bims-lysosi 2022-04-10 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022‒04‒10
twenty-six papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Mutation of SLC7A14 causes auditory neuropathy and retinitis pigmentosa mediated by lysosomal dysfunction.
Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair.
Mechanisms regulating the sorting of soluble lysosomal proteins.
Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells.
Niacin promotes the efflux of lysosomal cholesterol from macrophages via the CD38/NAADP signaling pathway.
Opposite physiological and pathological mTORC1-mediated roles of the CB1 receptor in regulating renal tubular function.
Targeting mTOR in the context of diet and whole-body metabolism.
mTOR Signaling in Kidney Diseases.
In Situ Analysis of mTORC1/C2 and Metabolism-Related Proteins in Pediatric Osteosarcoma.
Lysosome-Targeted Biosensor for the Super-Resolution Imaging of Lysosome-Mitochondrion Interaction.
Measurement of lysosomal enzyme activities: A technical standard of the American College of Medical Genetics and Genomics (ACMG).
Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation.
Enterococcus faecalis alters endo-lysosomal trafficking to replicate and persist within mammalian cells.
Rheb Promotes Triglyceride Secretion and Ameliorates Diet-Induced Steatosis in the Liver.
Blunting TRPML1 channels protects myocardial ischemia/reperfusion injury by restoring impaired cardiomyocyte autophagy.
Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling.
miRNAs in the regulation of mTOR signaling and host immune responses: the case of Leishmania infections.
The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.
S-adenosyl-L-homocysteine extends lifespan through methionine restriction effects.
EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma.
CB2R Activation Regulates TFEB-Mediated Autophagy and Affects Lipid Metabolism and Inflammation of Astrocytes in POCD.
Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation.
Coordinated regulation of TORC2 signaling by MCC/eisosome-associated proteins, Pil1 and tetraspan membrane proteins during the stress response.
Localized TPC1-mediated Ca2+ release from endolysosomes contributes to myoseptal junction development in zebrafish.
Rapamycin Promotes the Expansion of Myeloid Cells by Increasing G-CSF Expression in Mesenchymal Stem Cells.
A negative feedback loop of TOR signaling balances growth and stress-response trade-offs in plants.

Sci Adv. 2022 Apr 08. 8(14): eabk0942

Mutation of SLC7A14 causes auditory neuropathy and retinitis pigmentosa mediated by lysosomal dysfunction.

Kimberlee P Giffen, Yi Li, Huizhan Liu, Xiao-Chang Zhao, Chang-Jun Zhang, Ren-Juan Shen, Tianying Wang, Amanda Janesick, Bo-Bei Chen, Shu-Sheng Gong, Bechara Kachar, Zi-Bing Jin, David Z He.

Lysosomes contribute to cellular homeostasis via processes including macromolecule degradation, nutrient sensing, and autophagy. Defective proteins related to lysosomal macromolecule catabolism are known to cause a range of lysosomal storage diseases; however, it is unclear whether mutations in proteins involved in homeostatic nutrient sensing mechanisms cause syndromic sensory disease. Here, we show that SLC7A14, a transporter protein mediating lysosomal uptake of cationic amino acids, is evolutionarily conserved in vertebrate mechanosensory hair cells and highly expressed in lysosomes of mammalian cochlear inner hair cells (IHCs) and retinal photoreceptors. Autosomal recessive mutation of SLC7A14 caused loss of IHCs and photoreceptors, leading to presynaptic auditory neuropathy and retinitis pigmentosa in mice and humans. Loss-of-function mutation altered protein trafficking and increased basal autophagy, leading to progressive cell degeneration. This study implicates autophagy-lysosomal dysfunction in syndromic hearing and vision loss in mice and humans.

DOI: https://doi.org/10.1126/sciadv.abk0942
Nat Commun. 2022 Apr 06. 13(1): 1875

Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair.

Patrick Niekamp, Felix Scharte, Tolulope Sokoya, Laura Vittadello, Yeongho Kim, Yongqiang Deng, Elisabeth Südhoff, Angelika Hilderink, Mirco Imlau, Christopher J Clarke, Michael Hensel, Christopher G Burd, Joost C M Holthuis.

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

DOI: https://doi.org/10.1038/s41467-022-29481-4
Biosci Rep. 2022 Apr 08. pii: BSR20211856. [Epub ahead of print]

Mechanisms regulating the sorting of soluble lysosomal proteins.

İçten Meraş, Juliette Maes, Stephane Lefrancois.

  Lysosomes are key regulators of many fundamental cellular processes such as metabolism, autophagy, immune response, cell signalling and plasma membrane repair. These highly dynamic organelles are composed of various membrane and soluble proteins, which are essential for their proper functioning. The soluble proteins include numerous proteases, glycosidases and other hydrolases, along with activators, required for catabolism. The correct sorting of soluble lysosomal proteins is crucial to ensure the proper functioning of lysosomes, and is achieved through the coordinated effort of many sorting receptors, resident ER and Golgi proteins, and several cytosolic components. Mutations in a number of proteins involved in sorting soluble proteins to lysosomes result in human disease. These can range from rare diseases such as lysosome storage disorders, to more prevalent ones, such as Alzheimer's disease, Parkinson's disease and others, including rare neurodegenerative diseases that affect children. In this review, we discuss the mechanisms that regulate the sorting of soluble proteins to lysosomes, and highlight the effects of mutations in this pathway that cause human disease. More precisely, we will review the route taken by soluble lysosomal proteins from their translation into the ER, their maturation along the Golgi apparatus, and sorting at the trans-Golgi network. We will also highlight the effects of mutations in this pathway that cause human disease.

Keywords:  Lysosomes; endoplasmic reticulum; endosomes; intracellular transport; neurodegeneration; neuronal ceroid lipofuscinosis

DOI:  https://doi.org/10.1042/BSR20211856
Front Cell Dev Biol. 2022 ;10 874043

Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells.

Pawan Faris, Claudio Casali, Sharon Negri, Lara Iengo, Marco Biggiogera, Angela Serena Maione, Francesco Moccia.

  Nicotinic acid adenine dinucleotide phosphate (NAADP) is a newly discovered second messenger that gates two pore channels 1 (TPC1) and 2 (TPC2) to elicit endo-lysosomal (EL) Ca2+ release. NAADP-induced lysosomal Ca2+ release may be amplified by the endoplasmic reticulum (ER) through the Ca2+-induced Ca2+ release (CICR) mechanism. NAADP-induced intracellular Ca2+ signals were shown to modulate a growing number of functions in the cardiovascular system, but their occurrence and role in cardiac mesenchymal stromal cells (C-MSCs) is still unknown. Herein, we found that exogenous delivery of NAADP-AM induced a robust Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store with Gly-Phe β-naphthylamide, nigericin, and bafilomycin A1, and blocking TPC1 and TPC2, that are both expressed at protein level in C-MSCs. Furthermore, NAADP-induced EL Ca2+ release resulted in the Ca2+-dependent recruitment of ER-embedded InsP3Rs and SOCE activation. Transmission electron microscopy revealed clearly visible membrane contact sites between lysosome and ER membranes, which are predicted to provide the sub-cellular framework for lysosomal Ca2+ to recruit ER-embedded InsP3Rs through CICR. NAADP-induced EL Ca2+ mobilization via EL TPC was found to trigger the intracellular Ca2+ signals whereby Fetal Bovine Serum (FBS) induces C-MSC proliferation. Furthermore, NAADP-evoked Ca2+ release was required to mediate FBS-induced extracellular signal-regulated kinase (ERK), but not Akt, phosphorylation in C-MSCs. These finding support the notion that NAADP-induced TPC activation could be targeted to boost proliferation in C-MSCs and pave the way for future studies assessing whether aberrant NAADP signaling in C-MSCs could be involved in cardiac disorders.

Keywords:  cardiac mesenchymal stem cells; membrane contact sites; nicotinic acid adenine dinucleotide phosphate (NAADP); proliferation; store operated Ca2+ entry; two-pore channels (TPCs)

DOI:  https://doi.org/10.3389/fcell.2022.874043
Exp Biol Med (Maywood). 2022 Apr 02. 15353702221084632

Niacin promotes the efflux of lysosomal cholesterol from macrophages via the CD38/NAADP signaling pathway.

Shenghua Yang, Fan Zhang, Quanwen Li, Quanzhong Li.

  The accumulation of free cholesterol in macrophage lysosomes significantly enhances atherogenesis. Our recent study demonstrated that the cluster of differentiation 38 (CD38)/nicotinic acid adenine dinucleotide phosphate (NAADP)/Ca2+ signaling pathway plays a critical role in the efflux of lysosomal free cholesterol from macrophages in atherosclerosis. Niacin, known as nicotinic acid, is one of the oldest lipid-lowering medications showing unique anti-atherosclerotic activity. However, it is unknown whether this anti-atherosclerosis activity is associated with the efflux of lysosomal compartmentalized cholesterol in macrophages. In this study, we investigated the anti-atherosclerotic effects of niacin on the reduction of lysosomal free cholesterol via CD38/NAADP signaling in macrophages derived from low-density lipoprotein receptor (LDLr-/-) mice. Fluorescent filipin and Nile red labeling coupled with confocal microscopy demonstrated that niacin reduced free cholesterol accumulation in lysosomes in a concentration-dependent manner. Transmission electron microscopy also showed that niacin markedly decreased cholesterol crystal formation in lysosomes in oxidized LDL-containing LDLr-/- bone marrow-derived macrophages. Enzyme-linked immunosorbent assays showed that niacin increased NAADP production in a concentration-dependent manner, which was inhibited by small interfering RNA interference of CD38. Therefore, niacin may promote the efflux of lysosomal cholesterol from macrophages via the CD38/NAADP signaling pathway.

Keywords:  Niacin; atherosclerosis; cholesterol; cluster of differentiation 38/nicotinic acid adenine dinucleotide phosphate; low-density lipoprotein receptor −/− mice; lysosome

DOI:  https://doi.org/10.1177/15353702221084632
Nat Commun. 2022 Apr 04. 13(1): 1783

Opposite physiological and pathological mTORC1-mediated roles of the CB1 receptor in regulating renal tubular function.

Liad Hinden, Majdoleen Ahmad, Sharleen Hamad, Alina Nemirovski, Gergő Szanda, Sandra Glasmacher, Aviram Kogot-Levin, Rinat Abramovitch, Bernard Thorens, Jürg Gertsch, Gil Leibowitz, Joseph Tam.

Activation of the cannabinoid-1 receptor (CB1R) and the mammalian target of rapamycin complex 1 (mTORC1) in the renal proximal tubular cells (RPTCs) contributes to the development of diabetic kidney disease (DKD). However, the CB1R/mTORC1 signaling axis in the kidney has not been described yet. We show here that hyperglycemia-induced endocannabinoid/CB1R stimulation increased mTORC1 activity, enhancing the transcription of the facilitative glucose transporter 2 (GLUT2) and leading to the development of DKD in mice; this effect was ameliorated by specific RPTCs ablation of GLUT2. Conversely, CB1R maintained the normal activity of mTORC1 by preventing the cellular excess of amino acids during normoglycemia. Our findings highlight a novel molecular mechanism by which the activation of mTORC1 in RPTCs is tightly controlled by CB1R, either by enhancing the reabsorption of glucose and inducing kidney dysfunction in diabetes or by preventing amino acid uptake and maintaining normal kidney function in healthy conditions.

DOI: https://doi.org/10.1038/s41467-022-29124-8
Endocrinology. 2022 Apr 02. pii: bqac041. [Epub ahead of print]

Targeting mTOR in the context of diet and whole-body metabolism.

Nikos Koundouros, John Blenis.

  The mechanistic target of rapamycin (mTOR) signaling pathway is the central regulator of cell growth and proliferation by integrating growth factor and nutrient availability. Under healthy physiological conditions, this process is tightly coordinated and essential to maintain whole-body homeostasis. Not surprisingly, dysregulated mTOR signaling underpins several diseases with increasing incidence worldwide, including obesity, diabetes and cancer. Consequently, there is significant clinical interest in developing therapeutic strategies that effectively target this pathway. The transition of mTOR inhibitors from the bench to bedside, however, has largely been marked with challenges and shortcomings, such as the development of therapy resistance and adverse side effects in patients. In this review, we discuss the current status of first, second and third generation mTOR inhibitors as a cancer therapy in both pre-clinical and clinical settings, with a particular emphasis on the mechanisms of drug resistance. We focus especially on the emerging role of diet as an important environmental determinant of therapy response, and posit a conceptual framework that links nutrient availability and whole-body metabolic states such as obesity with many of the previously defined processes that drive resistance to mTOR-targeted therapies. Given the role of mTOR as a central integrator of cell metabolism and function, we propose that modulating nutrient inputs through dietary interventions may influence the signaling dynamics of this pathway and compensatory nodes. In doing so, new opportunities for exploiting diet/drug synergies are highlighted that may unlock the therapeutic potential of mTOR inhibitors as a cancer treatment.

Keywords:  diet; drug resistance; mTOR; metabolism

DOI:  https://doi.org/10.1210/endocr/bqac041
Kidney360. 2020 Nov 25. 1(11): 1319-1327

mTOR Signaling in Kidney Diseases.

Yuan Gui, Chunsun Dai.

  The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, is crucial in regulating cell growth, metabolism, proliferation, and survival. Under physiologic conditions, mTOR signaling maintains podocyte and tubular cell homeostasis. In AKI, activation of mTOR signaling in tubular cells and interstitial fibroblasts promotes renal regeneration and repair. However, constitutive activation of mTOR signaling in kidneys results in the initiation and progression of glomerular hypertrophy, interstitial fibrosis, polycystic kidney disease, and renal cell carcinoma. Here, we summarize the recent studies about mTOR signaling in renal physiology and injury, and discuss the possibility of its use as a therapeutic target for kidney diseases.

Keywords:  TOR serine-threonine kinases; acute kidney injury; acute kidney injury and ICU nephrology; glomerular hypertrophy; kidney fibrosis; mTOR; polycystic kidney disease; renal cell carcinoma

DOI:  https://doi.org/10.34067/KID.0003782020
Pathol Oncol Res. 2022 ;28 1610231

In Situ Analysis of mTORC1/C2 and Metabolism-Related Proteins in Pediatric Osteosarcoma.

Anna Mohás, Ildikó Krencz, Zsófia Váradi, Gabriella Arató, Luca Felkai, Dorottya Judit Kiss, Dorottya Moldvai, Anna Sebestyén, Monika Csóka.

  Activation of the mTOR pathway has been observed in osteosarcoma, however the inhibition of mammalian target of rapamycin (mTOR) complex 1 has had limited results in osteosarcoma treatment. Certain metabolic pathways can be altered by mTOR activation, which can affect survival. Our aim was to characterize the mTOR profile and certain metabolic alterations in pediatric osteosarcoma to determine the interactions between the mTOR pathway and metabolic pathways. We performed immunohistochemistry on 28 samples to analyze the expression of mTOR complexes such as phospho-mTOR (pmTOR), phosphorylated ribosomal S6 (pS6), and rapamycin-insensitive companion of mTOR (rictor). To characterize metabolic pathway markers, we investigated the expression of phosphofructokinase (PFK), lactate dehydrogenase-A (LDHA), β-F1-ATPase (ATPB), glucose-6-phosphate dehydrogenase (G6PDH), glutaminase (GLS), fatty acid synthetase (FASN), and carnitin-O-palmitoyltransferase-1 (CPT1A). In total, 61% of the cases showed low mTOR activity, but higher pmTOR expression was associated with poor histological response to chemotherapy and osteoblastic subtype. Rictor expression was higher in metastatic disease and older age at the time of diagnosis. Our findings suggest the importance of the Warburg-effect, pentose-phosphate pathway, glutamine demand, and fatty-acid beta oxidation in osteosarcoma cells. mTOR activation is linked to several metabolic pathways. We suggest performing a detailed investigation of the mTOR profile before considering mTORC1 inhibitor therapy. Our findings highlight that targeting certain metabolic pathways could be an alternative therapeutic approach.

Keywords:  mTOR; metabolic; metabolic adaptation; osteosarcoma; pathways; pediatric

DOI:  https://doi.org/10.3389/pore.2022.1610231
Front Pharmacol. 2022 ;13 865173

Lysosome-Targeted Biosensor for the Super-Resolution Imaging of Lysosome-Mitochondrion Interaction.

Han Wang, Guiqian Fang, Huimin Chen, Maomao Hu, Yajuan Cui, Boyang Wang, Yudong Su, Yu Liu, Bo Dong, Xintian Shao.

  Background: The interaction between lysosomes and mitochondria includes not only mitophagy but also mitochondrion-lysosome contact (MLC) that enables the two organelles to exchange materials and information. In our study, we synthesised a biosensor with fluorescence characteristics that can image lysosomes for structured illumination microscopy and, in turn, examined morphological changes in mitochondria and the phenomenon of MLC under pathological conditions. Methods: After designing and synthesising the biosensor, dubbed CNN, we performed an assay with a Cell Counting Kit-8 to detect CNN's toxicity in relation to H9C2 cardiomyocytes. We next analysed the co-localisation of CNN and the commercial lysosomal probe LTG in cells, qualitatively analysed the imaging characteristics of CNN in different cells (i.e. H9C2, HeLa and HepG2 cells) via structured illumination microscopy and observed how CNN entered cells at different temperatures and levels of endocytosis. Last, we treated the H9C2 cells with mannitol or glucose to observe the morphological changes of mitochondria and their positions relative to lysosomes. Results: After we endocytosed CNN, a lysosome-targeted biosensor with a wide, stable pH response range, into cells in an energy-dependent manner. SIM also revealed that conditions in high glucose induced stress in lysosomes and changed the morphology of mitochondria from elongated strips to round spheres. Conclusion: CNN is a new tool for tracking lysosomes in living cells, both physiologically and pathologically, and showcases new options for the design of similar biosensors.

Keywords:  lysosome; mitochondria; mitochondria-lysosome contact; nanoscopic; organelle; super-resolution imaging

DOI:  https://doi.org/10.3389/fphar.2022.865173
Genet Med. 2022 Apr;pii: S1098-3600(21)05472-1. [Epub ahead of print]24(4): 769-783

Measurement of lysosomal enzyme activities: A technical standard of the American College of Medical Genetics and Genomics (ACMG).

ACMG Laboratory Quality Assurance Committee. Electronic address: documents@acmg.net

  Assays that measure lysosomal enzyme activity are important tools for the screening and diagnosis of lysosomal storage disorders (LSDs). They are often ordered in combination with urine oligosaccharide and glycosaminoglycan analysis, additional biomarker assays, and/or DNA sequencing when an LSD is suspected. Enzyme testing in whole blood/leukocytes, serum/plasma, cultured fibroblasts, or dried blood spots demonstrating deficient enzyme activity remains a key component of LSD diagnosis and is often prompted by characteristic clinical findings, abnormal newborn screening, abnormal biochemical findings (eg, elevated glycosaminoglycans), or molecular results indicating pathogenic variants or variants of uncertain significance in a gene associated with an LSD. This document, which focuses on clinical enzyme testing for LSDs, provides a resource for laboratories to develop and implement clinical testing, to describe variables that can influence test performance and interpretation of results, and to delineate situations for which follow-up molecular testing is warranted.

Keywords:  Enzyme assays; Lysosomal enzymes; Lysosomal storage disorders; Mucopolysaccharidosis; Sphingolipidoses

DOI:  https://doi.org/10.1016/j.gim.2021.12.013
Mol Ther. 2022 Apr 05. pii: S1525-0016(22)00228-3. [Epub ahead of print]

Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation.

Rebecca C Ahrens-Nicklas, Luis Tecedor, Arron F Hall, Owen Kane, Richard J Chung, Elena Lysenko, Eric D Marsh, Colleen S Stein, Beverly L Davidson.

Although neurologic symptoms occur in two-thirds of lysosomal storage disorders (LSDs), for most we do not understand the mechanisms underlying brain dysfunction. A major unanswered question is if the pathogenic hallmark of LSDs, storage accumulation, induces functional defects directly or is a disease bystander. Also, for most LSDs we do not know the impact of loss-of-function in individual cell types. Understanding these critical questions are essential to therapy development. Here, we determined the impact of genetic rescue in distinct cell types on neural circuit dysfunction in CLN3 disease, the most common pediatric dementia and a paradigmatic neurodegenerative LSD. We restored Cln3 expression via AAV-mediated gene delivery and conditional genetic rescue in a CLN3 disease mouse model. Surprisingly, we found that low-level rescue of Cln3 expression in neurons alone normalized clinically-relevant electrophysiologic markers of network dysfunction, despite the presence of substantial residual histopathology, in contrast to restoring expression in astrocytes. Thus, loss of CLN3 function in neurons, not storage accumulation, underlies neurologic dysfunction in CLN3 disease, implying that storage clearance may be an inappropriate target for therapy development and an ineffectual biomarker.

DOI: https://doi.org/10.1016/j.ymthe.2022.03.025
PLoS Pathog. 2022 Apr 07. 18(4): e1010434

Enterococcus faecalis alters endo-lysosomal trafficking to replicate and persist within mammalian cells.

Ronni A G da Silva, Wei Hong Tay, Foo Kiong Ho, Frederick Reinhart Tanoto, Kelvin K L Chong, Pei Yi Choo, Alexander Ludwig, Kimberly A Kline.

Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using in vitro keratinocyte and macrophage infection models, we show that E. faecalis becomes internalized and a subpopulation of bacteria can survive and replicate intracellularly. E. faecalis are internalized into keratinocytes primarily via macropinocytosis into single membrane-bound compartments and can persist in late endosomes up to 24 h after infection in the absence of colocalization with the lysosomal protease Cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in heterotypic intracellular trafficking with partial or absent labelling of E. faecalis-containing compartments with Rab5 and Rab7, small GTPases required for the endosome-lysosome trafficking. In addition, E. faecalis infection results in marked reduction of Rab5 and Rab7 protein levels which may also contribute to attenuated Rab incorporation into E. faecalis-containing compartments. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.

DOI: https://doi.org/10.1371/journal.ppat.1010434
Front Cell Dev Biol. 2022 ;10 808140

Rheb Promotes Triglyceride Secretion and Ameliorates Diet-Induced Steatosis in the Liver.

Chongyangzi Du, Wanchun Yang, Zongyan Yu, Qiuyun Yuan, Dejiang Pang, Ping Tang, Wanxiang Jiang, Mina Chen, Bo Xiao.

  Hepatosteatosis, characterized by excessive accumulation of lipids in the liver, is a major health issue in modern society. Understanding how altered hepatic lipid metabolism/homeostasis causes hepatosteatosis helps to develop therapeutic interventions. Previous studies identify mitochondrial dysfunction as a contributor to hepatosteatosis. But, the molecular mechanisms of mitochondrial dysfunction leading to altered lipid metabolism remain incompletely understood. Our previous work shows that Rheb, a Ras-like small GTPase, not only activates mTORC1 but also promotes mitochondrial ATP production through pyruvate dehydrogenase (PDH). In this study, we further demonstrate that Rheb controls hepatic triglyceride secretion and reduces diet-induced lipid accumulation in a mouse liver. Genetic deletion of Rheb causes rapid and spontaneous steatosis in the liver, which is unexpected from the role of mTORC1 that enhances lipid synthesis, whereas Rheb transgene remarkably reduces diet-induced hepatosteatosis. Results suggest that the hepatosteatosis in Rheb KO is an outcome of impaired lipid secretion, which is linked to mitochondrial ATP production of hepatocytes. Our findings highlight an under-appreciated role of Rheb in the regulation of hepatic lipid secretion through mitochondrial energy production, with therapeutic implication.

Keywords:  ATP; Rheb; VLDL; hepatosteatosis; triglyceride

DOI:  https://doi.org/10.3389/fcell.2022.808140
Basic Res Cardiol. 2022 Apr 07. 117(1): 20

Blunting TRPML1 channels protects myocardial ischemia/reperfusion injury by restoring impaired cardiomyocyte autophagy.

Yanhong Xing, Zhongheng Sui, Yucheng Liu, Meng-Meng Wang, Xiangqing Wei, Qixia Lu, Xinyan Wang, Nan Liu, Chen Lu, Rong Chen, Mengmei Wu, Yuqing Wang, Yu-Hong Zhao, Feng Guo, Jun-Li Cao, Jiansong Qi, Wuyang Wang.

  Accumulating evidence suggests that autophagy dysfunction plays a critical role in myocardial ischemia/reperfusion (I/R) injury. However, the underling mechanism of malfunctional autophagy in the cardiomyocytes subjected to I/R has not been well defined. As a result, there is no effective therapeutic option by targeting autophagy to prevent myocardial I/R injury. Here, we used both an in vitro and an in vivo I/R model to monitor autophagic flux in the cardiomyocytes, by exposing neonatal rat ventricular myocytes to hypoxia/reoxygenation and by subjecting mice to I/R, respectively. We observed that the autophagic flux in the cardiomyocytes subjected to I/R was blocked in both in vitro and in vivo models. Down-regulating a lysosomal cationic channel, TRPML1, markedly restored the blocked myocardial autophagic flux induced by I/R, demonstrating that TRPML1 directly contributes to the blocked autophagic flux in the cardiomyocytes subjected to I/R. Mechanistically, TRPML1 is activated secondary to ROS elevation following ischemia/reperfusion, which in turn induces the release of lysosomal zinc into the cytosol and ultimately blocks the autophagic flux in cardiomyocytes, presumably by disrupting the fusion between autophagosomes and lysosomes. As a result, the inhibited myocardial autophagic flux induced by TRPML1 disrupted mitochondria turnover and resulted in mass accumulation of damaged mitochondria and further ROS release, which directly led to cardiomyocyte death. More importantly, pharmacological and genetic inhibition of TRPML1 channels greatly reduced infarct size and rescued heart function in mice subjected to I/R in vivo by restoring impaired myocardial autophagy. In summary, our study demonstrates that secondary to ROS elevation, activation of TRPML1 results in autophagy inhibition in the cardiomyocytes subjected to I/R, which directly leads to cardiomyocyte death by disrupting mitochondria turnover. Therefore, targeting TRPML1 represents a novel therapeutic strategy to protect against myocardial I/R injury.

Keywords:  Autophagy inhibition; Cardiomyocyte death; Ischemia/reperfusion injury; Mitochondria turnover

DOI:  https://doi.org/10.1007/s00395-022-00930-x
J Biol Chem. 2022 Mar 31. pii: S0021-9258(22)00328-3. [Epub ahead of print] 101888

Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling.

Adrienn Skopál, Tamás Kéki, Péter Á Tóth, Balázs Csóka, Balázs Koscsó, Zoltán H Német, Luca Antonioli, Andreas Ivessa, Francisco Ciruela, László Virág, György Haskó, Endre Kókai.

  Adenosine A2A receptor (A2AR)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A2AR-interacting partners have in macrophages. Here we aimed to identify A2AR interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A2AR as the "bait" and a macrophage expression library as the "prey". We found the lysosomal protease cathepsin D (CtsD) was a robust hit. The A2AR-CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPMΦ) cells. We also demonstrated that the A2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A2AR in macrophages. Conversely, we demonstrate A2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A2AR interacting partner, and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.

Keywords:  adenosine receptor; cathepsin D; inflammation; macrophage; protein interaction

DOI:  https://doi.org/10.1016/j.jbc.2022.101888
Acta Trop. 2022 Mar 31. pii: S0001-706X(22)00129-2. [Epub ahead of print] 106431

miRNAs in the regulation of mTOR signaling and host immune responses: the case of Leishmania infections.

Sajad Rashidi, Reza Mansouri, Mohammad Ali-Hassanzadeh, Esmaeel Ghani, Mohammadreza Karimazar, Antonio Muro, Paul Nguewa, Raúl Manzano-Román.

  Micro RNAs (miRNAs), as regulators of gene expression at the post-transcriptional level, can respond to/or interact with cell signaling and affect the pathogenesis of different diseases/infections. The interaction/crosstalk of miRNAs with various cellular signaling networks including mTOR (as a master regulator of signaling relevant to different cellular mechanisms) might lead to the initiation, progression or restriction of certain disease processes. There are numerous studies that have identified the crosstalk between regulatory miRNA expression and the mTOR pathway (or mTOR signaling regulated by miRNAs) in different diseases which has a dual function in pathogenesis. However, the corresponding information in parasitic infections remains scarce. miRNAs have been suggested as specific targets for therapeutic strategies in several disorders such as parasitic infections. Thus, the targeting of miRNAs (as the modulators/regulators of mTOR) by small molecules and RNA-based therapeutics and consequently managing and modulating mTOR signaling and the downstream/related cell signaling/pathways might shed some light on the design of new therapeutic strategies against parasitic diseases, including Leishmaniasis. Accordingly, the present study attempts to highlight the importance of the crosstalk between regulatory miRNAs and mTOR signaling, and to review the relevant insights into parasitic infections by focusing specifically on Leishmania.

Keywords:  Crosstalk; Leishmania; Parasites; mTOR signaling; miRNAs

DOI:  https://doi.org/10.1016/j.actatropica.2022.106431
PNAS Nexus. 2022 Mar;1(1): pgac013

The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.

Myungjun Ko, Monish R Makena, Paula Schiapparelli, Paola Suarez-Meade, Allatah X Mekile, Bachchu Lal, Hernando Lopez-Bertoni, Kristen L Kozielski, Jordan J Green, John Laterra, Alfredo Quiñones-Hinojosa, Rajini Rao.

  A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Keywords:  Na+/H+ exchanger; STAT3; endosomes; glioblastoma; receptor tyrosine kinase

DOI:  https://doi.org/10.1093/pnasnexus/pgac013
Aging Cell. 2022 Apr 07. e13604

S-adenosyl-L-homocysteine extends lifespan through methionine restriction effects.

Takafumi Ogawa, Koji Masumura, Yuki Kohara, Muneyoshi Kanai, Tomoyoshi Soga, Yoshikazu Ohya, T Keith Blackwell, Masaki Mizunuma.

  Methionine restriction (MetR) can extend lifespan and delay the onset of aging-associated pathologies in most model organisms. Previously, we showed that supplementation with the metabolite S-adenosyl-L-homocysteine (SAH) extends lifespan and activates the energy sensor AMP-activated protein kinase (AMPK) in the budding yeast Saccharomyces cerevisiae. However, the mechanism involved and whether SAH can extend metazoan lifespan have remained unknown. Here, we show that SAH supplementation reduces Met levels and recapitulates many physiological and molecular effects of MetR. In yeast, SAH supplementation leads to inhibition of the target of rapamycin complex 1 (TORC1) and activation of autophagy. Furthermore, in Caenorhabditis elegans SAH treatment extends lifespan by activating AMPK and providing benefits of MetR. Therefore, we propose that SAH can be used as an intervention to lower intracellular Met and confer benefits of MetR.

Keywords:   Caenorhabditis elegans ; Saccharomyces cerevisiae ; S-adenosyl-L-homocysteine (SAH); S-adenosyl-L-methionine (SAM); methionine restriction (MetR)

DOI:  https://doi.org/10.1111/acel.13604
Cell Death Discov. 2022 Apr 04. 8(1): 157

EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma.

Kai Voeltzke, Katerina Scharov, Cornelius Maximilian Funk, Alisa Kahler, Daniel Picard, Laura Hauffe, Martin F Orth, Marc Remke, Irene Esposito, Thomas Kirchner, Alexander Schramm, Barak Rotblat, Thomas G P Grünewald, Guido Reifenberger, Gabriel Leprivier.

Neuroblastoma (NB) accounts for 15% of cancer-related deaths in childhood despite considerable therapeutic improvements. While several risk factors, including MYCN amplification and alterations in RAS and p53 pathway genes, have been defined in NB, the clinical outcome is very variable and difficult to predict. Since genes of the mechanistic target of rapamycin (mTOR) pathway are upregulated in MYCN-amplified NB, we aimed to define the predictive value of the mTOR substrate-encoding gene eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) expression in NB patients. Using publicly available data sets, we found that EIF4EBP1 mRNA expression is positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. In addition, high EIF4EBP1 mRNA expression is associated with reduced overall and event-free survival in the entire group of NB patients in three cohorts, as well as in stage 4 and high-risk patients. This was confirmed by monitoring the clinical value of 4EBP1 protein expression, which revealed that high levels of 4EBP1 are significantly associated with prognostically unfavorable NB histology. Finally, functional analyses revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter in NB cells. Our data highlight that EIF4EBP1 is a direct transcriptional target of MYCN whose high expression is associated with poor prognosis in NB patients. Therefore, EIF4EBP1 may serve to better stratify patients with NB.

DOI: https://doi.org/10.1038/s41420-022-00963-0
Front Immunol. 2022 ;13 836494

CB2R Activation Regulates TFEB-Mediated Autophagy and Affects Lipid Metabolism and Inflammation of Astrocytes in POCD.

Lieliang Zhang, Xifeng Wang, Wen Yu, Jun Ying, Pu Fang, Qingcui Zheng, Xiaojin Feng, Jialing Hu, Fan Xiao, Shoulin Chen, Gen Wei, Yue Lin, Xing Liu, Danying Yang, Yang Fang, Guohai Xu, Fuzhou Hua.

  Evidence suggests that the accumulation of lipid drots (LDs) accelerates damage to mitochondria and increases the release of inflammatory factors. These have been implicated as a mechanism underlying neurodegenerative diseases or tumors and aging-related diseases such as postoperative cognitive dysfunction (POCD), nevertheless, accumulation of lipid droplets has not been extensively studied in the central nervous system (CNS). Here, we found that after surgery, there was activation of astrocytes and lipid accumulation in the hippocampus. However, cannabinoid receptor type II (CB2R) activation significantly reduced lipid accumulation in astrocytes and change the expression of genes related to lipid metabolism. CB2R reduces the release of the inflammatory factors interleukin-1 beta (IL-1β) and interleukin 6 (IL-6) in peripheral serum and simultaneously improves cognitive ability in mice with POCD. Further research on mechanisms indicates that CB2R activation promotes the nuclear entry of the bHLH-leucine zipper transcription factor, the transcription factor EB (TFEB), and which is a master transcription factor of the autophagy-lysosomal pathway, also reduces TFEB-S211 phosphorylation. When CB2R promotes TFEB into the nucleus, TFEB binds at two sites within promoter region of PGC1α, promoting PGC1α transcription and accelerating downstream lipid metabolism. The aforementioned process leads to autophagy activation and decreases cellular lipid content. This study uncovers a new mechanism allowing CB2R to regulate lipid metabolism and inflammation in POCD.

Keywords:  astrocytes; autophagy; cannabinoid type 2 receptor; inflammation; lipid accumulation; mitochondrial damage; postoperative cognitive dysfunction

DOI:  https://doi.org/10.3389/fimmu.2022.836494
Nat Commun. 2022 Apr 06. 13(1): 1874

Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation.

Aleksandra Levina, Kaelin D Fleming, John E Burke, Thomas A Leonard.

3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a 'master' kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer. We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3 binding drives switch-like activation of PDK1. These results imply that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3 or PI(3,4)P2.

DOI: https://doi.org/10.1038/s41467-022-29368-4
Mol Microbiol. 2022 Apr 05.

Coordinated regulation of TORC2 signaling by MCC/eisosome-associated proteins, Pil1 and tetraspan membrane proteins during the stress response.

Ken-Taro Sakata, Keisuke Hashii, Koushiro Yoshizawa, Yuhei O Tahara, Kaori Yae, Ryohei Tsuda, Naotaka Tanaka, Tatsuya Maeda, Makoto Miyata, Mitsuaki Tabuchi.

  MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC-invaginated structures. In addition, six-tetraspan membrane proteins (6-Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6-Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS-sensitive phenotype of pil1Δ 6-tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)-Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well-known downstream pathway of TORC2-Ypk1 signaling, did not rescue the SDS-sensitivity of pil1Δ 6-tspΔ cells. These results suggest that Pil1 and 6-Tsp cooperatively regulate TORC2 signaling during the stress response.

Keywords:  Saccharomyces cerevisiae; TORC2; eisosome; microdomain; plasma membrane; stress tolerance

DOI:  https://doi.org/10.1111/mmi.14903
J Cell Sci. 2022 Apr 08. pii: jcs.259564. [Epub ahead of print]

Localized TPC1-mediated Ca2+ release from endolysosomes contributes to myoseptal junction development in zebrafish.

Keira L Rice, Sarah E Webb, Andrew L Miller.

  In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), dynamic structures that connect the actin cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane protein complexes in the sarcolemma. Here, we report that the endolysosomal ion channel, two-pore channel type 1 (TPC1), generates highly localized, non-propagating Ca2+ transients that play a distinct and required role in the capture and attachment of superficial slow skeletal muscle cells (SMCs) at MJs. Use of antisense morpholinos or CRISPR/Cas9 gene editing to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes including SMCs detaching from or crossing the myosepta. We also report that TPC1-decorated endolysosomes are dynamically associated with MJs in a microtubule-dependent manner, and that attenuating tpcn1 expression or function disrupted endolysosomal trafficking and resulted in an abnormal distribution of β-dystroglycan (a key transmembrane component of the dystrophin-associated protein complex). Together, our data suggest that localized TPC1-generated Ca2+ signals facilitate essential endolysosomal trafficking and membrane contact events, which help form and maintain MJs following the onset of SMC contractile activity.

Keywords:  Ca2+ signaling; Endolysosomes; Myoseptal junctions; Skeletal slow muscle cells; Two-pore channel type 1; Zebrafish

DOI:  https://doi.org/10.1242/jcs.259564
Front Cell Dev Biol. 2022 ;10 779159

Rapamycin Promotes the Expansion of Myeloid Cells by Increasing G-CSF Expression in Mesenchymal Stem Cells.

Minghao Li, Yanjie Lan, Juan Gao, Shengnan Yuan, Shuaibing Hou, Tengxiao Guo, Fei Zhao, Yuxia Wang, Weiping Yuan, Xiaomin Wang.

  Rapamycin, also known as sirolimus, an inhibitor of mammalian target of rapamycin (mTOR), is a regulatory kinase responsible for multiple signal transduction pathways. Although rapamycin has been widely used in treating various hematologic diseases, the effects of rapamycin are still not fully understood. Here we found that both oral and intraperitoneal administration of rapamycin led to the expansion of myeloid lineage, while intraperitoneal administration of rapamycin impaired granulocyte differentiation in mice. Rapamycin induced bone marrow mesenchymal stem cells to produce more G-CSF in vitro and in vivo, and promoted the myeloid cells expansion. Our results thus demonstrated that intraperitoneal administration of rapamycin might promote the expansion of myeloid lineage while impair myeloid cell differentiation in vivo.

Keywords:  G-CSF; hematopoiesis; mTOR; myeloid cells; rapamycin

DOI:  https://doi.org/10.3389/fcell.2022.779159
Cell Rep. 2022 Apr 05. pii: S2211-1247(22)00383-7. [Epub ahead of print]39(1): 110631

A negative feedback loop of TOR signaling balances growth and stress-response trade-offs in plants.

Muhammed Jamsheer K, Sunita Jindal, Mohan Sharma, Prakhar Awasthi, Sreejath S, Manvi Sharma, Chanchal Thomas Mannully, Ashverya Laxmi.

  TOR kinase is a central coordinator of nutrient-dependent growth in eukaryotes. Maintaining optimal TOR signaling is critical for the normal development of organisms. In this study, we describe a negative feedback loop of TOR signaling helping in the adaptability of plants in changing environmental conditions. Using an interdisciplinary approach, we show that the plant-specific zinc finger protein FLZ8 acts as a regulator of TOR signaling in Arabidopsis. In sugar sufficiency, TOR-dependent and -independent histone modifications upregulate the expression of FLZ8. FLZ8 negatively regulates TOR signaling by promoting antagonistic SnRK1α1 signaling and bridging the interaction of SnRK1α1 with RAPTOR1B, a crucial accessory protein of TOR. This negative feedback loop moderates the TOR-growth signaling axis in the favorable condition and helps in the activation of stress signaling in unfavorable conditions, establishing its importance in the adaptability of plants.

Keywords:  CP: Metabolism; CP: Plants; FCS-like zinc finger; SNF1-related protein kinase 1; growth-stress response trade-offs; negative feedback loop; nutrient signaling; target of rapamycin

DOI:  https://doi.org/10.1016/j.celrep.2022.110631