bims-lysosi 2022-01-02 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022–01–02
thirty papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Phosphoproteomic responses of TORC1 target kinases reveal discrete and convergent mechanisms that orchestrate the quiescence program in yeast.
Alterations in Lysosome Homeostasis in Lipid-Related Disorders: Impact on Metabolic Tissues and Immune Cells.
A Highly Sensitive Fluorescent Akt Biosensor Reveals Lysosome-Selective Regulation of Lipid Second Messengers and Kinase Activity.
GPCR Signaling and mTORC1 Regulation.
Autophagy and lysosomal defects in cells expressing disease-associated tau.
Converging roles of PSENEN/PEN2 and CLN3 in the autophagy-lysosome system.
Simultaneous loss of TSC1 and DEPDC5 in skeletal and cardiac muscles produces early-onset myopathy and cardiac dysfunction associated with oxidative damage and SQSTM1/p62 accumulation.
Dissecting a neuron-to-liver crosstalk to modulate lipid metabolism in lisosomal storage diseases.
Behind every smile there's teeth: Cathepsin B's function in health and disease with a kidney view.
Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations.
Therapeutic Potential of Vital Transcription Factors in Alzheimer's and Parkinson's Disease With Particular Emphasis on Transcription Factor EB Mediated Autophagy.
The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.
Defective proteostasis in patient-derived iPSC-astrocytes and neurons carrying a MAPT IVS10+16 mutation.
Defining the role of PLD3 in Alzheimer's disease pathology.
Discovery of an Ultra-rapid and Sensitive Lysosomal Fluorescence Lipophagy Process.
Dorsal telencephalon-specific Nprl2- and Nprl3-knockout mice: novel mouse models for GATORopathy.
Plant TOR signaling network: Complexes, conservations and specificities.
Lysosome‑targeted drug combination induces multiple organelle dysfunctions and non‑canonical death in pancreatic cancer cells.
Lysosomal acid lipase promotes endothelial proliferation in cold-activated adipose tissue.
GSK-3β inhibition elicits a neuroprotection by restoring lysosomal dysfunction in neurons via facilitation of TFEB nuclear translocation after ischemic stroke.
CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells.
Conditional deletion of mTOR discloses its essential role in early B-cell development.
Glutamine promotes the generation of B10+ cells via the mTOR/GSK3 pathway.
Progression and metastasis of small cell lung carcinoma: the role of the PI3K/Akt/mTOR pathway and metabolic alterations.
Calcium sequestration by human platelet acidic organelles is regulated by the actin cytoskeleton and autocrine 5-hydroxytryptamine.
Demystifying Lysosomal α-l-Fucosidase in Liver Cancer-Bearing Mice by Specific Two-Photon Fluorescence Imaging.
Suppression of transient receptor potential melastatin 7 regulates pluripotency, proliferation, and differentiation of mouse embryonic stem cells via mechanistic target of rapamycin-extracellular signal-regulated kinase activation.
The Importance of Vacuolar Ion Homeostasis and Trafficking in Hyphal Development and Virulence in Candida albicans.
Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2.
AMPK-mTOR-mediated activation of autophagy promotes formation of dormant polyploid giant cancer cells.

Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01645-4. [Epub ahead of print]37(13): 110149

Phosphoproteomic responses of TORC1 target kinases reveal discrete and convergent mechanisms that orchestrate the quiescence program in yeast.

Ladislav Dokládal, Michael Stumpe, Zehan Hu, Malika Jaquenoud, Jörn Dengjel, Claudio De Virgilio.

  The eukaryotic TORC1 kinase assimilates diverse environmental cues, including growth factors and nutrients, to control growth by tuning anabolic and catabolic processes. In yeast, TORC1 stimulates protein synthesis in response to abundant nutrients primarily through its proximal effector kinase Sch9. Conversely, TORC1 inhibition following nutrient limitation unlocks various distally controlled kinases (e.g., Atg1, Gcn2, Npr1, Rim15, Slt2/Mpk1, and Yak1), which cooperate through poorly defined circuits to orchestrate the quiescence program. To better define the signaling landscape of the latter kinases, we use in vivo quantitative phosphoproteomics. Through pinpointing known and uncharted Npr1, Rim15, Slt2/Mpk1, and Yak1 effectors, our study examines the architecture of the distally controlled TORC1 kinase network. Accordingly, this is built on a combination of discrete, convergent, and multilayered feedback regulatory mechanisms, which likely ensure homeostatic control of and/or robust responses by TORC1 and its effector kinases under fluctuating nutritional conditions.

Keywords:  Atg9; Gis1; Npr1; Rim15; Slt2/Mpk1; TORC1; Yak1; autophagy; phosphoproteomics; quiescence program; target of rapamycin complex 1

DOI:  https://doi.org/10.1016/j.celrep.2021.110149
Front Cell Dev Biol. 2021 ;9 790568

Alterations in Lysosome Homeostasis in Lipid-Related Disorders: Impact on Metabolic Tissues and Immune Cells.

Fernanda Cabrera-Reyes, Claudia Parra-Ruiz, María Isabel Yuseff, Silvana Zanlungo.

  Lipid-related disorders, which primarily affect metabolic tissues, including adipose tissue and the liver are associated with alterations in lysosome homeostasis. Obesity is one of the more prevalent diseases, which results in energy imbalance within metabolic tissues and lysosome dysfunction. Less frequent diseases include Niemann-Pick type C (NPC) and Gaucher diseases, both of which are known as Lysosomal Storage Diseases (LSDs), where lysosomal dysfunction within metabolic tissues remains to be fully characterized. Adipocytes and hepatocytes share common pathways involved in the lysosome-autophagic axis, which are regulated by the function of cathepsins and CD36, an immuno-metabolic receptor and display alterations in lipid diseases, and thereby impacting metabolic functions. In addition to intrinsic defects observed in metabolic tissues, cells of the immune system, such as B cells can infiltrate adipose and liver tissues, during metabolic imbalance favoring inflammation. Moreover, B cells rely on lysosomes to promote the processing and presentation of extracellular antigens and thus could also present lysosome dysfunction, consequently affecting such functions. On the other hand, growing evidence suggests that cells accumulating lipids display defective inter-organelle membrane contact sites (MCSs) established by lysosomes and other compartments, which contribute to metabolic dysfunctions at the cellular level. Overall, in this review we will discuss recent findings addressing common mechanisms that are involved in lysosome dysregulation in adipocytes and hepatocytes during obesity, NPC, and Gaucher diseases. We will discuss whether these mechanisms may modulate the function of B cells and how inter-organelle contacts, emerging as relevant cellular mechanisms in the control of lipid homeostasis, have an impact on these diseases.

Keywords:  B cell activation and membrane contact sites (MCSs); CD36; cathepsins; gaucher disease (GD); lysosomal dysfunction; niemann-pick type C (NPC); obesity

DOI:  https://doi.org/10.3389/fcell.2021.790568
ACS Cent Sci. 2021 Dec 22. 7(12): 2009-2020

A Highly Sensitive Fluorescent Akt Biosensor Reveals Lysosome-Selective Regulation of Lipid Second Messengers and Kinase Activity.

Mingyuan Chen, Tengqian Sun, Yanghao Zhong, Xin Zhou, Jin Zhang.

The serine/threonine protein kinase Akt regulates a wide range of cellular functions via phosphorylation of various substrates distributed throughout the cell, including at the plasma membrane and endomembrane compartments. Disruption of compartmentalized Akt signaling underlies the pathology of many diseases such as cancer and diabetes. However, the specific spatial organization of Akt activity and the underlying regulatory mechanisms, particularly the mechanism controlling its activity at the lysosome, are not clearly understood. We developed a highly sensitive excitation-ratiometric Akt activity reporter (ExRai-AktAR2), enabling the capture of minute changes in Akt activity dynamics at subcellular compartments. In conjunction with super-resolution expansion microscopy, we found that growth factor stimulation leads to increased colocalization of Akt with lysosomes and accumulation of lysosomal Akt activity. We further showed that 3-phosphoinositides (3-PIs) accumulate on the lysosomal surface, in a manner dependent on dynamin-mediated endocytosis. Importantly, lysosomal 3-PIs are needed for growth-factor-induced activities of Akt and mechanistic target of rapamycin complex 1 (mTORC1) on the lysosomal surface, as targeted depletion of 3-PIs has detrimental effects. Thus, 3-PIs, a class of critical lipid second messengers that are typically found in the plasma membrane, unexpectedly accumulate on the lysosomal membrane in response to growth factor stimulation, to direct the multifaceted kinase Akt to organize lysosome-specific signaling.

DOI: https://doi.org/10.1021/acscentsci.1c00919
Mol Pharmacol. 2021 Dec 28. pii: MOLPHARM-MR-2021-000302. [Epub ahead of print]

GPCR Signaling and mTORC1 Regulation.

Chase H Melick, Tshering D Lama-Sherpa, Adna Curukovic, Jenna L Jewell.

  The mammalian target of rapamycin (mTOR) senses upstream stimuli to regulate numerous cellular functions such as metabolism, growth, and autophagy. The activation of mTOR complex 1 (mTORC1) is typically observed in human disease and continues to be an important therapeutic target. Understanding the upstream regulators of mTORC1 will provide a crucial link to targeting mTORC1 hyperactivated diseases. In this review, we will discuss the regulation of mTORC1 by upstream stimuli, with a specific focus on G-protein coupled receptor (GPCR) signaling to mTORC1. Significance Statement mTORC1 is a master regulator of many cellular processes and is often hyperactivated in human disease. Therefore, understanding the molecular underpinnings of these pathways will undoubtedly be promising to the mTORC1 field and human disease.

Keywords:  G protein coupled signaling; G proteins; Mammalian target of rapamycin (mTOR); Protein Kinase A (PKA); g protein-coupled receptors (GPCRS)

DOI:  https://doi.org/10.1124/molpharm.121.000302
Alzheimers Dement. 2021 Dec;17 Suppl 2 e058299

Autophagy and lysosomal defects in cells expressing disease-associated tau.

Saskia J Pollack, Dina Dakkak, Tong Guo, Patricia Gómez-Suaga, Wendy Noble, Diane P Hanger.

BACKGROUND: Intracellular accumulation of insoluble tau is an important hallmark of Alzheimer's disease (AD) and related tauopathies. We have previously identified in human tauopathy brain a truncated tau species (Tau35), comprising the C-terminal half with four microtubule-binding repeats. Minimal Tau35 expression in transgenic mice results in a progressive tauopathy phenotype including tau phosphorylation and aggregation, cognitive and behavioural abnormalities and impaired protein clearance. The autophagy-lysosomal pathway (ALP) plays a crucial role in the clearance of protein aggregates and defects in ALP are associated with the pathogenesis of AD. We sought to explore the effect of Tau35 expression on the ALP and whether autophagy is disrupted due to lysosomal dysfunction.
METHOD: Chinese hamster ovary (CHO) cells stably expressing Tau35 (CHO-Tau35) or full-length human 2N4R tau (CHO-FL) were generated. Primary cortical neurons from Tau35 transgenic and wild-type mice were cultured for 14 days in vitro(DIV) and brain homogenates were prepared from mice aged 4 and 12 months. The effect of Tau35 on the ALP was examined using immunofluorescence and western blots.
RESULT: Nuclear translocation of transcription factor EB (TFEB), a key mediator of lysosomal biogenesis, was significantly reduced in both CHO-FL and CHO-Tau35 cells. However, only CHO-Tau35 cells exhibited disrupted mammalian target of rapamycin complex 1 (mTORC1) activity and autophagic flux. Expression of ALP markers, including LC3-I/II, LAMP1, LAMP2 and cathepsin D, were also reduced in CHO-Tau35 cells.
CONCLUSION: Our findings suggest that N-terminally cleaved tau damages both lysosomal clearance of cellular proteins and lysosomal biogenesis. The Tau35-expressing cultured neurons will provide a useful tool to explore molecular mechanisms underlying tau-induced lysosomal dysfunction, which may lead to the identification of novel therapeutic targets for dementia.

DOI: https://doi.org/10.1002/alz.058299
Autophagy. 2021 Dec 29. 1-18

Converging roles of PSENEN/PEN2 and CLN3 in the autophagy-lysosome system.

Marcel Klein, Abuzar Kaleem, Sandra Oetjen, Daniela Wünkhaus, Lars Binkle, Sandra Schilling, Milena Gjorgjieva, Ralf Scholz, Doris Gruber-Schoffnegger, Stephan Storch, Stefan Kins, Gerard Drewes, Sabine Hoffmeister-Ullerich, Dietmar Kuhl, Guido Hermey.

  PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.

Keywords:  Alzheimer disease; cln3 disease; knockout cells; neuronal ceroid lipofuscinosis; transcription factor eb; γ-secretase

DOI:  https://doi.org/10.1080/15548627.2021.2016232
Autophagy. 2021 Dec 29. 1-20

Simultaneous loss of TSC1 and DEPDC5 in skeletal and cardiac muscles produces early-onset myopathy and cardiac dysfunction associated with oxidative damage and SQSTM1/p62 accumulation.

Chun-Seok Cho, Yongsung Kim, Sung-Rye Park, Boyoung Kim, Carol Davis, Irene Hwang, Susan V Brooks, Jun Hee Lee, Myungjin Kim.

  By promoting anabolism, MTORC1 is critical for muscle growth and maintenance. However, genetic MTORC1 upregulation promotes muscle aging and produces age-associated myopathy. Whether MTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effects of muscular MTORC1 hyperactivation, produced by simultaneous depletion of TSC1 and DEPDC5 (CKM-TD). CKM-TD mice produced myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac failure and bradypnea. These pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicated that genes mediating proteasomal and macroautophagic/autophagic pathways were highly upregulated in CKM-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. In CKM-TD muscle, autophagosome levels were increased, and the AMPK and ULK1 pathways were activated; in addition, autophagy induction was not completely blocked in CKM-TD myotubes. Despite the upregulation of autolysosomal markers, CKM-TD myofibers exhibited accumulation of autophagy substrates, such as SQSTM1/p62 and ubiquitinated proteins, suggesting that the autophagic activities were insufficient. Administration of a superoxide scavenger, tempol, normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. However, CKM-TD autophagy alterations were not normalized by rapamycin or tempol, suggesting that they may involve non-canonical targets other than MTORC1. These results collectively indicate that the concomitant muscle deficiency of TSC1 and DEPDC5 can produce early-onset myopathy through accumulation of oxidative stress, which dysregulates myocellular homeostasis.Abbreviations: AMPK: AMP-activated protein kinase; CKM: creatine kinase, M-type; COX: cytochrome oxidase; DEPDC5: DEP domain containing 5, GATOR1 subcomplex subunit; DHE: dihydroethidium; EDL: extensor digitorum longus; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase-activating protein; GTN: gastrocnemius; MTORC1: mechanistic target of rapamycin kinase complex 1; PLA: plantaris; QUAD: quadriceps; RPS6KB/S6K: ribosomal protein S6 kinase beta; SDH: succinate dehydrogenase; SOL: soleus; SQSTM1: sequestosome 1; TA: tibialis anterior; TSC1: TSC complex subunit 1; ULK1: unc-51 like autophagy activating kinase 1.

Keywords:  MTORC1; ULK1; myopathy; oxidative stress; tempol

DOI:  https://doi.org/10.1080/15548627.2021.2016255
Alzheimers Dement. 2021 Dec;17 Suppl 2 e058600

Dissecting a neuron-to-liver crosstalk to modulate lipid metabolism in lisosomal storage diseases.

Marina Garcia-Macia, Juan Pedro Bolaños.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL), known as Batten disease, are the most common of the rare neurodegenerative disorders in children. To date, defects in thirteen different genes have been identified in NCL patients. Despite the genetic heterogeneity, Batten diseases are grouped together based on clinical similarities and broadly uniform neuropathological features, including accumulation of lipofuscin in lysosomes, as well as profound neurodegeneration and widespread gliosis. Amongst these, the incidence of Cln7 disease, caused by mutation in MFSD8 gene, is the highest in southern and Mediterranean Europe. CLN7/MFSD8 encodes a lysosomal membrane glycoprotein with unknown function. Lysosomes are the only organelles able to hydrolyse triacylglycerols, which fuels the mitochondria for energy generation. The autophagic machinery provides TGs to the lysosomes through a process known as lipophagy. Although defective autophagy has been related with Cln7 disease, Cln7 role in lipid metabolism is unknown, particularly in lipophagy. Here, we hypothesized that disruption of lipophagy links neuronal death and Cln7-mediated Batten disease.
METHOD: To address this, we have studied lipophagy in Cln7 knockout (Cln7-KO) mice and investigated whether Cln7 loss in the hypothalamus disrupts liver lipophagy. We have obtained experimental data from different techniques in Cln7-KO mice fibroblasts, liver, brown fat and brain, to stablish a connection between the brain and the metabolism of the peripheral tissues.
RESULT: Thus, our data show that Cln7 deficiency in the hypothalamus damages liver lipophagy resulting in fat accumulation. Ongoing work is being developed to validate these observations using robust metabolic and in vivo uncoupling approaches.
CONCLUSION: Cln7 deficiency causes neuronal damages which seem to produce lipophagy impairment in the periphery tissues.

DOI: https://doi.org/10.1002/alz.058600
Biochim Biophys Acta Mol Cell Res. 2021 Dec 27. pii: S0167-4889(21)00244-5. [Epub ahead of print] 119190

Behind every smile there's teeth: Cathepsin B's function in health and disease with a kidney view.

Makhabbat Saudenova, Jessica Promnitz, Gerrit Ohrenschall, Nina Himmerkus, Martina Böttner, Madlen Kunke, Markus Bleich, Franziska Theilig.

Cathepsin B (CatB) is a very abundant lysosomal protease with endo- and carboxydipeptidase activities and even ligase features. In this review, we will provide a general characterization of CatB and describe structure, structure-derived properties and location-dependent proteolytic actions. We depict CatB action within lysosome and its important roles in lysosomal biogenesis, lysosomal homeostasis and autophagy rendering this protease a key player in orchestrating lysosomal functions. Lysosomal leakage and subsequent escape of CatB into the cytosol lead to harmful actions, e.g. the role in activating the NLPR3 inflammasome, affecting immune responses and cell death. The second focus of this review addresses CatB functions in the kidney, i.e. the glomerulus, the proximal tubule and collecting duct with strong emphasis of its role in pathology of the respective segment. Finally, observations regarding CatB functions that need to be considered in cell culture will be discussed. In conclusion, CatB a physiologically important molecule may, upon aberrant expression in different cellular context, become a harmful player effectively showing its teeth behind its smile.

DOI: https://doi.org/10.1016/j.bbamcr.2021.119190
Methods Mol Biol. 2022 ;2445 27-38

Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations.

Merve Kacal, Helin Vakifahmetoglu-Norberg.

  Accurate isolation of functional and intact lysosomes enables the quantification and analyses of abundances, dynamic changes and enrichment levels of lysosomal content, allowing specific lysosomal investigations induced by autophagy. In this protocol chapter, we describe detailed practical instructions and advices for an efficacious lysosomal enrichment and isolation procedure by differential multilayered density gradient centrifugations using human cancer cell lines. By this method, intact and autophagy competent lysosomes can be isolated from cancer cells based on their distinct density and obtained fractions can further be analyzed for functional lysosomal assays, as well as for protein or metabolic loads to identify select spatiotemporal changes by comparative quantitative measurement. This method has been used to enrich lysosomes from a variety of cancer cells with activated chaperone-mediated autophagy, but can be optimized for other cell lines and tissues for multiple autophagy-induced conditions.

Keywords:  Autophagy; Cancer; Chaperone-mediated autophagy; LAMP-2A; Lysosomes

DOI:  https://doi.org/10.1007/978-1-0716-2071-7_2
Front Neurosci. 2021 ;15 777347

Therapeutic Potential of Vital Transcription Factors in Alzheimer's and Parkinson's Disease With Particular Emphasis on Transcription Factor EB Mediated Autophagy.

Sachchida Nand Rai, Neeraj Tiwari, Payal Singh, Divya Mishra, Anurag Kumar Singh, Etrat Hooshmandi, Emanuel Vamanu, Mohan P Singh.

  Autophagy is an important cellular self-digestion and recycling pathway that helps in maintaining cellular homeostasis. Dysregulation at various steps of the autophagic and endolysosomal pathway has been reported in several neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington disease (HD) and is cited as a critically important feature for central nervous system (CNS) proteostasis. Recently, another molecular target, namely transcription factor EB (TFEB) has been explored globally to treat neurodegenerative disorders. This TFEB, is a key regulator of autophagy and lysosomal biogenesis pathway. Multiple research studies suggested therapeutic potential by targeting TFEB to treat human diseases involving autophagy-lysosomal dysfunction, especially neurodegenerative disorders. A common observation involving all neurodegenerative disorders is their poor efficacy in clearing and recycle toxic aggregated proteins and damaged cellular organelles due to impairment in the autophagy pathway. This dysfunction in autophagy characterized by the accumulation of toxic protein aggregates leads to a progressive loss in structural integrity/functionality of neurons and may even result in neuronal death. In recent years TFEB, a key regulator of autophagy and lysosomal biogenesis, has received considerable attention. It has emerged as a potential therapeutic target in numerous neurodegenerative disorders like AD and PD. In various neurobiology studies involving animal models, TFEB has been found to ameliorate neurotoxicity and rescue neurodegeneration. Since TFEB is a master transcriptional regulator of autophagy and lysosomal biogenesis pathway and plays a crucial role in defining autophagy activation. Studies have been done to understand the mechanisms for TFEB dysfunction, which may yield insights into how TFEB might be targeted and used for the therapeutic strategy to develop a treatment process with extensive application to neurodegenerative disorders. In this review, we explore the role of different transcription factor-based targeted therapy by some natural compounds for AD and PD with special emphasis on TFEB.

Keywords:  Alzheimer’s disease; NF-κB; Parkinson’s disease; TFEB; autophagy

DOI:  https://doi.org/10.3389/fnins.2021.777347
PLoS One. 2021 ;16(12): e0262180

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Rossana Segreto, Hoda Bazafkan, Julia Millinger, Martina Schenk, Lea Atanasova, Maria Doppler, Christoph Büschl, Mélanie Boeckstaens, Silvia Soto Diaz, Ulrike Schreiner, Fabiano Sillo, Raffaella Balestrini, Rainer Schuhmacher, Susanne Zeilinger.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

DOI: https://doi.org/10.1371/journal.pone.0262180
Alzheimers Dement. 2021 Dec;17 Suppl 2 e058727

Defective proteostasis in patient-derived iPSC-astrocytes and neurons carrying a MAPT IVS10+16 mutation.

Sidhartha Mahali, Celeste Karch.

BACKGROUND: Impaired proteostasis is associated with normal aging and is accelerated in neurodegeneration. This impairment may lead to the toxic protein accumulation. In a subset of frontotemporal dementia (FTD) cases, mutations in the microtubule-associated protein tau (MAPT) that alter the relative levels of specific tau isoforms are sufficient to cause tau inclusions in neurons and astroglia and neurodegeneration without the presence of mutated protein (e.g. MAPTIVS10+16). However, the pathogenic events triggered by the expression of the alternatively spliced tau remain poorly understood.
METHOD: To determine whether altered tau splicing induced from MAPT IVS10+16 mutations is sufficient to alter proteostasis in neurons and glia, we used human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes from patients carrying the MAPT IVS10+16 mutation and CRISPR/Cas9, isogenic corrected controls.
RESULT: We found that neurons from MAPT IVS10+16 carriers exhibited significantly higher levels of 4 repeat tau, deficits in lysosomal trafficking, and reduced lysosomal acidity relative to isogenic-control neurons. Additionally, MAPTIVS10+16 was sufficient to reduce genes associated with lysosomal biogenesis (regulated by TFEB). Interestingly, mutant astrocytes also produce elevated 4 repeat tau levels and exhibit several key hallmarks of cellular aging. Mutant astrocytes were larger in size with enlarged nuclei compared to isogenic controls. In addition to this hypertrophy phenotype, mutant astrocytes exhibited significantly elevated levels of senescence genes. Furthermore, markers of proteostasis were altered in mutant astrocytes: MAPT IVS10+16 carriers exhibited an increase in acidic lysosomes compared to isogenic-control astrocytes, and TFEB-regulated genes were upregulated MAPT IVS10+16 astrocytes.
CONCLUSION: Our findings suggest that altered tau splicing induced by the MAPT IVS10+16 mutation is sufficient to cause aging signatures, including hypertrophy, senescence, and altered proteostasis in a cell-type specific manner.

DOI: https://doi.org/10.1002/alz.058727
Alzheimers Dement. 2021 Dec;17 Suppl 2 e058730

Defining the role of PLD3 in Alzheimer's disease pathology.

Matthew J Rosene, Simon Hsu, Rita Martinez, Joanne Norton, Ping Yan, John R Cirrito, Jin-Moo Lee, Ana Maria Cuervo, Alison M Goate, Carlos Cruchaga, Celeste M Karch.

BACKGROUND: Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) in the brain. We recently identified coding variants in the phospholipase D3 (PLD3) gene that double the risk for late onset AD.
METHOD: We examined the impact of PLD3 risk variants on PLD3 and Aβ metabolism using CRISPR/Cas9 in induced pluripotent stem cells (iPSC). We then modeled the PLD3 expression patterns observed in AD brains in immortalized cell and AD mouse models. Lysosomal function was assessed in human brain tissue.
RESULT: PLD3 A442A disrupts a splicing enhancer binding site and reduces PLD3 splicing in human brains. Differentiation of PLD3 A442A and isogenic control iPSCs into cortical neurons produced cells that were morphologically similar. At the molecular level, PLD3 A442A neurons displayed a similar defect in PLD3 splicing as was observed in human brains and a significant increase in Aβ42/Aβ40 compared with isogenic control lines. Thus, PLD3 A442A is sufficient to alter PLD3 splicing and Aβ metabolism. PLD3 expression was significantly lower in AD brains compared with controls, and PLD3 expression was highly correlated with expression of lysosomal genes. Thus, we sought to determine whether PLD3 contributes to Aβ accumulation in AD via disrupted Aβ metabolism. We found that overexpression of PLD3 in immortalized cells decreased Aβ levels while shRNA silencing of Pld3 increased Aβ levels. In an AD mouse model, overexpression of PLD3 in hippocampal neurons produced decreased interstitial fluid (ISF) Aβ levels and accelerated Aβ turnover. Conversely, knocking out Pld3 increased ISF Aβ, reduced Aβ turnover, and increased APP protein levels. Knocking out Pld3 overtime lead to altered amyloid morphology. To begin to determine whether PLD3 influences Aβ turnover via the lysosome, we isolated lysosomal fractions from human AD and control brains. PLD3 was enriched in lysosomal subfractions and PLD3 distribution in these subfractions was altered in AD. Furthermore, PLD3 stability in the lysosomal fractions was disrupted in AD brains.
CONCLUSION: Together, our findings demonstrate that PLD3 promotes Aβ clearance through pathways involving lysosomal degradation.

DOI: https://doi.org/10.1002/alz.058730
Angew Chem Int Ed Engl. 2021 Dec 28.

Discovery of an Ultra-rapid and Sensitive Lysosomal Fluorescence Lipophagy Process.

Jong Seung Kim, Hong Zhang, Kun Li, Lei Shi, Xin Liu, Miae Won, Yan-Zhao Liu, Youmi Choe, Xin-Yao Liu, Yan-Hong Liu, Shan-Yong Chen, Kang-Kang Yu, Xiao-Qi Yu.

  Non-invasive dynamic tracking of lysosomes and their interactions with other organelles is important for the study of lysosomal function and related diseases. However, many fluorescent dyes developed so far to target lysosomes cannot be used to monitor these processes due to the high concentrations required for imaging, long cell penetration times, and non-ideal photostability. In this regard, we synthesized three lysosomal targeting probes with large Stokes shifts, good stability, and high brightness. The Q-P-ARh , developed by us for the first time, can stain lysosomes at ultra-low concentrations (1.0 nM) without affecting the physiological functions of the lysosomes. More importantly, its excellent anti-interference ability and ultrafast lysosomal staining ability (within 1.0 min) clearly monitored the entire dynamic process of lipophagy. Ultimately, this method can greatly contribute to the study of autophagy pathways. This novel fluorescence platform shows great promise for the development of biological probes for application in pathological environments.

Keywords:  lipophagy * lysosomes * near-infrared * large stokes shift

DOI:  https://doi.org/10.1002/anie.202116439
Hum Mol Genet. 2021 Nov 20. pii: ddab337. [Epub ahead of print]

Dorsal telencephalon-specific Nprl2- and Nprl3-knockout mice: novel mouse models for GATORopathy.

Saeko Ishida, Di Zhao, Yuta Sawada, Yuichi Hiraoka, Tomoji Mashimo, Kohichi Tanaka.

The most frequent genetic cause of focal epilepsies is variations in the GAP activity toward RAGs 1 complex genes DEP domain containing 5 (DEPDC5), nitrogen permease regulator 2-like protein (NPRL2) and nitrogen permease regulator 3-like protein (NPRL3). Because these variations are frequent and associated with a broad spectrum of focal epilepsies, a unique pathology categorized as GATORopathy can be conceptualized. Animal models recapitulating the clinical features of patients are essential to decipher GATORopathy. Although several genetically modified animal models recapitulate DEPDC5-related epilepsy, no models have been reported for NPRL2- or NPRL3-related epilepsies. Here, we conditionally deleted Nprl2 and Nprl3 from the dorsal telencephalon in mice [Emx1cre/+; Nprl2f/f (Nprl2-cKO) and Emx1cre/+; Nprl3f/f (Nprl3-cKO)] and compared their phenotypes with Nprl2+/-, Nprl3+/- and Emx1cre/+; Depdc5f/f (Depdc5-cKO) mice. Nprl2-cKO and Nprl3-cKO mice recapitulated the major abnormal features of patients-spontaneous seizures, and dysmorphic enlarged neuronal cells with increased mechanistic target of rapamycin complex 1 signaling-similar to Depdc5-cKO mice. Chronic postnatal rapamycin administration dramatically prolonged the survival period and inhibited seizure occurrence but not enlarged neuronal cells in Nprl2-cKO and Nprl3-cKO mice. However, the benefit of rapamycin after withdrawal was less durable in Nprl2- and Nprl3-cKO mice compared with Depdc5-cKO mice. Further studies using these conditional knockout mice will be useful for understanding GATORopathy and for the identification of novel therapeutic targets.

DOI: https://doi.org/10.1093/hmg/ddab337
J Integr Plant Biol. 2021 Dec 28.

Plant TOR signaling network: Complexes, conservations and specificities.

Yanlin Liu, Yan Xiong.

  Target of Rapamycin (TOR) is an evolutionarily conserved protein kinase that functions as a central signaling hub to integrate diverse internal and external cues to precisely orchestrate cellular and organismal physiology. During evolution, TOR both maintains the highly conserved TOR complex compositions, cellular and molecular functions, but also evolves distinctive roles and strategies to modulate cell growth, proliferation, metabolism, survival and stress responses in eukaryotes. Here, we review recent discoveries on the plant TOR signaling network. We present an overview of plant TOR complexes, analyze the signaling landscape of the plant TOR signaling network from the upstream signals that regulate plant TOR activation to the downstream effectors involved in various biological processes, and compare their conservation and specificities within different biological contexts. Finally, we summarize the impact of dysregulation of TOR signaling on every stage of plant growth and development, from embryogenesis and seedling growth, to flowering and senescence. This article is protected by copyright. All rights reserved.

Keywords:  Abiotic and biotic stress; hormone sensing; metabolism regulation; nutrient sensing; plant growth and development; target of rapamycin; transcriptional reprogramming; translational regulation

DOI:  https://doi.org/10.1111/jipb.13212
Oncol Rep. 2022 Feb;pii: 40. [Epub ahead of print]47(2):

Lysosome‑targeted drug combination induces multiple organelle dysfunctions and non‑canonical death in pancreatic cancer cells.

Sumire Suzuki, Masato Ogawa, Masaya Miyazaki, Kohki Ota, Hiromi Kazama, Ayako Hirota, Naoharu Takano, Masaki Hiramoto, Keisuke Miyazawa.

  Pancreatic cancer is one of the leading causes of cancer‑related mortality and has the lowest 5‑year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC‑3, KP‑4, PANC‑1 and MIA PaCa‑2, were examined. It was observed that FTY720 enhanced the lapatinib‑induced cytotoxic effect and caused non‑canonical and lysosome‑dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome‑targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

Keywords:  abemaciclib; calcium homeostasis; endoplasmic reticulum stress; fingolimod; hydroxychloroquine; lapatinib; lysosomal membrane permeabilization; lysosome‑targeted drug combination; mitochondrial depolarization; non‑canonical cell death; pancreatic cancer cells

DOI:  https://doi.org/10.3892/or.2021.8251
Adipocyte. 2022 Dec;11(1): 28-33

Lysosomal acid lipase promotes endothelial proliferation in cold-activated adipose tissue.

Alexander W Fischer, Michelle Y Jaeckstein, Joerg Heeren.

  Oxidative tissues such as brown adipose tissue and muscle internalize large amounts of circulating lipids and glucose as energy source. Endothelial cells (ECs) provide a platform for regulated transport and processing of blood-borne nutrients. Next to this role, it has become recognized that intercellular crosstalk between ECs and underlying parenchymal cells is indispensable for maintenance of tissue homoeostasis. Here, we comment on our recent observation that capillary ECs in thermogenic adipose tissues take up and metabolize entire triglyceride-rich lipoprotein (TRL) particles in response to cold exposure. This process is dependent on CD36, lipoprotein lipase (LPL) and lysosomal acid lipase (LAL). Remarkably, loss of LAL specifically in endothelial cells results in impaired endothelial proliferation and diminished thermogenic adaptation. Mechanistically, cell culture experiments indicate that LAL-mediated TRL processing leads to the generation of reactive oxygen species, which in turn activate hypoxia-induced factor (HIF)-mediated proliferative responses. In the current manuscript, we provide in vivo evidence that LAL-deficiency impairs proliferation of endothelial cells in thermogenic adipose tissue. In addition, we show uptake of nanoparticle-labelled TRL and LAL expression in cardiac endothelial cells, suggesting a physiological function of endothelial lipoprotein processing not only in thermogenic adipose tissue but also in cardiac muscle.

Keywords:  Adipose tissue; browning; endothelial cells; lipoprotein; lysosomal acid lipase; lysosome; proliferation; thermogenesis; triglyceride

DOI:  https://doi.org/10.1080/21623945.2021.2013416
Brain Res. 2021 Dec 27. pii: S0006-8993(21)00627-2. [Epub ahead of print] 147768

GSK-3β inhibition elicits a neuroprotection by restoring lysosomal dysfunction in neurons via facilitation of TFEB nuclear translocation after ischemic stroke.

Zhang Yongjie, Wu Zhiyuan, Huang Zhiwen, Liu Yuyuan, Chen Xuemei, Zhao Xiaoming, He Hongyun, Deng Yihao.

  Lysosomal dysfunction is an essential pathogenesis of autophagic neuronal injury after ischemic stroke. As a result of cerebral ischemia, transcription factor EB (TFEB) is greatly phosphorylated by prominently activated glycogen synthase kinase-3β (GSK-3β). This increased TFEB phosphorylation decreases its nuclear translocation and subsequently leads to reduced lysosomal biosynthesis, which ultimately results in lysosomal dysfunction. The present study is to investigate whether the lysosomal dysfunction in neurons can be restored to alleviate post-stroke damage by GSK-3β inhibition. The GSK-3β activity was inhibited by pre-treatment with CHIR-99021 (CHIR) for 3 days before middle cerebral artery occlusion (MCAO) surgery in rats. Besides, the lysosomal capacity was altered by pre-administration with Bafilomycin A1 (Baf-A1) and EN6, respectively. Twenty-four hours after MCAO/reperfusion, the penumbral tissues were obtained to detect the GSK-3β, cytoplasmic and nuclear TFEB, and proteins in autophagic/lysosomal pathway by western blot and immunofluorescence, respectively. Meanwhile, the infarct volume, neurological deficits and neuron survival were assessed to evaluate the neurological outcomes elicited by GSK-3β inhibition. The results demonstrated that the neurological injury could be significantly mitigated by GSK-3β inhibition in MCAO+CHIR group, compared with that in MCAO group. Moreover, CHIR-facilitated TFEB nuclear translocation in neurons was coupled with reinforced lysosomal activities and attenuated autophagic substrates. However, GSK-3β inhibition-induced neuroprotection was greatly counteracted by Baf-A1-weakened lysosomal capacity. Conversely, EN6-reinforced lysosomal activities further ameliorated the autophagic/lysosomal signaling, and synergistically alleviated the neurological damage upon GSK-3β inhibition after MCAO/reperfusion. Our data suggests that GSK-3β inhibition-augmented neuroprotection against ischemic stroke is elicited by restoring the lysosomal dysfunction in neurons.

Keywords:  GSK-3β inhibition; TFEB nuclear translocation; autophagic/lysosomal signaling; ischemic stroke; neuroprotection

DOI:  https://doi.org/10.1016/j.brainres.2021.147768
Methods Mol Biol. 2022 ;2445 39-50

CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells.

Ting-Ting Yu, Xun Zhou, Helin Vakifahmetoglu-Norberg.

  Chaperone-mediated autophagy (CMA) is a highly specific lysosomal-dependent protein degradation pathway. A critical molecular component of CMA is the lysosome-associated membrane protein (LAMP) type 2A, which is required for substrate uptake by the lysosome. Defects in the CMA pathway have been associated with various human pathologies, including malignancies, increasing the overall interest in methods to monitor this selective autophagy process. Yet isogenic LAMP-2A knockout cancer cell models are still lacking. This is likely to depend on challenges related to that human LAMP-2 gene undergoes alternative splicing of its pre-mRNA, generating three isoform variants, LAMP-2A, LAMP-2B, and LAMP-2C. However, without assessment of the impact of LAMP-2A loss of function specifically in human cells, the involvement of CMA in human pathologies, including carcinogenesis remains speculative. Here, we describe the generation of isoform-specific CRISPR-Cas9 genomic editing of LAMP-2A in human cancer cells, without affecting the other two isoforms, allowing for experimental evaluation of LAMP-2A, thus CMA in human cancer models.

Keywords:  Autophagy; CRISPR-Cas9; Cancer; Chaperone-mediated autophagy; Gene editing; LAMP-2A

DOI:  https://doi.org/10.1007/978-1-0716-2071-7_3
Mol Carcinog. 2021 Dec 29.

Conditional deletion of mTOR discloses its essential role in early B-cell development.

Shuling Zhang, Wendy Dubois, Xingmin Feng, Joe T Nguyen, Neal S Young, Beverly A Mock.

  Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and central regulator of cell growth, differentiation, and survival. mTOR is commonly hyperactivated in a diverse number of cancers and critical roles for mTOR in regulating immune cell differentiation and function have been demonstrated. However, there is little work investigating the roles of mTOR in early B-cell development. Here we demonstrate that conditional disruption of mTOR in developing mouse B cells results in reduced pre-B-cell proliferation and survival, as well as a developmental block at the pre-B-cell stage, with a corresponding lack of peripheral B cells. Upon immunization with NP-CGG antigen, mice with Mtor conditional disruption in early B cells lost their ability to form germinal centers and produce specific antibodies. In competitive BM repopulation assays, donor BM cells from conditional knock-out mice were completely impaired in their ability to reconstitute B cells. Our data reveal the essential role of mTOR in early pre-B-cell development and survival.

Keywords:  B-cell development; Mb1-Cre mice; antibody production; competitive BM transplantation; mTOR

DOI:  https://doi.org/10.1002/mc.23386
Eur J Immunol. 2021 Dec 27.

Glutamine promotes the generation of B10+ cells via the mTOR/GSK3 pathway.

Julie Mielle, Jacques Morel, Jamila ElHmioui, Bernard Combe, Laurence Macia, Valérie Dardalhon, Naomi Taylor, Rachel Audo, Claire Daien.

  Alterations in cell metabolism can shift the differentiation of immune cells towards a regulatory or inflammatory phenotype, thus opening up new therapeutic opportunities for immune-related diseases. Indeed, growing knowledge on T cell metabolism has revealed differences in the metabolic programs of suppressive regulatory T cells (Tregs) as compared to inflammatory Th1 and Th17 cells. In addition to Tregs, IL-10-producing regulatory B cells are crucial for maintaining tolerance, inhibiting inflammation and autoimmunity. Yet, the metabolic networks regulating diverse B lymphocyte responses are not well known. Here, we show that glutaminase blockade decreased downstream mTOR activation and attenuated IL-10 secretion. Direct suppression of mTOR activity by rapamycin selectively impaired IL-10 production by B cells whereas secretion was restored upon GSK3 inhibition. Mechanistically, we found mTORC1 activation leads to GSK3 inhibition, identifying a key signalling pathway regulating IL-10 secretion by B lymphocytes. Thus, our results identify glutaminolysis and the mTOR/GSK3 signalling axis, as critical regulators of the generation of IL-10 producing B cells with regulatory functions. This article is protected by copyright. All rights reserved.

Keywords:  B10; GSK3; IL-10; glutamine; immunotherapy; mTOR; metabolism; regulatory B cells

DOI:  https://doi.org/10.1002/eji.202149387
Cancer Metastasis Rev. 2021 Dec 27.

Progression and metastasis of small cell lung carcinoma: the role of the PI3K/Akt/mTOR pathway and metabolic alterations.

Ildiko Krencz, Daniel Sztankovics, Titanilla Danko, Anna Sebestyen, Andras Khoor.

  Small cell lung carcinoma (SCLC) is characterized by high metastatic rate and poor prognosis. The platinum-based chemotherapy still represents the backbone of the therapy; however, acquired resistance develops almost in all patients. Although SCLC has been formerly considered a homogeneous disease, recent advances in SCLC research have highlighted the importance of inter- and intratumoral heterogeneity and have resulted in the subclassification of SCLC. The newly described SCLC subtypes are characterized by distinct biological behavior and vulnerabilities that can be therapeutically exploited. The PI3K/Akt/mTOR pathway is frequently affected in SCLC, and its activation represents a promising therapeutic target. Since the mTOR pathway is a master regulator of cellular metabolism, its alterations may also influence the bioenergetic processes of SCLC cells. Despite the encouraging preclinical results, both mTOR and metabolic inhibitors have met limited clinical success so far. Patient selection for personalized therapy, the development of rational drug combinations, and a better understanding of heterogeneity and spatiotemporal evolution of the tumor cells may improve efficacy and can help to overcome acquired resistance. Here we provide a summary of current investigations regarding the role of the mTOR pathway and metabolic alterations in the progression and metastasis formation of SCLC.

Keywords:  Metabolism; Metastasis; Small cell lung carcinoma; mTOR

DOI:  https://doi.org/10.1007/s10555-021-10012-4
Cell Calcium. 2021 Dec 21. pii: S0143-4160(21)00176-7. [Epub ahead of print]101 102522

Calcium sequestration by human platelet acidic organelles is regulated by the actin cytoskeleton and autocrine 5-hydroxytryptamine.

Stewart O Sage, Alan G S Harper.

  Human platelets regulate agonist-evoked Ca2+ signalling through Ca2+ release from and sequestration into acidic organelles. Previous studies have pharmacologically characterised the presence of a Ca2+-H+ exchanger in these organelles. This exchanger appears to regulate a secondary plateau phase in agonist-evoked cytosolic Ca2+ signals in fura-2-loaded human platelets. Here we demonstrate that cytochalasin D treatment removes the secondary plateau in ADP-evoked Ca2+ signals elicited in the absence of external Ca2+. This effect was reversed by pretreatment with nigericin, a K+/H+ exchanger that short-circuits the Ca2+-H+ exchanger. Using Fluo-5N- or Lysosensor Green-loaded cells, cytochalasin D was found to enhance Ca2+ sequestration into acidic organelles by preventing their alkalinisation. Additional experiments demonstrated that ADP-evoked alkalinisation of acidic organelles and subsequent slowing of acidic organellar Ca2+ sequestration was mediated by autocrine 5-HT signalling. Enhancing this 5-HT signalling using fluoxetine overcame the inhibitory effect of cytochalasin D on ADP-evoked Ca2+ signals, indicating that cytochalasin D interferes with 5-HT autocrine signalling. The ability of Cytochalasin D to interfere with autocrine 5-HT signalling was downstream of the 5-HT2A receptor as secretion of [3H]-5-HT from ADP-stimulated human platelets was not reduced. These data provide the first evidence that the pH gradient across acidic organelles is dynamically regulated upon human platelet activation, and that this can play a significant role in controlling human platelet function by modulating Ca2+-H+ exchange and so [Ca2+]i.

Keywords:  5-hydroxytryptamine; ADP; Acidic organelle; Calcium sequestration; Human platelet; pH

DOI:  https://doi.org/10.1016/j.ceca.2021.102522
ACS Sens. 2021 Dec 30.

Demystifying Lysosomal α-l-Fucosidase in Liver Cancer-Bearing Mice by Specific Two-Photon Fluorescence Imaging.

Nannan Fan, Ping Li, Yongqing Zhou, Chuanchen Wu, Xin Wang, Zhenzhen Liu, Bo Tang.

  Liver cancer is one of the most frequently diagnosed cancers and has high mortality. However, the early treatment and prognosis can greatly prolong the survival time of patients, which depends on its early detection. α-l-Fucosidase (AFU), as a vital lysosomal hydrolase, is considered to be an ideal biomarker for early stage liver cancer. So, in vivo monitoring of AFU is essential for the early and accurate diagnosis of liver cancer. Hence, we designed the first two-photon turn-on fluorescent reporter, termed HcyCl-F, which localized to lysosomes for fast imaging of AFU. The 2-chloro-4-phenyl-α-l-fucoside bond of HcyCl-F could be effectively hydrolyzed by AFU and released the hydroxyl on the benzene ring, eventually obtaining a strong conjugated compound (HcyCl-OH) with shiny fluorescence. We demonstrated that HcyCl-F was able to rapidly and accurately respond to AFU. Using a two-photon fluorescence microscope, we successfully visualized the fluctuation of AFU in lysosomes. More importantly, a fascinatingly strong fluorescence signal was observed in the tumor tissue of liver cancer-bearing mice. Of note, we confirmed that HcyCl-F could clearly detect liver tumors in stage I. Altogether, our work provides a simple and convenient method for deciphering the critical pathological function of AFU in depth and facilitates the nondestructive and effective diagnosis of liver cancer in the early stage.

Keywords:  AFU; fast; in situ; liver cancer-bearing mice; lysosome; two-photon imaging

DOI:  https://doi.org/10.1021/acssensors.1c01630
J Cell Biochem. 2021 Dec 27.

Suppression of transient receptor potential melastatin 7 regulates pluripotency, proliferation, and differentiation of mouse embryonic stem cells via mechanistic target of rapamycin-extracellular signal-regulated kinase activation.

Wansoo Kim, Song Park, Wookbong Kwon, Daehwan Kim, Jin-Kyu Park, Jee Eun Han, Gil-Jae Cho, Se-Hyeon Han, Yonghun Sung, Jun-Koo Yi, Myoung Ok Kim, Zae Young Ryoo, Seong-Kyoon Choi.

  Mouse embryonic stem cells (mESCs) are a widely used model for their diverse availability in studying early embryonic development and their application in regenerative treatment of various intractable diseases. Transient receptor potential melastatin 7 (Trpm7) regulates Ca2+ as a nonselective ion channel and is essential for early embryonic development; however, the precise role of Trpm7 in mESCs has not been clearly elucidated. In this study, we showed that the inhibition of Trpm7 affects the pluripotency and self-renewal of mESCs. We found that short hairpin RNA (shRNA)-mediated suppression of Trpm7 resulted in decreased expression of transcriptional regulators, Oct4 and Sox2, which maintain stemness in mESCs. In addition, Trpm7 knockdown led to alterations in the basic properties of mESCs, such as decreased proliferation, cell cycle arrest at the G0/G1 phase, and increased apoptosis. Furthermore, embryoid body (EB) formation and teratoma formation assays revealed abnormal regulation of differentiation due to Trpm7 knockdown, including the smaller size of EBs, elevated ectodermal differentiation, and diminished endodermal and mesodermal differentiation. We found that EB Day 7 samples displayed decreased intracellular Ca2+ levels compared to those of the scrambled group. Finally, we identified that these alterations induced by Trpm7 knockdown occurred due to decreased phosphorylation of mechanistic target of rapamycin (mTOR) and subsequent activation of extracellular signal-regulated kinase (ERK) in mESCs. Our findings suggest that Trpm7 could be a novel regulator for maintaining stemness and modulating the differentiation of mESCs.

Keywords:  ERK; Trpm7; mESC; mESC differentiation; mTOR; pluripotency

DOI:  https://doi.org/10.1002/jcb.30199
Front Microbiol. 2021 ;12 779176

The Importance of Vacuolar Ion Homeostasis and Trafficking in Hyphal Development and Virulence in Candida albicans.

Quanzhen Lv, Lan Yan, Yuanying Jiang.

  The vacuole of Candida albicans plays a significant role in many processes including homeostasis control, cellular trafficking, dimorphic switching, and stress tolerance. Thus, understanding the factors affecting vacuole function is important for the identification of new drug targets needed in response to the world's increasing levels of invasive infections and the growing issue of fungal drug resistance. Past studies have shown that vacuolar proton-translocating ATPases (V-ATPases) play a central role in pH homeostasis and filamentation. Vacuolar protein sorting components (VPS) regulate V-ATPases assembly and at the same time affect hyphal development. As well, vacuolar calcium exchange systems like Yvc1 and Pmc1 maintain cytosolic calcium levels while being affected by V-ATPases function. All these proteins play a role in the virulence and pathogenesis of C. albicans. This review highlights the relationships among V-ATPases, VPS, and vacuolar calcium exchange proteins while summarizing their importance in C. albicans infections.

Keywords:  Candida albicans; vacuolar Ca2+ channel; vacuolar protein sorting components; vacuolar proton-translocating ATPases; virulence

DOI:  https://doi.org/10.3389/fmicb.2021.779176
PLoS Biol. 2021 Dec;19(12): e3001492

Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2.

Rona Aviram, Vaishnavi Dandavate, Gal Manella, Marina Golik, Gad Asher.

Rhythmicity of biological processes can be elicited either in response to environmental cycles or driven by endogenous oscillators. In mammals, the circadian clock drives about 24-hour rhythms of multitude metabolic and physiological processes in anticipation to environmental daily oscillations. Also at the intersection of environment and metabolism is the protein kinase-AKT. It conveys extracellular signals, primarily feeding-related signals, to regulate various key cellular functions. Previous studies in mice identified rhythmicity in AKT activation (pAKT) with elevated levels in the fed state. However, it is still unknown whether rhythmic AKT activation can be driven through intrinsic mechanisms. Here, we inspected temporal changes in pAKT levels both in cultured cells and animal models. In cultured cells, pAKT levels showed circadian oscillations similar to those observed in livers of wild-type mice under free-running conditions. Unexpectedly, in livers of Per1,2-/- but not of Bmal1-/- mice we detected ultradian (about 16 hours) oscillations of pAKT levels. Importantly, the liver transcriptome of Per1,2-/- mice also showed ultradian rhythms, corresponding to pAKT rhythmicity and consisting of AKT-related genes and regulators. Overall, our findings reveal ultradian rhythms in liver gene expression and AKT phosphorylation that emerge in the absence of environmental rhythms and Per1,2-/- genes.

DOI: https://doi.org/10.1371/journal.pbio.3001492
Cancer Res. 2021 Dec 29. pii: canres.2342.2021. [Epub ahead of print]

AMPK-mTOR-mediated activation of autophagy promotes formation of dormant polyploid giant cancer cells.

Bo You, Tian Xia, Miao Gu, Zhenxin Zhang, Qicheng Zhang, Jianhong Shen, Yue Fan, Hui Yao, Si Pan, Yingna Lu, Tianyi Cheng, Zhiyuan Yang, Xin He, Hao Zhang, Muqi Shi, Dong Liu, Yiwen You.

Dormant cancer cells that survive anti-cancer therapy can lead to cancer recurrence and disseminated metastases that prove fatal in most cases. Recently, specific dormant polyploid giant cancer cells (PGCC) have drawn our attention because of their association with the clinical risk of nasopharyngeal carcinoma (NPC) recurrence, as demonstrated by previous clinical data. In this study, we report the biological properties of PGCC, including mitochondrial alterations, and reveal that autophagy is a critical mechanism of PGCC induction. Moreover, pharmacological or genetic inhibition of autophagy greatly impaired PGCC formation, significantly suppressing metastasis and improving survival in a mouse model. Mechanistically, chemotherapeutic drugs partly damaged mitochondria, which then produced low ATP levels and activated autophagy via the AMPK-mTOR pathway to promote PGCC formation. Analysis of the transcriptional and epigenetic landscape of PGCC revealed overexpression of RIPK1, and the scaffolding function of RIPK1 was required for AMPK-mTOR pathway-induced PGCC survival. High numbers of PGCCs correlated with shorter recurrence time and worse survival outcomes in NPC patients. Collectively, these findings suggest a therapeutic approach of targeting dormant PGCCs in cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2342