bims-lysosi 2020-07-26 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2020–07–26
39 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Mitochondria-lysosome contacts regulate mitochondrial Ca2+ dynamics via lysosomal TRPML1.
N-myristoyltransferase-1 is necessary for lysosomal degradation and mTORC1 activation in cancer cells.
Lysosomes, Autophagy, and Hormesis in Cell Physiology, Pathology, and Age-Related Disease.
NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin.
Multifunctional TPP-PEG-biotin self-assembled nanoparticle drug delivery-based combination therapeutic approach for co-targeting of GRP78 and lysosome.
Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine.
Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury.
Enhanced lysosomal function is critical for paclitaxel resistance in cancer cells: reversed by artesunate.
A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca2+ during Autophagy Regulation.
Ramping up the autophagy-lysosome system to cope with osmotic stress.
The mechano-sensitive response of β1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein.
A lysosomal hydrolase receptor, CPBF2, is associated motility and invasion of the enteric protozoan parasite Entamoeba histolytica.
PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes.
The Lysosomal Diseases Testing Laboratory: A review of the past 47 years.
Quantifying lymphocyte vacuolization serves as a measure of CLN3 disease severity.
Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression.
Characterization of Fluid Biomarkers Reveals Lysosome Dysfunction and Neurodegeneration in Neuronopathic MPS II Patients.
3-Chloro-1, 2-Propanediol inhibits autophagic flux by impairment of lysosomal function in HepG2 cells.
Rare Etiology of Renal Failure in a 25-Year-Old Caucasian Man: Fabry Disease With a Novel Mutation of GLA Gene.
Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition.
Acidic Compartment Size, Positioning, and Function during Myogenesis and Their Modulation by the Wnt/Beta-Catenin Pathway.
Endosomal-Lysosomal Processing of Neurodegeneration-Associated Proteins in Astrocytes.
Distinct Roles for RAB10 and RAB29 in Pathogenic LRRK2-Mediated Endolysosomal Trafficking Alterations.
Endosomal Trafficking in Alzheimer's disease, Parkinson's Disease and Neuronal Ceroid Lipofuscinosis.
PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
Management of Hypersensitivity Reactions to Enzyme Replacement Therapy in Children with Lysosomal Storage Diseases.
Intracerebroventricular Cerliponase Alfa for Neuronal Ceroid Lipofuscinosis Type 2 Disease: Clinical Practice Considerations From US Clinics.
Cystinosis: Therapy adherence and metabolic monitoring in patients treated with immediate-release cysteamine.
Molecular logic of mTORC1 signalling as a metabolic rheostat.
Hepatic Peroxisomal β-Oxidation Suppresses Lipophagy via RPTOR Acetylation and MTOR Activation.
Metabolites Regulate Cell Signaling and Growth via Covalent Modification of Proteins.
TGF-β-activated kinase 1 (TAK1) mediates cadmium-induced autophagy in osteoblasts via the AMPK / mTORC1 / ULK1 pathway.
Nutrients men-TOR β-Cells to Adulthood.
p62: Friend or Foe? Evidences for OncoJanus and NeuroJanus Roles.
Characterization of mTOR Activity and Metabolic Profile in Pediatric Rhabdomyosarcoma.
The expression and significance of mTORC1 in diabetic retinopathy.
Discovery of new fluorescent thiazole-pyrazoline derivatives as autophagy inducers by inhibiting mTOR activity in A549 human lung cancer cells.
mTORC1 Signaling Controls TLR2-Mediated T-Cell Activation by Inducing TIRAP Expression.
Hematopoietic Stem Cell Metabolism during Development and Aging.

Proc Natl Acad Sci U S A. 2020 Jul 23. pii: 202003236. [Epub ahead of print]

Mitochondria-lysosome contacts regulate mitochondrial Ca2+ dynamics via lysosomal TRPML1.

Wesley Peng, Yvette C Wong, Dimitri Krainc.

  Mitochondria and lysosomes are critical for cellular homeostasis, and dysfunction of both organelles has been implicated in numerous diseases. Recently, interorganelle contacts between mitochondria and lysosomes were identified and found to regulate mitochondrial dynamics. However, whether mitochondria-lysosome contacts serve additional functions by facilitating the direct transfer of metabolites or ions between the two organelles has not been elucidated. Here, using high spatial and temporal resolution live-cell microscopy, we identified a role for mitochondria-lysosome contacts in regulating mitochondrial calcium dynamics through the lysosomal calcium efflux channel, transient receptor potential mucolipin 1 (TRPML1). Lysosomal calcium release by TRPML1 promotes calcium transfer to mitochondria, which was mediated by tethering of mitochondria-lysosome contact sites. Moreover, mitochondrial calcium uptake at mitochondria-lysosome contact sites was modulated by the outer and inner mitochondrial membrane channels, voltage-dependent anion channel 1 and the mitochondrial calcium uniporter, respectively. Since loss of TRPML1 function results in the lysosomal storage disorder mucolipidosis type IV (MLIV), we examined MLIV patient fibroblasts and found both altered mitochondria-lysosome contact dynamics and defective contact-dependent mitochondrial calcium uptake. Thus, our work highlights mitochondria-lysosome contacts as key contributors to interorganelle calcium dynamics and their potential role in the pathophysiology of disorders characterized by dysfunctional mitochondria or lysosomes.

Keywords:  TRPML1; calcium; lysosomal storage disorder; mitochondria–lysosome contacts; interorganelle membrane contact sites

DOI:  https://doi.org/10.1073/pnas.2003236117
Sci Rep. 2020 Jul 20. 10(1): 11952

N-myristoyltransferase-1 is necessary for lysosomal degradation and mTORC1 activation in cancer cells.

Yu-Chuan Chen, Marian S Navarrete, Ying Wang, Natalie C McClintock, Reiko Sakurai, Feng Wang, Kathryn T Chen, Tsui-Fen Chou, Virender K Rehan, Delphine J Lee, Begoña Diaz.

N-myristoyltransferase-1 (NMT1) catalyzes protein myristoylation, a lipid modification that is elevated in cancer cells. NMT1 sustains proliferation and/or survival of cancer cells through mechanisms that are not completely understood. We used genetic and pharmacological inhibition of NMT1 to further dissect the role of this enzyme in cancer, and found an unexpected essential role for NMT1 at promoting lysosomal metabolic functions. Lysosomes mediate enzymatic degradation of vesicle cargo, and also serve as functional platforms for mTORC1 activation. We show that NMT1 is required for both lysosomal functions in cancer cells. Inhibition of NMT1 impaired lysosomal degradation leading to autophagy flux blockade, and simultaneously caused the dissociation of mTOR from the surface of lysosomes leading to decreased mTORC1 activation. The regulation of lysosomal metabolic functions by NMT1 was largely mediated through the lysosomal adaptor LAMTOR1. Accordingly, genetic targeting of LAMTOR1 recapitulated most of the lysosomal defects of targeting NMT1, including defective lysosomal degradation. Pharmacological inhibition of NMT1 reduced tumor growth, and tumors from treated animals had increased apoptosis and displayed markers of lysosomal dysfunction. Our findings suggest that compounds targeting NMT1 may have therapeutic benefit in cancer by preventing mTORC1 activation and simultaneously blocking lysosomal degradation, leading to cancer cell death.

DOI: https://doi.org/10.1038/s41598-020-68615-w
Dose Response. 2020 Jul-Sep;18(3):18(3): 1559325820934227

Lysosomes, Autophagy, and Hormesis in Cell Physiology, Pathology, and Age-Related Disease.

Michael N Moore.

  Autophagy has been strongly linked with hormesis, however, it is only relatively recently that the mechanistic basis underlying this association has begun to emerge. Lysosomal autophagy is a group of processes that degrade proteins, protein aggregates, membranes, organelles, segregated regions of cytoplasm, and even parts of the nucleus in eukaryotic cells. These degradative processes are evolutionarily very ancient and provide a survival capability for cells that are stressed or injured. Autophagy and autophagic dysfunction have been linked with many aspects of cell physiology and pathology in disease processes; and there is now intense interest in identifying various therapeutic strategies involving its regulation. The main regulatory pathway for augmented autophagy is the mechanistic target of rapamycin (mTOR) cell signaling, although other pathways can be involved, such as 5'-adenosine monophosphate-activated protein kinase. Mechanistic target of rapamycin is a key player in the many highly interconnected intracellular signaling pathways and is responsible for the control of cell growth among other processes. Inhibition of mTOR (specifically dephosphorylation of mTOR complex 1) triggers augmented autophagy and the search is on the find inhibitors that can induce hormetic responses that may be suitable for treating many diseases, including many cancers, type 2 diabetes, and age-related neurodegenerative conditions.

Keywords:  AMPK; aging; autophagy; cancers; cell signaling; hormesis; lysosomes; mTOR; neurodegenerative diseases; therapeutics

DOI:  https://doi.org/10.1177/1559325820934227
Nat Metab. 2020 May;2(5): 387-396

NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin.

Rebecca George Tharyan, Andrea Annibal, Isabelle Schiffer, Raymond Laboy, Ilian Atanassov, Anna Luise Weber, Birgit Gerisch, Adam Antebi.

Mitochondria are multidimensional organelles whose activities are essential to cellular vitality and organismal longevity, yet underlying regulatory mechanisms spanning these different levels of organization remain elusive1-5. Here we show that Caenorhabditis elegans nuclear transcription factor Y, beta subunit (NFYB-1), a subunit of the NF-Y transcriptional complex6-8, is a crucial regulator of mitochondrial function. Identified in RNA interference (RNAi) screens, NFYB-1 loss leads to perturbed mitochondrial gene expression, reduced oxygen consumption, mitochondrial fragmentation, disruption of mitochondrial stress pathways, decreased mitochondrial cardiolipin levels and abolition of organismal longevity triggered by mitochondrial impairment. Multi-omics analysis reveals that NFYB-1 is a potent repressor of lysosomal prosaposin, a regulator of glycosphingolipid metabolism. Limiting prosaposin expression unexpectedly restores cardiolipin production, mitochondrial function and longevity in the nfyb-1 background. Similarly, cardiolipin supplementation rescues nfyb-1 phenotypes. These findings suggest that the NFYB-1-prosaposin axis coordinates lysosomal to mitochondria signalling via lipid pools to enhance cellular mitochondrial function and organismal health.

DOI: https://doi.org/10.1038/s42255-020-0200-2
J Nanobiotechnology. 2020 Jul 20. 18(1): 102

Multifunctional TPP-PEG-biotin self-assembled nanoparticle drug delivery-based combination therapeutic approach for co-targeting of GRP78 and lysosome.

Baskaran Purushothaman, Jeongmin Lee, Sera Hong, Joon Myong Song.

   BACKGROUND: In this study, a multifunctional tetraphenylporphyrin (TPP) conjugated polyethylene glycol with biotin (TPP-PEG-biotin) as a photo-dynamic therapy (PDT) material encapsulating a ruthenium complex 1 (Ru-1) was fabricated as self-assembled nanoparticle (Ru-1@TPP-PEG-biotin SAN) to co-target glucose-regulated protein 78 (GRP78) and the lysosome as a new anti-cancer therapeutic strategy.
RESULTS: The MTT assay results reveals the enhanced anticancer activity of the Ru-1@TPP-PEG-biotin SANs due to the co-targeting of the GRP78 and lysosome. The Ru-1@TPP-PEG-biotin reduced level of GRP78 and lysosomal ceramide that contributed to the stability of the lysosomal membrane. The endoplasmic reticulum (ER) stress concomitant with the inhibition of GRP78 was clearly monitored by the phosphorylation of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 α (IRE1α) kinases to indicate the activation of the unfolded protein response (UPR) signaling using immunofluorescence assay. On the other hand, the degradation of the lysosome was observed through PDT action by the Ru-1@TPP-PEG-biotin SAN treatment. This was confirmed by the co-localization assay showing the disappearance of cathepsin D and lysosomal-associated membrane protein 1 (LAMP1) in the lysosome.
CONCLUSIONS: Considering lysosome-mediated autophagy is an effective cancer cell survival mechanism, the degradation of the lysosome along with GRP78 inhibition by the Ru-1@TPP-PEG-biotin SAN combination therapy is suggested as a new co-targeting cancer treatment.

Keywords:  Autophagy; Co-targeting; Combination therapy; GRP78; TPP-PEG-biotin self-assembly

DOI:  https://doi.org/10.1186/s12951-020-00661-y
Proc Natl Acad Sci U S A. 2020 Jul 20. pii: 202010682. [Epub ahead of print]

Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine.

Michael N Trinh, Michael S Brown, Joseph L Goldstein, Jaeil Han, Gonçalo Vale, Jeffrey G McDonald, Joachim Seemann, Joshua T Mendell, Feiran Lu.

  Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid-PS-controls the movement of another lipid-cholesterol-between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.

Keywords:  CRISPR screen; PTDSS1; cholesterol; phosphatidylserine; plasma membrane

DOI:  https://doi.org/10.1073/pnas.2010682117
Circ Res. 2020 Jul 22.

Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury.

Shanshan Gu, Jiliang Tan, Qiang Li, Shenyan Liu, Jian Ma, Yanjun Zheng, Jinlong Liu, Wei Bi, Ping Sha, Xuxia Li, Meng Wei, Nan Cao, Huang-Tian Yang.

  Rationale: Impaired autophagic flux contributes to ischemia/reperfusion (I/R)-induced cardiomyocyte death, but the underlying molecular mechanisms remain largely unexplored. Objective: To determine the role of lysosomal-associated transmembrane protein 4B (LAPTM4B) in the regulation of autophagic flux and myocardial I/R injury. Methods and Results: LAPTM4B was expressed in murine hearts but downregulated in hearts with I/R (30 minutes/2 hours) injury and neonatal rat cardiomyocytes (NRCMs) with hypoxia/reoxygenation (6 hours/2 hours) injury. During myocardial reperfusion, LAPTM4B knockout (LAPTM4B-/-) mice had a significantly increased infarct size and lactate dehydrogenase release, while adenovirus-mediated LAPTM4B-overexpression was cardioprotective. Concomitantly, LAPTM4B-/- mice showed higher accumulation of the autophagy markers LC3-II, but not P62, in the I/R heart, while they did not alter chloroquine-induced further increases of LC3-II and P62 in both Sham and I/R hearts. Conversely, LAPTM4B-overexpression had opposite effects. The hypoxia/reoxygenation-reduced viability of NRCMs, ratio of autolysosomes/autophagosomes, and function of lysosomes were further decreased by LAPTM4B-knockdown but reversed by LAPTM4B-overexpression. Moreover, the LAPTM4B-overexpression-mediated benefits were abolished by knockdown of lysosome-associated membrane protein-2 (an autophagosome-lysosome fusion protein) in vitro and by the autophagy inhibitor bafilomycin A1 in vivo. In contrast, rapamycin successfully restored the impaired autophagic flux in LAPTM4B-/- mice and the subsequent myocardial I/R injury. Mechanistically, LAPTM4B regulated the activity of mammalian target of rapamycin complex 1 (mTORC1) via interacting with mTOR through its EC3 domain. Thus, mTORC1 was overactivated in LAPTM4B-/- mice, leading to the repression of transcription factor EB (TFEB), a master regulator of lysosomal and autophagic genes, during myocardial I/R. The mTORC1 inhibition or TFEB-overexpression rescued the LAPTM4B-/--induced impairment in autophagic flux and I/R injury, whereas TFEB-knockdown abolished the LAPTM4B-overexpression-mediated recovery of autophagic flux and cardioprotection. Conclusions: The downregulation of LAPTM4B contributes to myocardial I/R-induced impairment of autophagic flux via modulation of the mTORC1/TFEB pathway.

Keywords:  LAPTM4B; autophagic flux; autophagosome-lysosome fusion; mTORC1/TFEB pathway

DOI:  https://doi.org/10.1161/CIRCRESAHA.119.316388
Acta Pharmacol Sin. 2020 Jul 23.

Enhanced lysosomal function is critical for paclitaxel resistance in cancer cells: reversed by artesunate.

Zhe Li, Yu-Ting Zhu, Min Xiang, Jun-Lan Qiu, Shou-Qing Luo, Fang Lin.

  The mechanism underlying the resistance of cancer cells to chemotherapeutic drug varies with different cancer cells. Recent evidence shows that lysosomal function is associated with drug resistance of cancer cells. Artesunate, a derivative of artemisinin, displays broad antitumor activity and direct cytotoxicity on various tumor cells. Our previous study shows that artesunate increases autophagosome accumulation, while significantly decreases autolysosome number in cancer cells, suggesting that artesunate might impair the lysosomal function. In this study, we investigated the effects of artesunate on lysosomal function and its relationship with chemotherapeutic drug resistance in cancer cells. We found that the lysosomal function was significantly enhanced in two drug-resistant (A549/TAX and A549/DDP) cells. Furthermore, we showed that the enhanced lysosomal function by overexpression of transcription factor EB (TFEB) significantly increased MCF-7 cells resistance to doxorubicin (DOX), whereas the decreased lysosomal function by TFEB-knockdown or lysosome inhibitor chloroquine increased MCF-7 cells sensitivity to DOX. Treatment of A549/TAX cells with artesunate (2.5-50 μM) dose-dependently inhibited lysosomal function and the clearance of dysfunctional mitochondria, and induced cell apoptosis. Moreover, we demonstrated that artesunate exerted more potent inhibition on the resistant (A549/TAX and MCF-7/ADR) cells with higher activity of lysosomal function. Our results suggest that artesunate or other inhibitors of lysosomal function would be potential in the treatment of cancer cells with drug resistance caused by the enhanced lysosomal function.

Keywords:  TFEB; artesunate; cisplatin-resistant human lung adenocarcinoma cells (A549/DDP); doxorubicin-resistant human breast cancer cells (MCF-7/ADR); lysosome; paclitaxel-resistant human lung adenocarcinoma cells (A549/TAX)

DOI:  https://doi.org/10.1038/s41401-020-0445-z
iScience. 2020 Jul 04. pii: S2589-0042(20)30531-9. [Epub ahead of print]23(7): 101344

A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca2+ during Autophagy Regulation.

Zhonghui Zhang, Zhichao Liu, Yang Tian.

  pH and Ca2+ play important roles in regulating lysosomal activity and lysosome-mediated physiological and pathological processes. However, effective methods for simultaneous determination of pH and Ca2+ is the bottleneck. Herein, a single DNA-based FLIM reporter was developed for real-time imaging and simultaneous quantification of pH and Ca2+ in lysosomes with high affinity, in which a specific probe for recognition of Ca2+ was assembled onto a DNA nanostructure together with pH-responsive and lysosome-targeted molecules. The developed DNA reporter showed excellent biocompatibility and long-term stability up to ∼56 h in lysosomes. Using this powerful tool, it was discovered that pH was closely related to Ca2+ concentration in lysosome, whereas autophagy can be regulated by lysosomal pH and Ca2+. Furthermore, Aβ-induced neuronal death resulted from autophagy abnormal through lysosomal pH and Ca2+ changes. In addition, lysosomal pH and Ca2+ were found to regulate the transformation of NSCs, resulting in Rapamycin-induced antiaging.

Keywords:  Cellular Neuroscience; Optical Imaging; Technical Aspects of Cell Biology

DOI:  https://doi.org/10.1016/j.isci.2020.101344
Autophagy. 2020 Jul 20.

Ramping up the autophagy-lysosome system to cope with osmotic stress.

Tania López-Hernández, Tanja Maritzen, Volker Haucke.

  Osmotic stress is a critical challenge for mammalian cells as loss of water triggered by a hyperosmotic environment promotes harmful protein aggregation and impairs cell survival. How the degradative capacity of cells, in particular the macroautophagy/autophagy-lysosome system, is adapted to meet the proteolytic demands induced by osmotic challenge remains poorly understood. We have identified a hitherto unknown pathway that is activated by hyperosmotic stress and serves to link alterations in cellular ion homeostasis to the induction of autophagy and lysosomal gene expression and, thereby, to lysosome biogenesis.

Keywords:  CLEAR network; TFEB; endocytosis; ion honeostasis; lysosome; macroautophagy; osmotic stress; proteostasis

DOI:  https://doi.org/10.1080/15548627.2020.1798065
J Biol Chem. 2020 Jul 19. pii: jbc.RA120.013503. [Epub ahead of print]

The mechano-sensitive response of β1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein.

Marc R Block, Molly Brunner, Théo Ziegelmeyer, Dominique Lallemand, Mylène Pezet, Genevieve Chevalier, Philippe Rondé, Cecile Gauthier-Rouviere, Bernhard Wehrle-Haller, Daniel Bouvard.

  Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, non-receptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of a SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, β1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling, and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity.

Keywords:  LAMTOR complex; Late endosomes; Src; cell adhesion; cell signaling; extracellular matrix; mechanosensing; vesicular trafficking; yes-associated protein (YAP)

DOI:  https://doi.org/10.1074/jbc.RA120.013503
Mol Biochem Parasitol. 2020 Jul 21. pii: S0166-6851(20)30063-3. [Epub ahead of print] 111299

A lysosomal hydrolase receptor, CPBF2, is associated motility and invasion of the enteric protozoan parasite Entamoeba histolytica.

Kumiko Nakada-Tsukui, Konomi Marumo, Tomoyoshi Nozaki.

  Proper targeting and secretion of lysosomal hydrolases are regulated by transporting receptors. Entamoeba histolytica, the enteric protozoan parasite responsible for human amebiasis, has a unique family of lysosomal hydrolase receptors, cysteine protease binding protein family, CPBF. CPBFs, consisting of 11 members with conserved domain organization, bind to a wide range of cargos including cysteine proteases and glycosidases, which are also known to be involved in pathogenesis of this parasite. In this study, we characterized one of CPBFs, CPBF2, which is involved in cell motility and extracellular matrix invasion. Unexpectedly, these roles of CPBF were not related to its cargo, α-amylase. This is the first demonstration that a putative hydrolase receptor is involved in cell motility and invasion in parasitic protozoa.

Keywords:  Entamoeba histolytica; hydrolase receptor; invasion; lysosome; motility

DOI:  https://doi.org/10.1016/j.molbiopara.2020.111299
Autophagy. 2020 Jul 21.

PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes.

Hongjun Zhang, Jiao Zhou, Peng Xiao, Yong Lin, Xin Gong, Shiyan Liu, Qingjia Xu, Minjin Wang, Haiyan Ren, Mengji Lu, Yuan Wang, Jing Zhu, Zhiping Xie, Huihui Li, Kefeng Lu.

  Biogenesis of autophagosomes is the early step of macroautophagy/autophagy and requires membrane acquisition mainly from ER-Golgi-sourced precursor vesicles. Matured autophagosomes fuse with lysosomes for final degradation. However, how this selective fusion is determined remains elusive. Here, we identified Sac1 by a high throughput screen in Saccharomyces cerevisiae to show it was critical for autophagosome-lysosome fusion through its PtdIns4P phosphatase activity. Sac1 deficiency caused a dramatic increase of PtdIns4P at early Golgi apparatus and abnormal incorporation of PtdIns4P into Atg9 vesicles and autophagosomes, which caused failure to recruit SNARE proteins for autophagosome fusion with vacuoles. Sac1 function in autophagy was highly conserved from yeast to mammalian cells. Our work thus suggested that correct upstream lipid incorporation was important for downstream fusion step of autophagy and that Sac1 played a critical and ancient role in this surveillance of lipid integration.

Keywords:  Atg9; ER; Golgi; PtdIns3P; PtdIns4P; SNARE; Sac1; autophagosome-lysosome fusion; hydrolase

DOI:  https://doi.org/10.1080/15548627.2020.1796321
JIMD Rep. 2020 Jul;54(1): 61-67

The Lysosomal Diseases Testing Laboratory: A review of the past 47 years.

David A Wenger, Paola Luzi.

  Lysosomal disorders are diseases that involve mutations in genes responsible for the coding of lysosomal enzymes, transport proteins, activator proteins and protein processing enzymes. These defects lead to the storage of specific metabolites within lysosomes resulting in a great variety of clinical features depending on the tissues with the storage, the storage products and the extent of the storage. The methods for rapidly diagnosing patients started in the late 1960's when the enzyme defects were identified eliminating the need for tissue biopsies. The first requests for diagnostic help in this laboratory came in 1973. In that year, patients with Krabbe disease and Niemann-Pick type A were diagnosed. Since that time samples from about 62 000 individuals have been received for diagnostic studies, and 4900 diagnoses have been made. The largest number of diagnosed individuals had metachromatic leukodystrophy and Krabbe disease because of our research interest in leukodystrophies. A number of new disorders were identified and the primary defects in other disorders were clarified. With new methods for diagnosis, including newborn screening, molecular analysis, microarrays, there is still a need for biochemical confirmation before treatment is considered. With new treatments, including gene therapy, stem cell transplantation, enzyme replacement used alone or in combination becoming more available, the need for rapid, accurate diagnosis is critical.

Keywords:  GM1 gangliosidosis; Krabbe disease; genetic complementation; lysosomal diseases; metachromatic leukodystrophy; newborn screening; storage diseases

DOI:  https://doi.org/10.1002/jmd2.12117
JIMD Rep. 2020 Jul;54(1): 87-97

Quantifying lymphocyte vacuolization serves as a measure of CLN3 disease severity.

Willemijn F E Kuper, Marlies Oostendorp, Brigitte T A van den Broek, Karin van Veghel, Lourens J P Nonkes, Edward E S Nieuwenhuis, Sabine A Fuchs, Tineke Veenendaal, Judith Klumperman, Albert Huisman, Stefan Nierkens, Peter M van Hasselt.

   Background: The CLN3 disease spectrum ranges from a childhood-onset neurodegenerative disorder to a retina-only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well-known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity.
Methods: Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal-associated membrane protein 1 (LAMP-1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis.
Results: Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes (P = .0001). On immunofluorescence, classical CLN3 disease lymphocytes exhibited abundant vacuole-shaped LAMP-1 expression, suggesting the use of LAMP-1 as a proxy for lymphocyte vacuolization. Using flow cytometry in lymphocyte subsets, quantifying intracellular LAMP-1 expression additionally allowed to differentiate between infection and storage and to differentiate between CLN3 phenotypes even more in-depth revealing that intracellular LAMP-1 expression was most pronounced in T-cells of classical-protracted CLN3 disease while it was most pronounced in B-cells of "retina-only" CLN3 disease.
Conclusion: Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies.

Keywords:  CLN3 disease; ImageStream; flow cytometry; lymphocyte vacuolization; lysosomal membrane‐associated protein‐1 (LAMP‐1); neuronal ceroid lipofuscinosis (NCL)

DOI:  https://doi.org/10.1002/jmd2.12128
Cancers (Basel). 2020 Jul 22. pii: E2004. [Epub ahead of print]12(8):

Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression.

María Alejandra Parigiani, Anett Ketscher, Sylvia Timme, Peter Bronsert, Manuel Schlimpert, Bernd Kammerer, Arnaud Jacquel, Paul Chaintreuil, Thomas Reinheckel.

  Background: Cathepsin L (Ctsl) is a cysteine protease mainly located within the endosomal/lysosomal cell compartment. High expression of Ctsl indicates poor prognosis in human breast cancer. However, the cell type-specific Ctsl functions responsible for this association remain elusive. Methods: Because constitutive Ctsl-/- mice develop a complex phenotype, we developed a conditional model allowing for cell type-specific inactivation of Ctsl in mammary epithelium or myeloid cells in the transgenic mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) breast cancer model. Results: Ctsl ablation in mammary epithelial cells resulted in delayed initiation and end-stage of cancers. The latter displayed large dead cell areas. Inducible in vitro deletion of Ctsl in MMTV-PyMT-derived breast cancer cells revealed expansion of the acidic cell compartment, alteration of intracellular amino acid levels, and impaired mTOR signaling. In consequence, Ctsl-deficient cells exhibited slow growth rates and high apoptosis susceptibility. In contrast to Ctsl-deficient mammary epithelium, selective knockout of Ctsl in myeloid cells had no effects on primary tumors, but promoted lung metastasis formation. Conclusions: Our cell type-specific in vivo analysis provides strong evidence for a cancer cell-intrinsic, tumor-promoting role of Ctsl in primary breast cancer, whereas metastasis is negatively regulated by Ctsl expressed by bone marrow-derived cells.

Keywords:  breast cancer; genetically engineered mice; lysosome; proteolysis

DOI:  https://doi.org/10.3390/cancers12082004
Int J Mol Sci. 2020 Jul 22. pii: E5188. [Epub ahead of print]21(15):

Characterization of Fluid Biomarkers Reveals Lysosome Dysfunction and Neurodegeneration in Neuronopathic MPS II Patients.

Akhil Bhalla, Ritesh Ravi, Meng Fang, Annie Arguello, Sonnet S Davis, Chi-Lu Chiu, Jessica R Blumenfeld, Hoang N Nguyen, Timothy K Earr, Junhua Wang, Giuseppe Astarita, Yuda Zhu, Damian Fiore, Kimberly Scearce-Levie, Dolores Diaz, Heather Cahan, Matthew D Troyer, Jeffrey M Harris, Maria L Escolar.

  Mucopolysaccharidosis type II is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and characterized by the accumulation of the primary storage substrate, glycosaminoglycans (GAGs). Understanding central nervous system (CNS) pathophysiology in neuronopathic MPS II (nMPS II) has been hindered by the lack of CNS biomarkers. Characterization of fluid biomarkers has been largely focused on evaluating GAGs in cerebrospinal fluid (CSF) and the periphery; however, GAG levels alone do not accurately reflect the broad cellular dysfunction in the brains of MPS II patients. We utilized a preclinical mouse model of MPS II, treated with a brain penetrant form of IDS (ETV:IDS) to establish the relationship between markers of primary storage and downstream pathway biomarkers in the brain and CSF. We extended the characterization of pathway and neurodegeneration biomarkers to nMPS II patient samples. In addition to the accumulation of CSF GAGs, nMPS II patients show elevated levels of lysosomal lipids, neurofilament light chain, and other biomarkers of neuronal damage and degeneration. Furthermore, we find that these biomarkers of downstream pathology are tightly correlated with heparan sulfate. Exploration of the responsiveness of not only CSF GAGs but also pathway and disease-relevant biomarkers during drug development will be crucial for monitoring disease progression, and the development of effective therapies for nMPS II.

Keywords:  ETV:IDS; GM3; Hunter syndrome; biomarkers; cerebrospinal fluid; dermatan sulfate (DS); gangliosides; glycosaminoglycans (GAGs); heparan sulfate (HS); inflammation; lysosome dysfunction; mucopolysaccharidosis type II; neurodegeneration; neurofilament light chain (Nf-L)

DOI:  https://doi.org/10.3390/ijms21155188
Food Chem Toxicol. 2020 Jul 20. pii: S0278-6915(20)30465-8. [Epub ahead of print] 111575

3-Chloro-1, 2-Propanediol inhibits autophagic flux by impairment of lysosomal function in HepG2 cells.

Jing Lu, Jianing Lu, Yan Chen, Zhe Feng, Shuang Liu, Shuang Guan.

  3-chloro-1, 2-propanediol (3-MCPD) is a well-known contaminant that was produced in the thermal processing of food. Dietary intake represents the greatest source of exposure to 3-MCPD. Autophagy is an important catabolic pathway that plays an important role in liver physiological function. Evidence suggests that 3-MCPD exposure causes toxicity in liver, but the mechanism remains unknown. Here, we explored the effects of 3-MCPD on autophagic flux and traced the molecular mechanism in HepG2 cells. The data showed 3-MCPD exposure promoted the accumulation of autophagosomes in HepG2 cells. Subsequently, by detected te expression of LC3-II and P62 and transfection of mRFP-GFP-LC3 adenovirus, we found that the accumulation of autophagosomes was caused by inhibition of autophagic flux. After that, we investigate lysosomal function and found that 3-MCPD induced lysosomal alkalinization. Further, we detected the expression of TFEB, which is a key nuclear transcription factor in control of lysosome biogenesis and function. We found that 3-MCPD inhibited the nuclear expression of TFEB and mRNA levels of some target genes of TFEB. In order to further verify the role of TFEB in autophagic flux blockage in HepG2 cells induced by 3-MCPD, we overexpressed TFEB by transfection with adenovirus and found that both autophagy inhibition and lysosomal alkalization induced by 3-MCPD were alleviated. These results suggested that 3-MCPD could induce the autophagic flux blockage in HepG2 cells. The possible mechanism was due to the destruction of lysosomal function.

Keywords:  3-MCPD; Autophagic flux; Lysosome; TFEB

DOI:  https://doi.org/10.1016/j.fct.2020.111575
Cureus. 2020 Jul 11. 12(7): e9136

Rare Etiology of Renal Failure in a 25-Year-Old Caucasian Man: Fabry Disease With a Novel Mutation of GLA Gene.

Salem Gaballa, Avan AlJaf, Jane Lindsay, Kashyap Patel, Kyaw M Hlaing.

  Fabry disease (FD) is an X-linked recessive lysosomal storage disease caused by a mutation of the galactosidase alpha (GLA) gene, leading to deficiency of α-galactosidase A (alpha-Gal A). This deficiency results in a progressive, multiorgan accumulation of glycolipids, most notably globotriaosylceramide (Gb3), leading to multiorgan failure and subsequently premature death. Gb3 accumulation in the podocytes, epithelial, and mesangial cells of the glomeruli results in progressive renal disease and eventually renal failure and hemodialysis (HD). There are two types of FD: early-onset classical type 1 and late-onset type 2. Although nearly a thousand mutations of the GLA gene have been identified, the majority of them are of unknown significance. Herein we report the case of a 25-year-old Caucasian male with no significant medical history who presented with peripheral neuropathy and end-stage renal failure, requiring HD. He was diagnosed with FD based on the electron microscopy findings of renal biopsy and severely reduced alpha-Gal A activity (<0.4 nmol/mL/hour). A novel mutation of c.281G>T; p.Cys94Phe was identified. On discharge from our facility, he was referred to a renal transplant center and genetic counseling.

Keywords:  enzyme replacement therapy; fabry's disease; lysosomal storage disease; painful neuropathy; renal failure; α-galactosidase a activity

DOI:  https://doi.org/10.7759/cureus.9136
Sci Adv. 2020 Jun;6(25): eabb2210

Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition.

Anne Fassl, Christopher Brain, Monther Abu-Remaileh, Iga Stukan, Deborah Butter, Piotr Stepien, Avery S Feit, Johann Bergholz, Wojciech Michowski, Tobias Otto, Qing Sheng, Alice Loo, Walter Michael, Ralph Tiedt, Carmine DeAngelis, Rachel Schiff, Baishan Jiang, Bojana Jovanovic, Karolina Nowak, Maria Ericsson, Michael Cameron, Nathanael Gray, Deborah Dillon, Jean J Zhao, David M Sabatini, Rinath Jeselsohn, Myles Brown, Kornelia Polyak, Piotr Sicinski.

Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

DOI: https://doi.org/10.1126/sciadv.abb2210
Biomed Res Int. 2020 ;2020 6404230

Acidic Compartment Size, Positioning, and Function during Myogenesis and Their Modulation by the Wnt/Beta-Catenin Pathway.

Kayo M Bagri, Ivone A Rosa, Stephany Corrêa, Aline Yamashita, José Brito, Flavia Bloise, Manoel Luis Costa, Claudia Mermelstein.

Lysosomes and acidic compartments are involved in breaking down of macromolecules, membrane recycling, and regulation of signaling pathways. Here, we analyzed the role of acidic compartments during muscle differentiation and the involvement of the Wnt/beta-catenin pathway in lysosomal function during myogenesis. Acridine orange was used to localize and quantify acidic cellular compartments in primary cultures of embryonic muscle cells from Gallus gallus. Our results show an increase in acidic compartment size and area, as well as changes in their positioning during the initial steps of myogenesis. The inhibition of lysosomal function by either the chloroquine Lys05 or the downregulation of LAMP-2 with siRNA impaired chick myogenesis, by inhibiting myoblast fusion. Two activators of the Wnt/beta-catenin pathway, BIO and Wnt3a, were able to rescue the inhibitory effects of Lys05 in myogenesis. These results suggest a new role for the Wnt/beta-catenin pathway in the regulation of acidic compartment size, positioning, and function in muscle cells.

DOI: https://doi.org/10.1155/2020/6404230
Int J Mol Sci. 2020 Jul 21. pii: E5149. [Epub ahead of print]21(14):

Endosomal-Lysosomal Processing of Neurodegeneration-Associated Proteins in Astrocytes.

Ching-On Wong.

  Most common neurodegenerative diseases (NDs) are characterized by deposition of protein aggregates that are resulted from misfolding, dysregulated trafficking, and compromised proteolytic degradation. These proteins exert cellular toxicity to a broad range of brain cells and are found in both neurons and glia. Extracellular monomeric and oligomeric ND-associated proteins are taken up by astrocytes, the most abundant glial cell in the brain. Internalization, intracellular trafficking, processing, and disposal of these proteins are executed by the endosomal-lysosomal system of astrocytes. Endosomal-lysosomal organelles thus mediate the cellular impact and metabolic fate of these toxic protein species. Given the indispensable role of astrocytes in brain metabolic homeostasis, the endosomal-lysosomal processing of these proteins plays a fundamental role in altering the trajectory of neurodegeneration. This review aims at summarizing the mounting evidence that has established the essential role of astrocytic endosomal-lysosomal organelles in the processing of amyloid precursor proteins, Apolipoprotein E (ApoE), tau, alpha synuclein, and huntingtin, which are associated with NDs such as Alzheimer's, Parkinson's, and Huntington diseases.

Keywords:  APP; ApoE; alpha synuclein; amyloid beta; astrocytes; endosome; huntingtin; lysosome; neurodegenerative diseases; tau

DOI:  https://doi.org/10.3390/ijms21145149
Cells. 2020 Jul 17. pii: E1719. [Epub ahead of print]9(7):

Distinct Roles for RAB10 and RAB29 in Pathogenic LRRK2-Mediated Endolysosomal Trafficking Alterations.

Pilar Rivero-Ríos, Maria Romo-Lozano, Belén Fernández, Elena Fdez, Sabine Hilfiker.

  Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease, and sequence variations are associated with the sporadic form of the disease. LRRK2 phosphorylates a subset of RAB proteins implicated in secretory and recycling trafficking pathways, including RAB8A and RAB10. Another RAB protein, RAB29, has been reported to recruit LRRK2 to the Golgi, where it stimulates its kinase activity. Our previous studies revealed that G2019S LRRK2 expression or knockdown of RAB8A deregulate epidermal growth factor receptor (EGFR) trafficking, with a concomitant accumulation of the receptor in a RAB4-positive recycling compartment. Here, we show that the G2019S LRRK2-mediated EGFR deficits are mimicked by knockdown of RAB10 and rescued by expression of active RAB10. By contrast, RAB29 knockdown is without effect, but expression of RAB29 also rescues the pathogenic LRRK2-mediated trafficking deficits independently of Golgi integrity. Our data suggest that G2019S LRRK2 deregulates endolysosomal trafficking by impairing the function of RAB8A and RAB10, while RAB29 positively modulates non-Golgi-related trafficking events impaired by pathogenic LRRK2.

Keywords:  Golgi; LRRK2; Parkinson’s disease; RAB10; RAB29; endolysosome

DOI:  https://doi.org/10.3390/cells9071719
Mol Cell Biol. 2020 Jul 20. pii: MCB.00262-20. [Epub ahead of print]

Endosomal Trafficking in Alzheimer's disease, Parkinson's Disease and Neuronal Ceroid Lipofuscinosis.

Yasir H Qureshi, Penelope Baez, Christiane Reitz.

Neuronal Ceroid Lipofuscinosis (NCL) is one of the most prevalent neurodegenerative disorders of early-life, Parkinson's Disease (PD) the most common neurodegenerative disorder of mid-life, while Alzheimer's disease (AD) is the most common neurodegenerative disorders of late-life. While phenotypically distinct, recent studies suggest that they might share a common biological pathway-retromer-dependent endosomal trafficking. 'Retromer' is a multi-modular protein assembly critical for sorting and trafficking cargo out of the endosome. As a lysosomal storage disease, all 13 of NCL's causative genes affect endolysosomal function, and at least four have been directly linked to retromer. PD has several known causative genes, with one directly linked to retromer, and others causing endolysosomal dysfunction. AD has over twenty-five causative genes/risk factors with several of them linked to retromer or endosomal trafficking dysfunction. In this article, we summarize the emerging evidence on the association of genes causing NCL to retromer function and endosomal trafficking, review the recent evidence linking NCL genes to AD and discuss how NCL, AD and PD converge onto a shared molecular pathway. We will also discuss this pathway's role in microglia and neurons - cell populations critical to proper brain homeostasis, and dysfunction of which plays a key role in neurodegeneration.

DOI: https://doi.org/10.1128/MCB.00262-20
Nat Commun. 2020 Jul 20. 11(1): 3645

PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.

Yael Elbaz-Alon, Yuting Guo, Nadav Segev, Michal Harel, Daniel E Quinnell, Tamar Geiger, Ori Avinoam, Dong Li, Jodi Nunnari.

Endosomes are compositionally dynamic organelles that regulate signaling, nutrient status and organelle quality by specifying whether material entering the cells will be shuttled back to the cell surface or degraded by the lysosome. Recently, membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and endosomes have emerged as important players in endosomal protein sorting, dynamics and motility. Here, we show that PDZD8, a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain-containing ER transmembrane protein, utilizes distinct domains to interact with Rab7-GTP and the ER transmembrane protein Protrudin and together these components localize to an ER-late endosome MCS. At these ER-late endosome MCSs, mitochondria are also recruited to form a three-way contact. Thus, our data indicate that PDZD8 is a shared component of two distinct MCSs and suggest a role for SMP-mediated lipid transport in the regulation of endosome function.

DOI: https://doi.org/10.1038/s41467-020-17451-7
Ann Allergy Asthma Immunol. 2020 Jul 17. pii: S1081-1206(20)30487-7. [Epub ahead of print]

Management of Hypersensitivity Reactions to Enzyme Replacement Therapy in Children with Lysosomal Storage Diseases.

Irem Turgay Yagmur, Ozlem Unal Uzun, Aynur Kucukcongar Yavas, Ilknur Kulhas Celik, Muge Toyran, Mehmet Gunduz, Ersoy Civelek, Emine Dibek Misirlioglu.

   BACKGROUND: Intravenous recombinant enzyme replacement therapy (ERT) is currently available for eight lysosomal diseases. Hypersensitivity reactions (HSR) may be observed during this long-term treatment.
OBJECTIVE: The aim of our study was to evaluate the frequency, clinical-treatment features of ERT HSRs, management of desensitizations in children.
METHODS: Medical records were reviewed retrospectively for patients who received ERT. Those who had developed HSRs to ERT were included in the study. Demographic characteristics of the patients, culprit enzyme, signs-symptoms, diagnostic tests, management of the reaction, the protocol employed for the maintenance of ERT were recorded.
RESULTS: During the study period 54 patients received ERT in our institute. Eleven patients (20.4%) developed HSR to ERT. All reactions were immediate type. The most common symptoms were cutaneous manifestations. Nine patients developed urticaria, two patients had anaphylaxis as initial reaction. Patients who had isolated cutaneous symptoms continued their treatments with antihistamines, corticosteroids premedication and/or slower infusion rate. Patients who had recurrent urticaria with this modalities and/or those who had anaphylaxis continued their ERT with desensitization (n=8). Three patients required revisions in desensitization protocols because of recurren anaphylaxis.
CONCLUSION: The reactions that develop during this long-term treatment may be treated by premedication-prolonged infusion but in some patients, desensitization protocols is necessary for the continuation of therapy. Revisions in desensitization protocols may be required.

Keywords:  Desensitization; enzyme replacement therapy; gaucher; hypersensitivity; lysosomal disease; mucopolysaccharidosis; pompe; premedication; wolman

DOI:  https://doi.org/10.1016/j.anai.2020.07.010
Pediatr Neurol. 2020 May 04. pii: S0887-8994(20)30149-1. [Epub ahead of print]

Intracerebroventricular Cerliponase Alfa for Neuronal Ceroid Lipofuscinosis Type 2 Disease: Clinical Practice Considerations From US Clinics.

Emily de Los Reyes, Lenora Lehwald, Erika F Augustine, Elizabeth Berry-Kravis, Karen Butler, Natalie Cormier, Scott Demarest, Sam Lu, Jacqueline Madden, Joffre Olaya, Susan See, Amy Vierhile, James W Wheless, Amy Yang, Jessica Cohen-Pfeffer, Dorna Chu, Fernanda Leal-Pardinas, Raymond Y Wang.

   BACKGROUND: Neuronal ceroid lipofuscinosis type 2 or CLN2 disease is a rare, autosomal recessive, neurodegenerative lysosomal storage disorder caused by tripeptidyl peptidase 1 deficiency. Cerliponase alfa, a recombinant human tripeptidyl peptidase 1 enzyme, is the first and only approved treatment for CLN2 disease and the first approved enzyme replacement therapy administered via intracerebroventricular infusion.
METHODS: A meeting of health care professionals from US institutions with experience in cerliponase alfa treatment of children with CLN2 disease was held in November 2018. Key common practices were identified, and later refined during the drafting of this article, that facilitate safe chronic administration of cerliponase alfa.
RESULTS: Key practices include developing a multidisciplinary team of clinicians, pharmacists, and coordinators, and institution-specific processes. Infection risk may be reduced through strict aseptic techniques and minimizing connections and disconnections during infusion. The impact of intracerebroventricular device design on port needle stability during extended intracerebroventricular infusion is a critical consideration in device selection. Monitoring for central nervous system infection is performed at each patient contact, but with flexibility in the degree of monitoring. Although few institutions had experienced positive cerebrospinal fluid test results, the response to a positive cerebrospinal fluid culture should be determined on a case-by-case basis, and the intracerebroventricular device should be removed if cerebrospinal fluid infection is confirmed.
CONCLUSIONS: The key common practices and flexible practices used by institutions with cerliponase alfa experience may assist other institutions in process development. Continued sharing of experiences will be essential for developing standards and patient care guidelines.

Keywords:  Batten disease; CLN2 disease; Cerliponase alfa; Enzyme replacement therapy; Intracerebroventricular; Late infantile neuronal ceroid lipofuscinosis; Neuronal ceroid lipofuscinosis

DOI:  https://doi.org/10.1016/j.pediatrneurol.2020.04.018
Mol Genet Metab Rep. 2020 Sep;24 100620

Cystinosis: Therapy adherence and metabolic monitoring in patients treated with immediate-release cysteamine.

Simone Linden, Sabrina Klank, Erik Harms, Marianne Grüneberg, Julien H Park, Thorsten Marquardt.

   Background: Cystinosis is a metabolic disease caused by intracellular accumulation of cystine within lysosomes. Development of symptoms can be delayed significantly by a life-long therapy with cysteamine, a drug that enters the lysosome and reacts with cystine thereby enabling its export from the organelle.
Methods: During a period of 16 years, blood samples of 330 cystinosis patients were analyzed to investigate therapeutic adherence and metabolic control in patients treated with immediate-release cysteamine. The accepted therapeutic goal is to measure intracellular cystine levels in white blood cells every 3 months and to keep them below 0.5 nmol cystine/mg protein (= 1 nmol hemicystine/mg protein).
Results: 42% of measurements were within the desired 3-month interval, 38% were done every 3-5 months, 11% every 6-8 months, 5% every 9-12 months and 4% after a 12-month interval only. 64.4% of the measurements were higher than the therapeutic target value. Median cystine levels increased with longer control intervals.
Conclusions: The majority of the cystinosis patients showed insufficient metabolic adjustment. Intracellular cystine levels were not done as often as recommended and were not within therapeutic range. Poor therapy adherence is likely to be caused by gastrointestinal side effects of immediate-release cysteamine. Incorrect intervals between drug intake and blood sampling could contribute to the results.

Keywords:  Adherence; Cysteamine; Cystine level; Cystinosis; Metabolic monitoring

DOI:  https://doi.org/10.1016/j.ymgmr.2020.100620
Nat Metab. 2019 Mar;1(3): 321-333

Molecular logic of mTORC1 signalling as a metabolic rheostat.

Alexander J Valvezan, Brendan D Manning.

The protein kinase complex mechanistic target of rapamycin complex 1 (mTORC1) serves as a key conduit between growth signals and the metabolic processes underlying cell growth. The activation state of mTORC1 is controlled by intracellular nutrients and energy, as well as exogenous hormones and growth factors, thereby integrating local and systemic growth signals. Here we discuss the molecular logic of the mTORC1 signalling network and its importance in coupling growth signals to the control of cellular metabolism. After activation, mTORC1 promotes the conversion of available nutrients and energy into the major macromolecular species contributing to cellular mass, including proteins, nucleic acids and lipids, while suppressing the autophagic recycling of these macromolecules back into their nutrient constituents. Given that uncoupling of mTORC1 from its normal regulatory inputs contributes to many diseases-including cancer, genetic tumour syndromes, metabolic diseases, autoimmune diseases and neurological disorders-understanding the molecular logic of the mTORC1 network and how to modulate it may present therapeutic opportunities for treatment of a broad range of diseases and potentially even for the extension of lifespan.

DOI: https://doi.org/10.1038/s42255-019-0038-7
Autophagy. 2020 Jul 20.

Hepatic Peroxisomal β-Oxidation Suppresses Lipophagy via RPTOR Acetylation and MTOR Activation.

Anyuan He, John M Dean, Dongliang Lu, Yali Chen, Irfan J Lodhi.

  Hepatic lipid homeostasis is controlled by a coordinated regulation of various metabolic pathways involved in de novo synthesis, uptake, storage, and catabolism of lipids. Disruption of this balance could lead to hepatic steatosis. Peroxisomes play an essential role in lipid metabolism, yet their importance is often overlooked. In a recent study, we demonstrated a role for hepatic peroxisomal β-oxidation in autophagic degradation of lipid droplets. ACOX1 (acyl-Coenzyme A oxidase 1, palmitoyl), the rate-limiting enzyme of peroxisomal β-oxidation, increases with fasting or high-fat diet (HFD). Liver-specific acox1 knockout (acox1-LKO) protects mice from hepatic steatosis induced by starvation or HFD via induction of lipophagy. Mechanistically, we showed that hepatic ACOX1 deficiency decreases the total cytosolic acetyl-CoA levels, which leads to reduced acetylation of RPTOR/RAPTOR, a component of MTORC1, which is a key regulator of macroautophagy/autophagy. These results identify peroxisome-derived acetyl-CoA as a critical metabolic regulator of autophagy that controls hepatic lipid homeostasis.

Keywords:  ACOX1; MTORC1; NAFLD; autophagy; lipids; lipophagy; lysosome; peroxisome

DOI:  https://doi.org/10.1080/15548627.2020.1797288
Dev Cell. 2020 Jul 20. pii: S1534-5807(20)30544-X. [Epub ahead of print]54(2): 156-170

Metabolites Regulate Cell Signaling and Growth via Covalent Modification of Proteins.

Gianluca Figlia, Philipp Willnow, Aurelio A Teleman.

  Metabolites affect cell growth in two different ways. First, they serve as building blocks for biomass accumulation. Second, metabolites regulate the activity of growth-relevant signaling pathways. They do so in part by covalently attaching to proteins, thereby generating post-translational modifications (PTMs) that affect protein function, the focus of this Perspective. Recent advances in mass spectrometry have revealed a wide variety of such metabolites, including lipids, amino acids, Coenzyme-A, acetate, malonate, and lactate to name a few. An active area of research is to understand which modifications affect protein function and how they do so. In many cases, the cellular levels of these metabolites affect the stoichiometry of the corresponding PTMs, providing a direct link between cell metabolism and the control of cell signaling, transcription, and cell growth.

Keywords:  O-GlcNAc; YAP; acetylation; autophagy; crotonylation; glutathionylation; hippo; mTORC1; malonylation; methylation; palmitoylation

DOI:  https://doi.org/10.1016/j.devcel.2020.06.036
Toxicology. 2020 Jul 18. pii: S0300-483X(20)30177-3. [Epub ahead of print] 152538

TGF-β-activated kinase 1 (TAK1) mediates cadmium-induced autophagy in osteoblasts via the AMPK / mTORC1 / ULK1 pathway.

Di Ran, Yonggang Ma, Wei Liu, Tongwang Luo, Jiaming Zheng, Dedong Wang, Ruilong Song, Hongyan Zhao, Hui Zou, Jianhong Gu, Yan Yuan, Jianchun Bian, Zongping Liu.

  Cadmium (Cd) is one of worldwide environmental pollutants that causes bone homeostasis, which depends on the resorption of bones by osteoclasts and formation of bones by the osteoblasts (OB). However, the Cd toxicity on OB and its mechanism are unclear. Autophagy is an evolutionarily conserved degradation process in which domestic intracellular components are selectively digested for the recycling of nutrients and energy. This process is indispensable for cell homeostasis maintenance and stress responses. Dysregulation at the level of autophagic activity consequently disturbs the balance between bone formation and bone resorption and mediates the onset and progression of multiple bone diseases, including osteoporosis. TAK1 has been recently emerged as an activator of AMPK and hence an autophagy inducer. AMPK is a key molecule that induces autophagy and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by mTORC1. In this study, we found that Cd treatment caused the formation of autophagosomes, LC3-II lipidation and p62 downregulation, and the increased autophagic flux, indicating that Cd treatment induced autophagy in OBs. Cd treatment induced TAK1 activation mediated AMPK phosphorylation, which promoted autophagy via phosphorylation of ULK1 at S317. Meanwhile, Cd treatment dramatically decreased mTORC1, S6K1, 4E-BP1, S6, ULK1S555 and ULK1S757 phosphorylation, suggesting that mTORC1 activity was inhibited and inactive mTORC1 prevents ULK1 activation by phosphorylating ULK1 at SerS555 and Ser757. Our data strongly suggest that TAK1 mediates AMPK activation, which activates ULK1 by phosphorylating ULK1S317 and suppressing mTORC1-mediated ULK1S555 and ULK1S757 phosphorylation. Our study has revealed a signaling mechanism for TAK1 in Cd-induced autophagy in OBs.

Keywords:  AMPK/mTORC1/ULK1 pathway; Autophagy; Cadmium; Osteoblast; TAK1

DOI:  https://doi.org/10.1016/j.tox.2020.152538
Dev Cell. 2020 Jul 20. pii: S1534-5807(20)30507-4. [Epub ahead of print]54(2): 140-141

Nutrients men-TOR β-Cells to Adulthood.

Keiichi Katsumoto, Anne Grapin-Botton.

A major trigger of adult β-cell insulin secretion is glucose. In a recent issue of Cell Metabolism, Helman and colleagues show that in fetuses insulin secretion depends on the activation of mTOR by amino acids and that reducing amino acids promotes maturation of β-cells derived from pluripotent stem cells.

DOI: https://doi.org/10.1016/j.devcel.2020.06.028
Int J Mol Sci. 2020 Jul 16. pii: E5029. [Epub ahead of print]21(14):

p62: Friend or Foe? Evidences for OncoJanus and NeuroJanus Roles.

Sonia Emanuele, Marianna Lauricella, Antonella D'Anneo, Daniela Carlisi, Anna De Blasio, Diana Di Liberto, Michela Giuliano.

  p62 is a versatile protein involved in the delicate balance between cell death and survival, which is fundamental for cell fate decision in the context of both cancer and neurodegenerative diseases. As an autophagy adaptor, p62 recognizes polyubiquitin chains and interacts with LC3, thereby targeting the selected cargo to the autophagosome with consequent autophagic degradation. Beside this function, p62 behaves as an interactive hub in multiple signalling including those mediated by Nrf2, NF-κB, caspase-8, and mTORC1. The protein is thus crucial for the control of oxidative stress, inflammation and cell survival, apoptosis, and metabolic reprogramming, respectively. As a multifunctional protein, p62 falls into the category of those factors that can exert opposite roles in the cells. Chronic p62 accumulation was found in many types of tumors as well as in stress granules present in different forms of neurodegenerative diseases. However, the protein seems to have a Janus behaviour since it may also serve protective functions against tumorigenesis or neurodegeneration. This review describes the diversified roles of p62 through its multiple domains and interactors and specifically focuses on its oncoJanus and neuroJanus roles.

Keywords:  apoptosis; autophagy; cancer; neurodegenerative diseases; p62

DOI:  https://doi.org/10.3390/ijms21145029
Cancers (Basel). 2020 Jul 17. pii: E1947. [Epub ahead of print]12(7):

Characterization of mTOR Activity and Metabolic Profile in Pediatric Rhabdomyosarcoma.

Luca Felkai, Ildikó Krencz, Dorottya Judit Kiss, Noémi Nagy, Gábor Petővári, Titanilla Dankó, Tamás Micsík, András Khoor, Tamás Tornóczky, Zoltán Sápi, Anna Sebestyén, Monika Csóka.

  mTOR activation has been observed in rhabdomyosarcoma (RMS); however, mTOR complex (mTORC) 1 inhibition has had limited success thus far. mTOR activation alters the metabolic pathways, which is linked to survival and metastasis. These pathways have not been thoroughly analyzed in RMSs. We performed immunohistochemistry on 65 samples to analyze the expression of mTOR complexes (pmTOR, pS6, Rictor), and several metabolic enzymes (phosphofructokinase, lactate dehydrogenase-A, β-F1-ATPase, glucose-6-phosphate dehydrogenase, glutaminase). RICTOR amplification, as a potential mechanism of Rictor overexpression, was analyzed by FISH and digital droplet PCR. In total, 64% of the studied primary samples showed mTOR activity with an mTORC2 dominance (82%). Chemotherapy did not cause any relevant change in mTOR activity. Elevated mTOR activity was associated with a worse prognosis in relapsed cases. RICTOR amplification was not confirmed in any of the cases. Our findings suggest the importance of the Warburg effect and the pentose-phosphate pathway beside a glutamine demand in RMS cells. The expression pattern of the studied mTOR markers can explain the inefficacy of mTORC1 inhibitor therapy. Therefore, we suggest performing a detailed investigation of the mTOR profile before administering mTORC1 inhibitor therapy. Furthermore, our findings highlight that targeting the metabolic plasticity could be an alternative therapeutic approach.

Keywords:  mTOR; metabolism; pediatric; rhabdomyosarcoma

DOI:  https://doi.org/10.3390/cancers12071947
BMC Ophthalmol. 2020 Jul 20. 20(1): 297

The expression and significance of mTORC1 in diabetic retinopathy.

Yanli Liu, Yarong Zheng, Yekai Zhou, Yi Liu, Mengxuan Xie, Wenjing Meng, Meixia An.

   BACKGROUND: To investigate the expression and significance of mechanistic target of rapamycin complex 1(mTORC1) in diabetic retinopathy (DR), and to find new targets and new methods for the treatment of DR.
METHODS: A DR rat model was prepared by general feeding combined with intraperitoneal injection of 10% streptozotocin (60 mg/kg). The rats were randomly divided into a control group (NDM group) and a diabetes group (DM group). Three months later, the degrees of retinopathy was determined using hematoxylin and eosin staining, and the levels of p-S6, VEGF, and PEDF proteins were detected by immunohistochemistry and western blotting. Human retinal capillary endothelial cells (HRCECs) were cultured in high glucose (HG) conditions, then treated with rapamycin or transfected with siTSC1.The protein levels of p-S6 were assessed by western blotting. The 5-ethynyl-2'-deoxyuridine assay was used to detect cell proliferation, and the Transwell assay was used to detect cell migration.
RESULTS: A DM rat model was successfully developed. The expressions of p-S6 and VEGF proteins were significantly increased in the DM group (p < 0.05), and the expression of PEDF protein was significantly decreased compared with the NDM group (p < 0.05). In vitro, the p-S6 protein, as well as cell proliferation and migration, in HG induced HRCECs were increased (p < 0.05) compared with the control (normal glucose) group (p < 0.05). After transfection with siTSC1 to activate mTORC1, the expression of p-S6, as well as cell proliferation and migration, were increased. In contrast, rapamycin decreased p-S6 expression, as well as proliferation and migration, in HG induced HRCECs compared to the control group (p < 0.05).
CONCLUSION: mTORC1 plays an important role in DR. After activation, mTORC1 induced expression of the p-S6 protein, regulated the expressions of VEGF and PEDF proteins, and changed the proliferation and migration of endothelial cells. The mTORC1 can therefore be used as a new target,as well as in the treatment of DR.

Keywords:  Cell proliferation and migration; Diabetic retinopathy; P-S6; PEDF; VEGF; mTORC1

DOI:  https://doi.org/10.1186/s12886-020-01553-3
Cell Death Dis. 2020 Jul 20. 11(7): 551

Discovery of new fluorescent thiazole-pyrazoline derivatives as autophagy inducers by inhibiting mTOR activity in A549 human lung cancer cells.

ZhaoMin Lin, ZhaoYang Wang, XueWen Zhou, Ming Zhang, DongFang Gao, Lu Zhang, Peng Wang, Yuan Chen, YuXing Lin, BaoXiang Zhao, JunYing Miao, Feng Kong.

A series of fluorescent thiazole-pyrazoline derivatives was synthesized and their structures were characterized by 1H NMR, 13C NMR, and HRMS. Biological evaluation demonstrated that these compounds could effectively inhibit the growth of human non-small cell lung cancer (NSCLC) A549 cells in a dose- and time-dependent manner in vitro and inhibit tumor growth in vivo. The structure-activity relationship (SAR) of the compounds was analyzed. Further mechanism research revealed they could induce autophagy and cell cycle arrest while had no influence on cell necrosis. Compound 5e inhibited the activity of mTOR via FKBP12, which could be reversed by 3BDO, an mTOR activator and autophagy inhibitor. Compound 5e inhibited growth, promoted autophagy of A549 cells in vivo. Moreover, compound 5e showed good selectivity with no influence on normal vascular endothelial cell growth and the normal chick embryo chorioallantoic membrane (CAM) capillary formation. Therefore, our research provides potential lead compounds for the development of new anticancer drugs against human lung cancer.

DOI: https://doi.org/10.1038/s41419-020-02746-w
Cell Rep. 2020 Jul 21. pii: S2211-1247(20)30892-5. [Epub ahead of print]32(3): 107911

mTORC1 Signaling Controls TLR2-Mediated T-Cell Activation by Inducing TIRAP Expression.

Takayuki Imanishi, Midori Unno, Wakana Kobayashi, Natsumi Yoneda, Shizuo Akira, Takashi Saito.

Effector, but not naïve, T cells are activated by toll-like receptor-2 (TLR2) stimulation, leading to cytokine production and proliferation. We found that the differential response is attributable to the lack of expression of the adaptor protein TIRAP in naive T cells. TIRAP expression is induced upon T-cell receptor (TCR) stimulation and sustained by strong interleukin-2 (IL-2) signals. Expression of TIRAP requires TCR- and IL-2-induced mTORC1 activation. TLR2 stimulation induced the activation of nuclear factor κB (NF-κB) and ERK, leading to much higher production of interferon-γ (IFN-γ) by T helper 1 (Th1) cells cultured in a high concentration of IL-2 than by those cultured in a low concentration of IL-2. In contrast, TLR2 stimulation induces mTORC1 activation through TIRAP, which is essential for TLR2-mediated IFN-γ production. These data demonstrate that the mTORC1 signal confers the response to TLR2 signaling by inducing TIRAP expression and that the TIRAP-mTORC1 axis is critical for TLR2-mediated IFN-γ production by effector T cells.

DOI: https://doi.org/10.1016/j.celrep.2020.107911
Dev Cell. 2020 Jul 20. pii: S1534-5807(20)30537-2. [Epub ahead of print]54(2): 239-255

Hematopoietic Stem Cell Metabolism during Development and Aging.

Ayako Nakamura-Ishizu, Keisuke Ito, Toshio Suda.

Cellular metabolism in hematopoietic stem cells (HSCs) is an area of intense research interest, but the metabolic requirements of HSCs and their adaptations to their niches during development have remained largely unaddressed. Distinctive from other tissue stem cells, HSCs transition through multiple hematopoietic sites during development. This transition requires drastic metabolic shifts, insinuating the capacity of HSCs to meet the physiological demand of hematopoiesis. In this review, we highlight how mitochondrial metabolism determines HSC fate, and especially focus on the links between mitochondria, endoplasmic reticulum (ER), and lysosomes in HSC metabolism.

DOI: https://doi.org/10.1016/j.devcel.2020.06.029