis, TNFSF10/TRAIL signaling, and the anti-diabetes drugs metformin. The LGALS9-ubiquitin system activating AMPK represents a novel signal transduction system contributing to various physiological outputs that are under the control of AMPK, including autophagy, MTOR, lysosomal maintenance and biogenesis, immunity, defense against microbes, and metabolic reprograming.

Keywords:   Mycobacterium tuberculosis ; AMPK; TAK1; TRAIL; USP9X; autophagy; diabetes; lysosome; metabolism; metformin

DOI:  https://doi.org/10.1080/15548627.2020.1788890
Nat Cell Biol. 2020 Jun 29.

Endocytic regulation of cellular ion homeostasis controls lysosome biogenesis.

Tania López-Hernández, Dmytro Puchkov, Eberhard Krause, Tanja Maritzen, Volker Haucke.

Lysosomes serve as cellular degradation and signalling centres that coordinate metabolism in response to intracellular cues and extracellular signals. Lysosomal capacity is adapted to cellular needs by transcription factors, such as TFEB and TFE3, which activate the expression of lysosomal and autophagy genes. Nuclear translocation and activation of TFEB are induced by a variety of conditions such as starvation, lysosome stress and lysosomal storage disorders. How these various cues are integrated remains incompletely understood. Here, we describe a pathway initiated at the plasma membrane that controls lysosome biogenesis via the endocytic regulation of intracellular ion homeostasis. This pathway is based on the exo-endocytosis of NHE7, a Na+/H+ exchanger mutated in X-linked intellectual disability, and serves to control intracellular ion homeostasis and thereby Ca2+/calcineurin-mediated activation of TFEB and downstream lysosome biogenesis in response to osmotic stress to promote the turnover of toxic proteins and cell survival.

DOI: https://doi.org/10.1038/s41556-020-0535-7
J Clin Invest. 2020 Jun 29. pii: 130955. [Epub ahead of print]

A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.

Lakshya Bajaj, Jaiprakash Sharma, Alberto di Ronza, Pengcheng Zhang, Aiden Eblimit, Rituraj Pal, Dany Roman, John R Collette, Clarissa Booth, Kevin T Chang, Richard N Sifers, Sung Y Jung, Jill M Weimer, Rui Chen, Randy W Schekman, Marco Sardiello.

  Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.

Keywords:  Cell Biology; Genetic diseases; Lysosomes; Molecular pathology

DOI:  https://doi.org/10.1172/JCI130955
Autophagy. 2020 Jun 28.

The Unfolded Protein Response Regulates Hepatic Autophagy by sXBP1-mediated Activation of TFEB.

Zeyuan Zhang, Qingwen Qian, Mark Li, Fan Shao, Wen-Xing Ding, Vitor A Lira, Sophia X Chen, Sara C Sebag, Gökhan S Hotamisligil, Huojun Cao, Ling Yang.

  Defective macroautophagy/autophagy and a failure to initiate the adaptive unfolded protein response (UPR) in response to the endoplasmic reticulum (ER) stress contributes to obesity-associated metabolic dysfunction. However, whether and how unresolved ER stress leads to defects in the autophagy pathway and to the progression of obesity-associated hepatic pathologies remains unclear. Obesity suppresses the expression of hepatic spliced XBP1 (X-box binding protein 1; sXBP1), the key transcription factor that promotes the adaptive UPR. Our RNA-seq analysis revealed that sXBP1 regulates genes involved in lysosomal function in the liver under fasting conditions. Chromatin immunoprecipitation (ChIP) analyses of both primary hepatocytes and whole liver further showed that sXBP1 occupies the -743 to -523 site of the promoter of Tfeb (transcription factor EB), a master regulator of autophagy and lysosome biogenesis. Notably, this occupancy was significantly reduced in livers from patients with steatosis. In mice, hepatic deletion of Xbp1 (xbp1 LKO) suppressed the transcription of Tfeb as well as autophagy, whereas hepatic overexpression of sXbp1 enhanced Tfeb transcription and autophagy. Moreover, overexpression of Tfeb in the xbp1 LKO mouse liver ameliorated glucose intolerance and steatosis in mice with diet-induced obesity (DIO). Conversely, loss of TFEB function impaired the protective role of sXBP1 in hepatic steatosis in mice with DIO. These data indicate that sXBP1-Tfeb signaling has direct functional consequences in the context of obesity. Collectively, our data provide novel insight into how two organelle stress responses are integrated to protect against obesity-associated metabolic dysfunction.

Keywords:  autophagy; endoplasmic reticulum; liver; obesity; spliced X-box-binding protein 1; transcription factor EB

DOI:  https://doi.org/10.1080/15548627.2020.1788889
J Biol Chem. 2020 Jul 01. pii: jbc.RA120.013222. [Epub ahead of print]

TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast.

Oliver Schmidt, Yannick Weyer, Simon Sprenger, Michael A Widerin, Sebastian Eising, Verena Baumann, Mihaela Angelova, Robbie Loewith, Christopher J Stefan, Michael W Hess, Florian Fröhlich, David Teis.

  The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

Keywords:  Endosome and Golgi-associated degradation (EGAD); ORMDL family; TORC2; calcineurin; endosomal sorting complexes required for transport (ESCRT); mTOR complex (mTORC); membrane; membrane stress; sphingolipid; stress

DOI:  https://doi.org/10.1074/jbc.RA120.013222
BMC Biol. 2020 Jul 03. 18(1): 81

AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions.

Nathalie Bouquier, Enora Moutin, Lionel A Tintignac, Amandine Reverbel, Elodie Jublanc, Michael Sinnreich, Yan Chastagnier, Julien Averous, Pierre Fafournoux, Chiara Verpelli, Tobias Boeckers, Gilles Carnac, Julie Perroy, Vincent Ollendorff.

  BACKGROUND: mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells.RESULTS: As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder.
CONCLUSIONS: Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.

Keywords:  Autism spectrum disorder; BRET; Muscle differentiation; Neuronal activity; mTORC1 Biosensor; mTor signaling; mToropathies

DOI:  https://doi.org/10.1186/s12915-020-00790-8
J Med Chem. 2020 Jul 01.

Amino acids bearing aromatic or heteroaromatic substituents as a new class of ligands for the lysosomal sialic acid transporter sialin (SLC17A5).

Lilian Dubois, Nicolas Pietrancosta, Alexandre Cabaye, Isabelle Fanget, Cécile Debacker, Pierre-André Gilormini, Patrick M Dansette, Julien Dairou, Christophe Biot, Roseline Froissart, Anne Goupil-Lamy, Hugues-Olivier Bertrand, Francine Clemy Acher, Isabelle McCort-Tranchepain, Bruno Gasnier, Christine Anne.

Sialin is a lysosomal sialic acid transporter defective in Salla disease, a rare inherited leukodystrophy. It also enables metabolic incorporation of exogenous sialic acids, leading to autoantibodies against N-glycolylneuraminic acid in humans. Here we identified a novel class of human sialin ligands by virtual screening and structure-activity relationship studies. The ligand scaffold is characterized by an amino acid backbone with a free carboxylate, an N-linked aromatic or heteroaromatic substituent and a hydrophobic side chain. The most potent compound, 45 (LSP12-3129), inhibited N-acetylneuraminic acid 1 (Neu5Ac) transport in a non-competitive manner with IC50 ≈ 2.5 µM, a value 400-fold lower than the KM for Neu5Ac. In vitro and molecular docking studies attributed the non-competitive character to selective inhibitor binding to the Neu5Ac site in cytosol-facing conformation. Moreover, compound 45 rescued the trafficking defect of the pathogenic mutant (R39C) causing Salla disease. This new class of cell-permeant inhibitors provide tools to investigate the physiological roles of sialin and to help develop pharmacological chaperones for Salla disease.

DOI: https://doi.org/10.1021/acs.jmedchem.9b02119
Sci Rep. 2020 Jul 02. 10(1): 10940

ULK1 and ULK2 are less redundant than previously thought: computational analysis uncovers distinct regulation and functions of these autophagy induction proteins.

Amanda Demeter, Mari Carmen Romero-Mulero, Luca Csabai, Márton Ölbei, Padhmanand Sudhakar, Wilfried Haerty, Tamás Korcsmáros.

Macroautophagy, the degradation of cytoplasmic content by lysosomal fusion, is an evolutionary conserved process promoting homeostasis and intracellular defence. Macroautophagy is initiated primarily by a complex containing ULK1 or ULK2 (two paralogs of the yeast Atg1 protein). To understand the differences between ULK1 and ULK2, we compared the human ULK1 and ULK2 proteins and their regulation. Despite the similarity in their enzymatic domain, we found that ULK1 and ULK2 have major differences in their autophagy-related interactors and their post-translational and transcriptional regulators. We identified 18 ULK1-specific and 7 ULK2-specific protein motifs serving as different interaction interfaces. We found that interactors of ULK1 and ULK2 all have different tissue-specific expressions partially contributing to diverse and ULK-specific interaction networks in various tissues. We identified three ULK1-specific and one ULK2-specific transcription factor binding sites, and eight sites shared by the regulatory region of both genes. Importantly, we found that both their post-translational and transcriptional regulators are involved in distinct biological processes-suggesting separate functions for ULK1 and ULK2. Unravelling differences between ULK1 and ULK2 could lead to a better understanding of how ULK-type specific dysregulation affects autophagy and other cellular processes that have been implicated in diseases such as inflammatory bowel disease and cancer.

DOI: https://doi.org/10.1038/s41598-020-67780-2
Nature. 2020 Jul 01.

Systematic quantitative analysis of ribosome inventory during nutrient stress.

Heeseon An, Alban Ordureau, Maria Körner, Joao A Paulo, J Wade Harper.

Mammalian cells reorganize their proteomes in response to nutrient stress through translational suppression and degradative mechanisms using the proteasome and autophagy systems1,2. Ribosomes are central targets of this response, as they are responsible for translation and subject to lysosomal turnover during nutrient stress3-5. The abundance of ribosomal (r)-proteins (around 6% of the proteome; 107 copies per cell)6,7 and their high arginine and lysine content has led to the hypothesis that they are selectively used as a source of basic amino acids during nutrient stress through autophagy4,7. However, the relative contributions of translational and degradative mechanisms to the control of r-protein abundance during acute stress responses is poorly understood, as is the extent to which r-proteins are used to generate amino acids when specific building blocks are limited7. Here, we integrate quantitative global translatome and degradome proteomics8 with genetically encoded Ribo-Keima5 and Ribo-Halo reporters to interrogate r-protein homeostasis with and without active autophagy. In conditions of acute nutrient stress, cells strongly suppress the translation of r-proteins, but, notably, r-protein degradation occurs largely through non-autophagic pathways. Simultaneously, the decrease in r-protein abundance is compensated for by a reduced dilution of pre-existing ribosomes and a reduction in cell volume, thereby maintaining the density of ribosomes within single cells. Withdrawal of basic or hydrophobic amino acids induces translational repression without differential induction of ribophagy, indicating that ribophagy is not used to selectively produce basic amino acids during acute nutrient stress. We present a quantitative framework that describes the contributions of biosynthetic and degradative mechanisms to r-protein abundance and proteome remodelling in conditions of nutrient stress.

DOI: https://doi.org/10.1038/s41586-020-2446-y
Elife. 2020 Jun 30. pii: e58281. [Epub ahead of print]9

Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes.

Yohei Ohashi, Shirley Tremel, Glenn Robert Masson, Lauren McGinney, Jerome Boulanger, Ksenia Rostislavleva, Christopher M Johnson, Izabella Niewczas, Jonathan Clark, Roger L Williams.

  The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

Keywords:  autophagy; biochemistry; chemical biology; endocytosis; human; membrane defects; molecular biophysics; phosphoinositide 3-kinase; phospholipids; sorting; structural biology

DOI:  https://doi.org/10.7554/eLife.58281
Dev Cell. 2020 Jun 26. pii: S1534-5807(20)30461-5. [Epub ahead of print]

Metabolic Communication and Healthy Aging: Where Should We Focus Our Energy?

Hannah J Smith, Arpit Sharma, William B Mair.

  Aging is associated with a loss of metabolic homeostasis and plasticity, which is causally linked to multiple age-onset pathologies. The majority of the interventions-genetic, dietary, and pharmacological-that have been found to slow aging and protect against age-related disease in various organisms do so by targeting central metabolic pathways. However, targeting metabolic pathways chronically and ubiquitously makes it difficult to define the downstream effects responsible for lifespan extension and often results in negative effects on growth and health, limiting therapeutic potential. Insight into how metabolic signals are relayed between tissues, cells, and organelles opens up new avenues to target metabolic regulators locally rather than globally for healthy aging. In this review, we discuss the pro-longevity effects of targeting metabolic pathways in specific tissues and how these interventions communicate with distal cells to modulate aging. These studies may be crucial in designing interventions that promote longevity without negative health consequences.

Keywords:  AMPK; NAD+; aging; extracellular vesicles; insulin signaling; mTOR; metabolism; microRNA; sirtuins; tissue-specificity

DOI:  https://doi.org/10.1016/j.devcel.2020.06.011
CNS Drugs. 2020 Jul 01.

Enhancing the Activity of Glucocerebrosidase as a Treatment for Parkinson Disease.

Elisa Menozzi, Anthony H V Schapira.

Mutations in the glucocerebrosidase (GBA1) gene are the most common genetic risk factor for Parkinson disease (PD). Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disorder Gaucher disease (GD), characterized by deficient activity of the glucocerebrosidase enzyme (GCase). Both individuals with GD type I and heterozygous carriers of pathogenic variants of GBA1 have an increased risk of developing PD, by approximately ten- to 20-fold compared to non-carriers. GCase activity is also reduced in PD patients without GBA1 mutations, suggesting that the GCase lysosomal pathway might be involved in PD pathogenesis. Available evidence indicates that GCase can affect α-synuclein pathology in different ways. Misfolded GCase proteins are retained in the endoplasmic reticulum, altering the lysosomal trafficking of the enzyme and disrupting protein trafficking. Also, deficient GCase leads to accumulation of substrates that in turn may bind α-synuclein and promote pathological formation of aggregates. Furthermore, α-synuclein itself can lower the enzymatic activity of GCase, indicating that a bidirectional interaction exists between GCase and α-synuclein. Targeted therapies aimed at enhancing GCase activity, augmenting the trafficking of misfolded GCase proteins by small molecule chaperones, or reducing substrate accumulation, have been tested in preclinical and clinical trials. This article reviews the molecular mechanisms linking GCase to α-synuclein and discusses the therapeutic drugs that by targeting the GCase pathway can influence PD progression.

DOI: https://doi.org/10.1007/s40263-020-00746-0
Int J Mol Sci. 2020 Jun 26. pii: E4564. [Epub ahead of print]21(12):

Neuropathophysiology, Genetic Profile, and Clinical Manifestation of Mucolipidosis IV-A Review and Case Series.

Aleksandra Jezela-Stanek, Elżbieta Ciara, Karolina M Stepien.

  Mucolipidosis type IV (MLIV) is an ultra-rare lysosomal storage disorder caused by biallelic mutations in MCOLN1 gene encoding the transient receptor potential channel mucolipin-1. So far, 35 pathogenic or likely pathogenic MLIV-related variants have been described. Clinical manifestations include severe intellectual disability, speech deficit, progressive visual impairment leading to blindness, and myopathy. The severity of the condition may vary, including less severe psychomotor delay and/or ocular findings. As no striking recognizable facial dysmorphism, skeletal anomalies, organomegaly, or lysosomal enzyme abnormalities in serum are common features of MLIV, the clinical diagnosis may be significantly improved because of characteristic ophthalmological anomalies. This review aims to outline the pathophysiology and genetic defects of this condition with a focus on the genotype-phenotype correlation amongst cases published in the literature. The authors will present their own clinical observations and long-term outcomes in adult MLIV cases.

Keywords:  MCOLN1; corneal clouding; gastrin; mucolipidosis type IV; myopathy; neurodegenerative

DOI:  https://doi.org/10.3390/ijms21124564
Mol Ther. 2020 Jun 19. pii: S1525-0016(20)30311-7. [Epub ahead of print]

Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice.

Hannah G Lahey, Chelsea J Webber, Diane Golebiowski, Cassandra M Izzo, Erin Horn, Toloo Taghian, Paola Rodriguez, Ana Rita Batista, Lauren E Ellis, Misako Hwang, Douglas R Martin, Heather Gray-Edwards, Miguel Sena-Esteves.

  The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme β-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

Keywords:  AAV9; GM2 gangliosidosis; Sandhoff disease; Tay-Sachs disease; gene therapy; intravenous delivery

DOI:  https://doi.org/10.1016/j.ymthe.2020.06.021
Nat Commun. 2020 Jul 03. 11(1): 3327

Engineering monocyte/macrophage-specific glucocerebrosidase expression in human hematopoietic stem cells using genome editing.

Samantha G Scharenberg, Edina Poletto, Katherine L Lucot, Pasqualina Colella, Adam Sheikali, Thomas J Montine, Matthew H Porteus, Natalia Gomez-Ospina.

Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebroside activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current therapies for Gaucher disease include life-long intravenous administration of recombinant glucocerebroside and orally-available glucosylceramide synthase inhibitors. An alternative approach is to engineer the patient's own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected cells, and constituting a potential one-time therapy for this disease. Here, we report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The targeted cells generate glucocerebroside-expressing macrophages and maintain long-term repopulation and multi-lineage differentiation potential with serial transplantation. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells. Furthermore, it constitutes an adaptable platform for other lysosomal enzyme deficiencies.

DOI: https://doi.org/10.1038/s41467-020-17148-x
Int J Mol Sci. 2020 Jun 24. pii: E4502. [Epub ahead of print]21(12):

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Anne Gläser, Franziska Hammerl, Markus H Gräler, Sina M Coldewey, Christin Völkner, Moritz J Frech, Fan Yang, Jiankai Luo, Eric Tönnies, Oliver von Bohlen Und Halbach, Nicola Brandt, Diana Heimes, Anna-Maria Neßlauer, Georg Christoph Korenke, Marta Owczarek-Lipska, John Neidhardt, Arndt Rolfs, Andreas Wree, Martin Witt, Anja Ursula Bräuer.

  Niemann-Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1-/- mice and evaluated specific effects of treatment with 2-hydroxypropyl-β-cyclodextrin (HPβCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1-/- mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1-/- mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

Keywords:  HPTLC; Niemann–Pick disease type C1; S1P; brain; fibroblasts; mass spectrometry; qRT-PCR; sphingolipids; sphingosine-1-phosphate receptors; white matter

DOI:  https://doi.org/10.3390/ijms21124502
Mol Ther. 2020 Jun 15. pii: S1525-0016(20)30301-4. [Epub ahead of print]

miR-143 Regulates Lysosomal Enzyme Transport across the Blood-Brain Barrier and Transforms CNS Treatment for Mucopolysaccharidosis Type I.

Yi Lin, Xiaohong Wang, Kevin P Rose, Mei Dai, Jingfen Han, Mei Xin, Dao Pan.

  During brain maturation, cation-independent mannose-6-phosphate receptor (CI-MPR), a key transporter for lysosomal hydrolases, decreases significantly on the blood-brain barrier (BBB). Such a phenomenon leads to poor brain penetration of therapeutic enzymes and subsequent failure in reversing neurological complications in patients with neuropathic lysosomal storage diseases (nLSDs), such as Hurler syndrome (severe form of mucopolysaccharidosis type I [MPS I]). In this study, we discover that upregulation of microRNA-143 (miR-143) contributes to the decline of CI-MPR on the BBB during development. Gain- and loss-of-function studies showed that miR-143 inhibits CI-MPR expression and its transport function in human endothelial cells in vitro. Genetic removal of miR-143 in MPS I mice enhances CI-MPR expression and improves enzyme transport across the BBB, leading to brain metabolic correction, pathology normalization, and correction of neurological functional deficits 5 months after peripheral protein delivery at clinically relevant levels that derived from erythroid/megakaryocytic cells via hematopoietic stem cell-mediated gene therapy, when otherwise no improvement was observed in MPS I mice at a parallel setting. These studies not only uncover a novel role of miR-143 as an important modulator for the developmental decline of CI-MPR on the BBB, but they also demonstrate the functional significance of depleting miR-143 for "rescuing" BBB-anchored CI-MPR on advancing CNS treatment for nLSDs.

Keywords:  blood-brain barrier; brain drug delivery; cation-independent mannose 6-phosphate receptor; enzyme replacement therapy; gene therapy; in vivo evaluation; lysosomal storage diseases; microRNA-143; mucopolysaccharidosis type I; neurological diseases

DOI:  https://doi.org/10.1016/j.ymthe.2020.06.011
Biochim Biophys Acta Mol Basis Dis. 2020 Jun 24. pii: S0925-4439(20)30231-3. [Epub ahead of print] 165883

Loss of CLN3, the gene mutated in juvenile neuronal ceroid lipofuscinosis, leads to metabolic impairment and autophagy induction in retina pigment epithelium.

Yu Zhong, Kabhilan Mohan, Jinpeng Liu, Ahmad Al-Attar, Penghui Lin, Robert M Flight, Qiushi Sun, Marc O Warmoes, Rahul R Deshpande, Huijuan Liu, Kyung Sik Jung, Mihail I Mitov, Nianwei Lin, D Allan Butterfield, Shuyan Lu, Jinze Liu, Hunter N B Moseley, Teresa W M Fan, Mark E Kleinman, Qing Jun Wang.

  Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: JNCL often first presents as vision loss; and vision loss has also been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J backgroun. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also report for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.

Keywords:  Autophagy; CLN3; Glycogen; Metabolism; Retinal pigment epithelium; Vision loss

DOI:  https://doi.org/10.1016/j.bbadis.2020.165883
Sci Rep. 2020 Jun 29. 10(1): 10591

A tailored Cln3Q352X mouse model for testing therapeutic interventions in CLN3 Batten disease.

Logan Langin, Tyler B Johnson, Attila D Kovács, David A Pearce, Jill M Weimer.

CLN3 Batten disease (CLN3 disease) is a pediatric lysosomal storage disorder that presents with progressive blindness, motor and cognitive decline, seizures, and premature death. CLN3 disease results from mutations in CLN3 with the most prevalent mutation, a 966 bp deletion spanning exons 7-8, affecting ~ 75% of patients. Mouse models with complete Cln3 deletion or Cln3Δex7/8 mutation have been invaluable for learning about both the basic biology of CLN3 and the underlying pathological changes associated with CLN3 disease. These models, however, vary in their disease presentation and are limited in their utility for studying the role of nonsense mediated decay, and as a consequence, in testing nonsense suppression therapies and read-through compounds. In order to develop a model containing a disease-causing nonsense point mutation, here we describe a first-of-its-kind Cln3Q352X mouse model containing a c.1054C > T (p.Gln352Ter) point mutation. Similar to previously characterized Cln3 mutant mouse lines, this novel model shows pathological deficits throughout the CNS including accumulation of lysosomal storage material and glial activation, and has limited perturbation in behavioral measures. Thus, at the molecular and cellular level, this mouse line provides a valuable tool for testing nonsense suppression therapies or read through compounds in CLN3 disease in the future.

DOI: https://doi.org/10.1038/s41598-020-67478-5
Epileptic Disord. 2020 Jun 01. 22(3): 317-322

ATP6V1B2-related epileptic encephalopathy.

Luciana Midori Inuzuka, Lúcia Inês Macedo-Souza, Bruno Della-Rippa, Fabiola Paoli Monteiro, Daniel de Souza Delgado, Luis Filipe Godoy, Luiza Ramos, Larissa Sampaio de Athayde Costa, Eliana Garzon, Fernando Kok.

  ATP6V1B2 encodes a subunit of the lysosomal transmembrane proton pump necessary for adequate functioning of several acid hydrolases. De novo monoallelic variants of this gene have been associated with two distinct phenotypes: Zimmermann-Laband syndrome 2 (ZLS2), an intellectual deficiency/multiple malformation syndrome, and dominant deafness onychodystrophy (DDOD), a multiple malformation syndrome without cognitive involvement. Epilepsy is not observed in DDOD, is variably present in ZLS2, but is a common feature in Zimmermann-Laband syndrome 1 (ZLS1) (caused by monoallelic pathogenic variants in KCNH1) and Zimmermann-Laband syndrome-like (ZLSL) (associated with KCNK4 variants). Herein, we report a case of an infant with severe epileptic encephalopathy with microcephaly and profound developmental delay, associated with a novel de novo loss-of-function variant in ATP6V1B2, diagnosed by whole-exome sequencing. This finding expands the spectrum of ATP6V1B2-associated disorders and adds ATP6V1B2 as a new member for the growing list of early-onset epileptic encephalopathy genes. [Published with video sequence].

Keywords:  ATP6V1B2; Zimmermann-Laband syndrome 1; Zimmermann-Laband syndrome 2; dominant deafness onychodystrophy; epilepsy; epileptic encephalopathy

DOI:  https://doi.org/10.1684/epd.2020.1166