bims-lysosi 2022-03-06 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022–03–06
37 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Lysosomes and lysosome-related organelles in immune responses.
The FACT complex facilitates expression of lysosomal and antioxidant genes through binding to TFEB and TFE3.
Protocols to monitor TFEB activation following lysosomal damage in cultured cells using microscopy and immunoblotting.
Activation of Liver mTORC1 Protects Against NASH via Dual Regulation of VLDL-TAG Secretion and De Novo Lipogenesis.
mTOR signaling as a molecular target for the alleviation of Alzheimer's disease pathogenesis.
Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients.
P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.
Ubr1-induced selective endophagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress.
Inhibition of MEK-ERK signaling reduces seizures in two mouse models of tuberous sclerosis complex.
Deficiency in Lyst function leads to accumulation of secreted proteases and reduced retinal adhesion.
The mechanistic target of rapamycin as a regulator of metabolic function in oligodendroglia during remyelination.
O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.
Modeling Parkinson's disease in LRRK2 mice: focus on synaptic dysfunction and the autophagy-lysosomal pathway.
Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.
Cholesterol biosynthesis defines oligodendrocyte precursor heterogeneity between brain and spinal cord.
Targeting macrophage TFEB-14-3-3 epsilon Interface by naringenin inhibits abdominal aortic aneurysm.
The Alzheimer's gene SORL1 is a regulator of endosomal traffic and recycling in human neurons.
Up-regulated YB-1 protein promotes glioblastoma growth through an YB-1/CCT4/mLST8/mTOR pathway.
Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.
The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking.
Tuberous sclerosis complex is a novel, amyloid-independent tauopathy associated with elevated phosphorylated 3R/4R tau aggregation.
Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling.
Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations.
Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling.
CLC anion/proton exchangers regulate secretory vesicle filling and granule exocytosis in chromaffin cells.
Reduced ech-6 expression attenuates fat-induced lifespan shortening in C. elegans.
Molecular architecture determines brain delivery of a transferrin receptor-targeted lysosomal enzyme.
The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast.
Reduced α-galactosidase A activity in zebrafish (Danio rerio) mirrors distinct features of Fabry nephropathy phenotype.
ROS-mediated activation of p38 protects hepatocellular carcinoma cells from caspase-independent death elicited by lysosomal damage.
Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) expression in glioblastoma is driven by ETS1- and MYBL2-dependent transcriptional activation.
Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila.
In Situ Artificial Contact Sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein tethering activities.
Compartmentation and functions of sphingolipids.
Dynamin 1 controls vesicle size and endocytosis at hippocampal synapses.
Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases.
Amino acid sensor conserved from bacteria to humans.

FEBS Open Bio. 2022 Feb 26.

Lysosomes and lysosome-related organelles in immune responses.

Colin Watts.

  The perception of lysosomes has undergone a remarkable change in recent years. Formerly viewed as end-stage compartments dedicated to macromolecule catabolism enabled by a cohort of hydrolytic enzymes, which if missing or mutated could lead to lysosomal storage diseases (LSDs), they now occupy a prominent position in many other aspects of cellular and organismal physiology. In particular, lysosomes have emerged as key signalling platforms, as regulators of transcription, as agents of plasma membrane repair, in regulated cell death and in autophagy. These exciting developments have been extensively reviewed elsewhere (1-3). Here we confine the discussion to the key roles that lysosomes and lysosome related organelles (LROs) play in immunity. Even with this restricted view their functions are broad. They play a key role in pathogen detection and signalling, in processing and presentation of antigens to T lymphocytes and are closely integrated into the different life stages of dendritic cells (DC) which are key cells that link innate and adaptive immunity (4-6). Some leukocytes assemble specialised lysosome-related organelles (LROs) which can be discharged at the cell surface to achieve distinct, mostly toxic, effector functions. Proteolytic enzymes, most with an acidic pH optimum, are important for many lysosomal functions in immunity. LROs accumulate distinct proteases and other toxic agents and we will see that potential toxicity from these organelles is countered by expression of members of the cystatin and serpin families of protease inhibitors. The term endo-lysosomes rather than lysosomes is often used to refer broadly to later stage endocytic pathway organelles.

Keywords:  antigen processing; endocytosis; immune response; lysosome-related organelles; lysosomes; proteases

DOI:  https://doi.org/10.1002/2211-5463.13388
Autophagy. 2022 Mar 01. 1-17

The FACT complex facilitates expression of lysosomal and antioxidant genes through binding to TFEB and TFE3.

Eutteum Jeong, José A Martina, Pablo S Contreras, Juhyung Lee, Rosa Puertollano.

  TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.

Keywords:  Autophagy; FACT; TFE3; TFEB; chaperone; histone; lysosomes

DOI:  https://doi.org/10.1080/15548627.2022.2029671
STAR Protoc. 2022 Mar 18. 3(1): 101018

Protocols to monitor TFEB activation following lysosomal damage in cultured cells using microscopy and immunoblotting.

Monami Ogura, Takayuki Shima, Tamotsu Yoshimori, Shuhei Nakamura.

  Following lysosomal damage, activation and nuclear translocation of transcription factor EB (TFEB) is the key event to maintain lysosomal homeostasis. Here, we describe steps to induce lysosomal damage in HeLa cells. This can be followed by monitoring the changes in TFEB localization using widefield fluorescence microscopy. As a complementary approach, we describe the use of immunoblotting to follow the activation and localization of TFEB in cell lysates. These protocols enable quantitative analysis of TFEB. For complete details on the use and execution of this protocol, please refer to Nakamura et al. (2020).

Keywords:  Antibody; Cell Biology; Cell culture; Cell separation/fractionation; Cell-based Assays; Microscopy

DOI:  https://doi.org/10.1016/j.xpro.2021.101018
Cell Mol Gastroenterol Hepatol. 2022 Feb 28. pii: S2352-345X(22)00042-X. [Epub ahead of print]

Activation of Liver mTORC1 Protects Against NASH via Dual Regulation of VLDL-TAG Secretion and De Novo Lipogenesis.

Kahealani Uehara, Jaimarie Sostre-Colón, Matthew Gavin, Dominic Santoleri, Kelly-Ann Leonard, René L Jacobs, Paul M Titchenell.

   BACKGROUND & AIMS: Dysregulation of liver lipid metabolism is associated with the development and progression of non-alcoholic fatty liver disease (NAFLD), a spectrum of liver diseases including non-alcoholic steatohepatitis (NASH). In the liver, insulin controls lipid homeostasis by increasing triglyceride (TAG) synthesis, suppressing fatty acid oxidation, and enhancing TAG export via very low-density lipoproteins (VLDL). Downstream of insulin signaling, the mechanistic target of rapamycin complex 1 (mTORC1), is a key regulator of lipid metabolism. Here, we define the role of mTORC1 activity in mouse models of NASH and investigate the mTORC1-dependent mechanisms responsible for protection against liver damage in NASH.
METHODS: Utilizing two rodent NASH-promoting diets, we demonstrate that hepatic mTORC1 activity was reduced in mice with NASH, whereas under conditions of insulin resistance and benign fatty liver, mTORC1 activity was elevated. To test the beneficial effects of hepatic mTORC1 activation in mouse models of NASH, we employed an acute, liver-specific knockout model of TSC1 (L-TSC-KO), a negative regulator of mTORC1.
RESULTS: L-TSC-KO mice are protected from and have improved markers of NASH including reduced steatosis, decreased circulating transaminases, and reduced expression of inflammation and fibrosis genes. Mechanistically, protection from hepatic inflammation and fibrosis by constitutive mTORC1 occurred via promotion of the phosphatidylcholine synthesizing enzyme, CCTα, and enhanced VLDL-TAG export. Additionally, activation of mTORC1 protected from hepatic steatosis via negative feedback of the mTORC2-AKT-FOXO-SREBP1c lipogenesis axis.
CONCLUSIONS: Collectively, this study identifies a protective role for mTORC1 signaling in the initiation and progression of NASH in mice via dual control of lipid export and synthesis.

Keywords:  CCTα; FOXO1; insulin; non-alcoholic fatty liver disease; phosphatidylcholine

DOI:  https://doi.org/10.1016/j.jcmgh.2022.02.015
Neurochem Int. 2022 Feb 23. pii: S0197-0186(22)00036-5. [Epub ahead of print]155 105311

mTOR signaling as a molecular target for the alleviation of Alzheimer's disease pathogenesis.

Deepthi Rapaka, Veera Raghavulu Bitra, Siva Reddy Challa, Paul C Adiukwu.

  Mechanistic/mammalian target of rapamycin (mTOR) belongs to the phosphatidylinositol kinase-related kinase (PIKK) family. mTOR signaling is required for the commencement of essential cell functions including autophagy. mTOR primarily governs cell growth in response to favourable nutrients and other growth stimuli. However, it also influences aging and other aspects of nutrient-related physiology such as protein synthesis, ribosome biogenesis, and cell proliferation in adults with very limited growth. The major processes for survival such as synaptic plasticity, memory storage and neuronal recovery involve a significant mTOR activity. mTOR dysregulation is becoming a prevalent motif in a variety of human diseases, including cancer, neurological disorders, and other metabolic syndromes. The use of rapamycin to prolong life in different animal models may be attributable to the multiple roles played by mTOR signaling in various processes involved in ageing, protein translation, autophagy, stem cell pool turnover, inflammation, and cellular senescence. mTOR activity was found to be altered in AD brains and rodent models, supporting the notion that aberrant mTOR activity is one of the key events contributing to the onset and progression of AD hallmarks This review assesses the molecular association between the mTOR signaling pathway and pathogenesis of Alzheimer's disease. The research data supporting this theme are also reviewed.

Keywords:  Alzheimer's disease; Apoptosis; Cognition; Macroautophagy; Rapamycin; mTOR

DOI:  https://doi.org/10.1016/j.neuint.2022.105311
Mol Genet Metab Rep. 2022 Mar;30 100843

Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients.

Richard W D Welford, Herve Farine, Michel Steiner, Marco Garzotti, Kostantin Dobrenis, Claudia Sievers, Daniel S Strasser, Yasmina Amraoui, Peter M A Groenen, Roberto Giugliani, Eugen Mengel.

  GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients.

Keywords:  Biomarker; CLN2, neuronal ceroid lipofuscinosis type 2; CNS, central nervous system; CSF, cerebrospinal fluid; EDTA, Ethylenediaminetetraacetic acid; GBA2, non-lysosomal glucocerebrosidase; GCS, glucosylceramide synthase; GD3, Gaucher disease type 3; GFAP, Glial fibrillary acidic protein; Gangliosidosis; GlcSph, glucosylsphingosine; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; LLOQ, lower limit of quantification; LSD, lysosomal storage disorders; Lysosome; Lysosphingolipid; MRI, magnetic resonance imaging; MS, multiple sclerosis; NF-L, Neurofilament light chain; NPC, Niemann Pick disease type C; Neurofilament; QC, quality control; lysoGb3, globotriaosylsphingosine

DOI:  https://doi.org/10.1016/j.ymgmr.2022.100843
Front Immunol. 2022 ;13 752105

P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.

Stephanie Alexia Cristina Silva Santos, Pedro Muanis Persechini, Bianca Monteiro Henriques-Santos, Victória Gabriela Bello-Santos, Newton G Castro, Júlia Costa de Sousa, Fernando Ariel Genta, Marcelo Felippe Santiago, Robson Coutinho-Silva, Luiz Eduardo Baggio Savio, Eleonora Kurtenbach.

  The P2X7 receptor is a critical purinergic receptor in immune cells. Its activation was associated with cathepsin release into macrophage cytosol, suggesting its involvement in lysosomal membrane permeabilization (LMP) and leakage. Nevertheless, the mechanisms by which P2X7 receptor activation induces LMP and leakage are unclear. This study investigated cellular mechanisms associated with endosomal and lysosomal leakage triggered by P2X7 receptor activation. We found that ATP at 500 μM and 5 mM (but not 50 μM) induced LMP in non-stimulated peritoneal macrophages. This effect was not observed in P2X7-deficient or A740003-pretreated macrophages. We found that the P2X7 receptor and pannexin-1 channels mediate calcium influx that might be important for activating specific ion channels (TRPM2 and two-pore channels) on the membranes of late endosomes and lysosomes leading to LMP leakage and consequent cathepsin release. These findings suggest the critical role of the P2X7 receptor in inflammatory and infectious diseases via lysosomal dysfunction.

Keywords:  P2 receptor; cathepsins; extracellular ATP; lysosomal permeabilization; lysosomes

DOI:  https://doi.org/10.3389/fimmu.2022.752105
Cell Mol Life Sci. 2022 Mar 01. 79(3): 167

Ubr1-induced selective endophagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress.

Ben B Wang, Haijin Xu, Sandra Isenmann, Cheng Huang, Xabier Elorza-Vidal, Grigori Y Rychkov, Raúl Estévez, Ralf B Schittenhelm, Gergely L Lukacs, Pirjo M Apaja.

  The cellular defense mechanisms against cumulative endo-lysosomal stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) E3 ubiquitin-ligase that counteracts proteostasis stresses by facilitating endosomal cargo-selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations cause endosomal compartment stress by fusion and enlargement. Partial lysosomal clearance of mutant endosomal MLC1 is accomplished by the endosomal QC ubiquitin ligases, CHIP and Ubr1 via ESCRT-dependent route. As a consequence of the endosomal stress, a supportive QC mechanism, dependent on both Ubr1 and SQSTM1/p62 activities, targets ubiquitinated and arginylated MLC1 mutants for selective endosomal autophagy (endophagy). This QC pathway is also activated for arginylated Ubr1-SQSTM1/p62 autophagy cargoes during cytosolic Ca2+-assault. Conversely, the loss of Ubr1 and/or arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1 and arginylation-dependent endophagy/autophagy during endo-lysosomal proteostasis perturbations and suggest a link of Ubr1 to Ca2+ homeostasis and proteins implicated in various diseases including cancers and brain disorders.

Keywords:  Lysosome; Protein homeostasis network; Protein stability; Regeneration; Reprogramming; Stress response

DOI:  https://doi.org/10.1007/s00018-022-04191-8
Epilepsy Res. 2022 Feb 18. pii: S0920-1211(22)00041-9. [Epub ahead of print]181 106890

Inhibition of MEK-ERK signaling reduces seizures in two mouse models of tuberous sclerosis complex.

Lena H Nguyen, Steven C Leiser, Dekun Song, Daniela Brunner, Steven L Roberds, Michael Wong, Angelique Bordey.

  Tuberous sclerosis complex (TSC) is a monogenic disorder characterized by hyperactivation of the mTOR signaling pathway and developmental brain malformations leading to intractable epilepsy. Although treatment with the recently approved mTOR inhibitor, everolimus, results in clinically relevant seizure suppression in up to 40% of TSC patients, seizures remain uncontrolled in a large number of cases, underscoring the need to identify novel treatment targets. The MEK-ERK signaling pathway has been found to be aberrantly activated in TSC and inhibition of MEK-ERK activity independently of mTOR rescued neuronal dendrite overgrowth in mice modeling TSC neuropathology. Here, we evaluated the efficacy of MEK-ERK inhibition on seizures in two mouse models of TSC. We found that treatment with the MEK inhibitor PD0325901 (mirdametinib) significantly reduced seizure activity in both TSC mouse models. These findings support inhibiting MEK-ERK activity as a potential alternative strategy to treat seizures in TSC.

Keywords:  Epilepsy; MAPK; MEK inhibitor; MEK-ERK signaling; Seizures; Tuberous sclerosis complex

DOI:  https://doi.org/10.1016/j.eplepsyres.2022.106890
PLoS One. 2022 ;17(3): e0254469

Deficiency in Lyst function leads to accumulation of secreted proteases and reduced retinal adhesion.

Xiaojie Ji, Lihong Zhao, Ankita Umapathy, Bernard Fitzmaurice, Jieping Wang, David S Williams, Bo Chang, Jürgen K Naggert, Patsy M Nishina.

Chediak-Higashi syndrome, caused by mutations in the Lysosome Trafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak-Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak-Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium of Lyst mutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused by Lyst deficiency. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak-Higashi syndrome pathology.

DOI: https://doi.org/10.1371/journal.pone.0254469
Curr Opin Pharmacol. 2022 Mar 01. pii: S1471-4892(22)00019-4. [Epub ahead of print]63 102193

The mechanistic target of rapamycin as a regulator of metabolic function in oligodendroglia during remyelination.

Marie L Mather, Marisa A Jeffries, Teresa L Wood.

Despite evidence for prominent metabolic dysfunction within multiple sclerosis (MS) lesions, the mechanisms controlling metabolic shifts in oligodendroglia are poorly understood. The cuprizone model of demyelination and remyelination is a valuable tool for assessing metabolic insult during oligodendrocyte death and myelin degradation, closely resembling the distal oligodendrogliopathy seen in Pattern III MS lesions. In this review we discuss how metabolic processes in oligodendrocytes are disrupted in both MS and the cuprizone model, as well as the evidence for mechanistic target of rapamycin (mTOR) signaling as a key regulator of oligodendroglial metabolic function and efficient remyelination.

DOI: https://doi.org/10.1016/j.coph.2022.102193
Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2107453119

O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.

Liming Wu, Yaxian Cheng, Didi Geng, Zhiya Fan, Bingyi Lin, Qiang Zhu, Jingchao Li, Weijie Qin, Wen Yi.

  SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.

Keywords:  EGFR; O-GlcNAcylation; endosomal sorting; membrane receptors

DOI:  https://doi.org/10.1073/pnas.2107453119
Biochem Soc Trans. 2022 Feb 28. 50(1): 621-632

Modeling Parkinson's disease in LRRK2 mice: focus on synaptic dysfunction and the autophagy-lysosomal pathway.

Federica Albanese, Chiara Domenicale, Mattia Volta, Michele Morari.

  Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with familial and sporadic forms of Parkinson's disease (PD), for which the LRRK2 locus itself represents a risk factor. Idiopathic and LRRK2-related PD share the main clinical and neuropathological features, thus animals harboring the most common LRRK2 mutations, i.e. G2019S and R1441C/G, have been generated to replicate the parkinsonian phenotype and investigate the underlying pathological mechanisms. Most LRRK2 rodent models, however, fail to show the main neuropathological hallmarks of the disease i.e. the degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of Lewy bodies or Lewy body-like aggregates of α-synuclein, lacking face validity. Rather, they manifest dysregulation in cellular pathways and functions that confer susceptibility to a variety of parkinsonian toxins/triggers and model the presymptomatic/premotor stages of the disease. Among such susceptibility factors, dysregulation of synaptic activity and proteostasis are evident in LRRK2 mutants. These abnormalities are also manifest in the PD brain and represent key events in the development and progression of the pathology. The present minireview covers recent articles (2018-2021) investigating the role of LRRK2 and LRRK2 mutants in the regulation of synaptic activity and autophagy-lysosomal pathway. These articles confirm a perturbation of synaptic vesicle endocytosis and glutamate release in LRRK2 mutants. Likewise, LRRK2 mutants show a marked impairment of selective forms of autophagy (i.e. mitophagy and chaperone-mediated autophagy) and lysosomal function, with minimal perturbations of nonselective autophagy. Thus, LRRK2 rodents might help understand the contribution of these pathways to PD.

Keywords:  Parkinson's disease; autophagy; leucine rich repeat kinase; lysosomes; synaptic transmission

DOI:  https://doi.org/10.1042/BST20211288
Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2107357119

Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.

Xiaolong Fu, Peipei Li, Linqing Zhang, Yuning Song, Yachun An, Aizhen Zhang, Wenwen Liu, Chao Ye, Yuan Zhang, Rongyu Yue, Xiaoyang Sun, Renjie Chai, Haibo Wang, Jiangang Gao.

  Significance The mechanistic target of rapamycin (mTOR) plays a central role in growth, metabolism, and aging. It is assembled into two multiprotein complexes, namely, mTORC1 and mTORC2. We previously demonstrated the efficacy of sirolimus in ARHL in mice by decreasing mTORC1. However, the aspect of mTORC2 regulation in the cochlea is poorly characterized. Herein, based on pharmacological and genetic interventions, we found that a high dose of sirolimus resulted in severe hearing loss by reducing the mTORC2/AKT signaling pathway in the cochlea. Furthermore, selective activation of mTORC2 could protect against hearing loss induced by acoustic trauma and cisplatin-induced ototoxicity. Hence, the therapeutic activation of mTORC2 in conjunction with decreasing mTORC1 might represent a promising and effective strategy in preventing hearing loss.

Keywords:  hair cells; hearing; mTORC2

DOI:  https://doi.org/10.1073/pnas.2107357119
Cell Rep. 2022 Mar 01. pii: S2211-1247(22)00147-4. [Epub ahead of print]38(9): 110423

Cholesterol biosynthesis defines oligodendrocyte precursor heterogeneity between brain and spinal cord.

Luipa Khandker, Marisa A Jeffries, Yun-Juan Chang, Marie L Mather, Angelina V Evangelou, Jennifer N Bourne, Azadeh K Tafreshi, Isis M Ornelas, Ozlem Bozdagi-Gunal, Wendy B Macklin, Teresa L Wood.

  Brain and spinal cord oligodendroglia have distinct functional characteristics, and cell-autonomous loss of individual genes can result in different regional phenotypes. However, a molecular basis for these distinctions is unknown. Using single-cell analysis of oligodendroglia during developmental myelination, we demonstrate that brain and spinal cord precursors are transcriptionally distinct, defined predominantly by cholesterol biosynthesis. We further identify the mechanistic target of rapamycin (mTOR) as a major regulator promoting cholesterol biosynthesis in oligodendroglia. Oligodendroglia-specific loss of mTOR decreases cholesterol biosynthesis in both the brain and the spinal cord, but mTOR loss in spinal cord oligodendroglia has a greater impact on cholesterol biosynthesis, consistent with more pronounced deficits in developmental myelination. In the brain, mTOR loss results in a later adult myelin deficit, including oligodendrocyte death, spontaneous demyelination, and impaired axonal function, demonstrating that mTOR is required for myelin maintenance in the adult brain.

Keywords:  cholesterol; mTOR; myelin; myelin maintenance; myelination; oligodendrocyte; oligodendrocyte heterogeneity; oligodendrocyte precursor; single-cell sequencing

DOI:  https://doi.org/10.1016/j.celrep.2022.110423
Cell Discov. 2022 Mar 01. 8(1): 21

Targeting macrophage TFEB-14-3-3 epsilon Interface by naringenin inhibits abdominal aortic aneurysm.

Yiting Jia, Lu Zhang, Ziyi Liu, Chenfeng Mao, Zihan Ma, Wenqiang Li, Fang Yu, Yingbao Wang, Yaqian Huang, Weizhen Zhang, Jingang Zheng, Xian Wang, Qingbo Xu, Jian Zhang, Wei Feng, Caihong Yun, Chuanju Liu, Jinpeng Sun, Yi Fu, Qinghua Cui, Wei Kong.

Abdominal aortic aneurysm (AAA) is a lethal cardiovascular disease, and there is no proven drug treatment for this condition. In this study, by using the Connectivity Map (CMap) approach, we explored naringenin, a naturally occurring citrus flavonoid, as a putative agent for inhibiting AAA. We then validated the prediction with two independent mouse models of AAA, calcium phosphate (CaPO4)-induced C57BL/6J mice and angiotensin II-infused ApoE-/- mice. Naringenin effectively blocked the formation of AAAs and the progression of established AAAs. Transcription factor EB (TFEB) is the master regulator of lysosome biogenesis. Intriguingly, the protective role of naringenin on AAA was abolished by macrophage-specific TFEB depletion in mice. Unbiased interactomics, combined with isothermal titration calorimetry (ITC) and cellular thermal shift assays (CETSAs), further revealed that naringenin is directly bound to 14-3-3 epsilon blocked the TFEB-14-3-3 epsilon interaction, and therefore promoted TFEB nuclear translocation and activation. On one hand, naringenin activated lysosome-dependent inhibition of the NLRP3 inflammasome and repressed aneurysmal inflammation. On the other hand, naringenin induced TFEB-dependent transcriptional activation of GATA3, IRF4, and STAT6 and therefore promoted reparative M2 macrophage polarization. In summary, naturally derived naringenin or macrophage TFEB activation shows promising efficacy for the treatment of AAA.

DOI: https://doi.org/10.1038/s41421-021-00363-1
Cell Mol Life Sci. 2022 Feb 28. 79(3): 162

The Alzheimer's gene SORL1 is a regulator of endosomal traffic and recycling in human neurons.

Swati Mishra, Allison Knupp, Marcell P Szabo, Charles A Williams, Chizuru Kinoshita, Dale W Hailey, Yuliang Wang, Olav M Andersen, Jessica E Young.

   BACKGROUND: Loss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimer's disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, AD's cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell-derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1).
METHODS: We used isogenic hiPSCs engineered to have SORL1 depleted or to have enhanced SORL1 expression. We differentiated neurons from these cell lines and mapped the trafficking of APP, TRKB and GLUA1 within the endosomal network using confocal microscopy. We also performed cell surface recycling and lysosomal degradation assays to assess the functionality of the endosomal network in both SORL1-depleted and -overexpressing neurons. The functional impact of GLUA1 recycling was determined by measuring synaptic activity. Finally, we analyzed alterations in gene expression in SORL1-depleted neurons using RNA sequencing.
RESULTS: We find that as with APP, endosomal trafficking of GLUA1 and TRKB is impaired by loss of SORL1. We show that trafficking of all three cargoes to late endosomes and lysosomes is affected by manipulating SORL1 expression. We also show that depletion of SORL1 significantly impacts the endosomal recycling pathway for APP and GLUA1 at the level of the recycling endosome and trafficking to the cell surface. This has a functional effect on neuronal activity as shown by multi-electrode array (MEA). Conversely, increased SORL1 expression enhances endosomal recycling for APP and GLUA1. Our unbiased transcriptomic data further support SORL1's role in endosomal recycling. We observe altered expression networks that regulate cell surface trafficking and neurotrophic signaling in SORL1-depleted neurons.
CONCLUSION: Collectively, and together with other recent observations, these findings suggest that one role for SORL1 is to contribute to endosomal degradation and recycling pathways in neurons, a conclusion that has both pathogenic and therapeutic implications for Alzheimer's disease.

Keywords:  Alzheimer’s disease; Endosomal network; Human induced pluripotent stem cells; Neurons; SORL1

DOI:  https://doi.org/10.1007/s00018-022-04182-9
J Clin Invest. 2022 Mar 03. pii: e146536. [Epub ahead of print]

Up-regulated YB-1 protein promotes glioblastoma growth through an YB-1/CCT4/mLST8/mTOR pathway.

Jin-Zhu Wang, Hong Zhu, Pu You, Hui Liu, Wei-Kang Wang, Xiaojuan Fan, Yun Yang, Keren Xu, Yingfeng Zhu, Qunyi Li, Ping Wu, Chao Peng, Catherine Cl Wong, Kaicheng Li, Yufeng Shi, Nu Zhang, Xiuxing Wang, Rong Zeng, Ying Huang, Liusong Yang, Zefeng Wang, Jingyi Hui.

  The Y-box binding protein 1 (YB-1) is a multi-functional RNA binding protein involved in virtually each step of RNA metabolism. However, the functions and mechanisms of YB-1 in one of the most aggressive cancers, glioblastoma, are not well understood. In this study, we identified that YB-1 protein was markedly overexpressed in glioblastoma and acted as a critical activator of both mTORC1 and mTORC2 signaling. Mechanistically, YB-1 bound the 5' untranslated region (UTR) of the CCT4 mRNA to promote the translation of CCT4, a component of CCT chaperone complex, that in turn activated the mTOR signal pathway by promoting mLST8 folding. In addition, YB-1 autoregulated its own translation by binding to its 5' UTR, leading to sustained activation of mTOR signaling. In glioblastoma patients, the protein level of YB-1 positively correlated with CCT4 and mLST8 expression as well as activated mTOR signaling. Importantly, the administration of RNA decoys specifically targeting YB-1 in a mouse xenograft model resulted in slower tumor growth and better survival. Taken together, these findings uncover a disrupted proteostasis pathway involving YB-1/CCT4/mLST8/mTOR axis in promoting glioblastoma growth, suggesting that YB-1 is a potential therapeutic target for the treatment of glioblastoma.

Keywords:  Molecular biology; Oncology; Signal transduction; Translation

DOI:  https://doi.org/10.1172/JCI146536
Cell Rep. 2022 Mar 01. pii: S2211-1247(22)00168-1. [Epub ahead of print]38(9): 110441

Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

Mario A Shields, Christina Spaulding, Anastasia E Metropulos, Mahmoud G Khalafalla, Thao N D Pham, Hidayatullah G Munshi.

  Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

Keywords:  E-cadherin; Gα13; KC mouse model; KPC mouse model; human PDAC tumors; mTOR; rapamycin

DOI:  https://doi.org/10.1016/j.celrep.2022.110441
J Vis Exp. 2022 Feb 12.

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking.

Anshul Milap Bhatt, Subba Rao Gangi Setty.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

DOI: https://doi.org/10.3791/63087
Acta Neuropathol Commun. 2022 Mar 03. 10(1): 27

Tuberous sclerosis complex is a novel, amyloid-independent tauopathy associated with elevated phosphorylated 3R/4R tau aggregation.

Andy J Liu, Jay B Lusk, John Ervin, James Burke, Richard O'Brien, Shih-Hsiu J Wang.

  Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 and TSC2 genes and autosomal dominantly inherited. These mutations cause hyperactivation of the mammalian Target of Rapamycin (mTOR) pathway, leading to the development of nonmalignant masses involving various organ systems. Patients with TSC also experience neuropsychiatric symptoms collectively termed Tuberous Sclerosis Complex Associated Neuropsychiatric Disorder (TAND). Due to research advancements in TSC, patients now live well beyond the age of 50. Many experience objective impairment of memory and executive function, supported by formal neuropsychological testing, beginning in their late 40s. Biomarker analysis has described elevated levels of phosphorylated tau-181 in the cerebrospinal fluid of patients with TAND. Tau-PET imaging has also shown focal accumulation of the radiotracer flortaucipir (AV1451), suggesting that TSC may be a neurodegenerative disorder arising from accumulation of phosphorylated tau. However, the flortaucipir tracer has been reported to have significant off-target binding, preventing definitive conclusions from being drawn about the molecular etiology of neurodegeneration in TSC. Therefore, we initiated the Colocalization of AV1451 and Phosphorylated Tau in Adult brain tissue (CAPA) study. This study aimed to determine if flortaucipir is bound to phosphorylated tau in brains of patients with TSC and further sought to determine the specific tau isoform seen in TSC. Our results show that flortaucipir labels the 3R/4R isoform of phosphorylated tau, commonly seen in Alzheimer's disease. However, amyloid staining was negative in brains of adult patients with TSC. Therefore, we conclude that TAND symptoms are due to the accumulation of the phosphorylated tau isoform seen in Alzheimer's disease. This study suggests that hyperactivation of the mammalian Target of Rapamycin pathway may play a role in the amyloid-independent development of 3R/4R tau aggregation. Our findings could lead to a new era of anti-tau therapies used to treat both disorders.

Keywords:  3R tauopathy; 4R tauopathy; Alzheimer’s disease; Phosphorylated tau; Tauopathy; Tuberous Sclerosis Complex

DOI:  https://doi.org/10.1186/s40478-022-01330-x
iScience. 2022 Mar 18. 25(3): 103932

Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling.

Federica Agliano, Timofey A Karginov, Antoine Ménoret, Anthony Provatas, Anthony T Vella.

  Nicotinamide (NAM) shapes T cell responses but its precise molecular mechanism of action remains elusive. Here, we show that NAM impairs naive T cell effector transition but also effector T cells themselves. Although aerobic glycolysis is a hallmark of activated T cells, CD8+ T cells exposed to NAM displayed enhanced glycolysis, yet producing significantly less IFNγ. Mechanistically, NAM reduced mTORC1 activity independently of NAD+ metabolism, decreasing IFNγ translation and regulating T cell transcriptional factors critical to effector/memory fate. Finally, the role of NAM in a biomedically relevant model of lung injury was tested. Specifically, a NAM-supplemented diet reduced systemic IL-2, antigen-specific T cell clonal expansion, and effector function after inhalation of Staphylococcus aureus enterotoxin A. These findings identify NAM as a potential therapeutic supplement that uncouples glycolysis from effector cytokine production and may be a powerful treatment for diseases associated with T cell hyperactivation.

Keywords:  Biological sciences; Immunology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2022.103932
Autophagy. 2022 Feb 28. 1-14

Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations.

Jing Xu, Kevin C Yang, Nancy Erro Go, Shane Colborne, Cally J Ho, Elham Hosseini-Beheshti, Alf H Lystad, Anne Simonsen, Emma Tomlinson Guns, Gregg B Morin, Sharon M Gorski.

  Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4',6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell.

Keywords:  ATG16L1; Atg8; CD63; MAP1LC3B; SDCBP/syntenin-1; autophagy; chloroquine; endosome; extracellular vesicle; lysosome

DOI:  https://doi.org/10.1080/15548627.2022.2039535
J Cell Biol. 2022 Apr 04. pii: e201808181. [Epub ahead of print]221(4):

Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling.

Rebecca Cabral-Dias, Stefanie Lucarelli, Karolina Zak, Sadia Rahmani, Gurjeet Judge, John Abousawan, Laura F DiGiovanni, Dafne Vural, Karen E Anderson, Michael G Sugiyama, Gizem Genc, Wanjin Hong, Roberto J Botelho, Gregory D Fairn, Peter K Kim, Costin N Antonescu.

The epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology. EGF binding to EGFR elicits the membrane recruitment and activation of phosphatidylinositol-3-kinase, leading to Akt phosphorylation and activation. Concomitantly, EGFR is recruited to clathrin-coated pits (CCPs), eventually leading to receptor endocytosis. Previous work uncovered that clathrin, but not receptor endocytosis, is required for EGF-stimulated Akt activation, and that some EGFR signals are enriched in CCPs. Here, we examine how CCPs control EGFR signaling. The signaling adaptor TOM1L1 and the Src-family kinase Fyn are enriched within a subset of CCPs with unique lifetimes and protein composition. Perturbation of TOM1L1 or Fyn impairs EGF-stimulated phosphorylation of Akt2 but not Akt1. EGF stimulation also triggered the TOM1L1- and Fyn-dependent recruitment of the phosphoinositide 5-phosphatase SHIP2 to CCPs. Thus, the recruitment of TOM1L1 and Fyn to a subset of CCPs underlies a role for these structures in the support of EGFR signaling leading to Akt activation.

DOI: https://doi.org/10.1083/jcb.201808181
J Neurosci. 2022 Mar 02. pii: JN-RM-2439-21. [Epub ahead of print]

CLC anion/proton exchangers regulate secretory vesicle filling and granule exocytosis in chromaffin cells.

Maddalena Comini, Juan Sierra-Marquez, Gustavo A Guzman, Arne Franzen, Antje Willuweit, Istvan Katona, Patricia Hidalgo, Christoph Fahlke, Raul E Guzman.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knockout animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock-out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent down-regulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENTCl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells, however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

DOI: https://doi.org/10.1523/JNEUROSCI.2439-21.2022
Sci Rep. 2022 Mar 01. 12(1): 3350

Reduced ech-6 expression attenuates fat-induced lifespan shortening in C. elegans.

Yasmine J Liu, Arwen W Gao, Reuben L Smith, Georges E Janssens, Daan M Panneman, Aldo Jongejan, Michel van Weeghel, Frédéric M Vaz, Melissa J Silvestrini, Louis R Lapierre, Alyson W MacInnes, Riekelt H Houtkooper.

Deregulated energy homeostasis represents a hallmark of aging and results from complex gene-by-environment interactions. Here, we discovered that reducing the expression of the gene ech-6 encoding enoyl-CoA hydratase remitted fat diet-induced deleterious effects on lifespan in Caenorhabditis elegans, while a basal expression of ech-6 was important for survival under normal dietary conditions. Lipidomics revealed that supplementation of fat in ech-6-silenced worms had marginal effects on lipid profiles, suggesting an alternative fat utilization for energy production. Transcriptomics further suggest a causal relation between the lysosomal pathway, energy production, and the longevity effect conferred by the interaction between ech-6 and fat diets. Indeed, enhancing energy production from endogenous fat by overexpressing lysosomal lipase lipl-4 recapitulated the lifespan effects of fat diets on ech-6-silenced worms. Collectively, these results suggest that the gene ech-6 is potential modulator of metabolic flexibility and may be a target for promoting metabolic health and longevity.

DOI: https://doi.org/10.1038/s41598-022-07397-9
J Exp Med. 2022 Mar 07. pii: e20211057. [Epub ahead of print]219(3):

Molecular architecture determines brain delivery of a transferrin receptor-targeted lysosomal enzyme.

Annie Arguello, Cathal S Mahon, Meredith E K Calvert, Darren Chan, Jason C Dugas, Michelle E Pizzo, Elliot R Thomsen, Roni Chau, Lorna A Damo, Joseph Duque, Meng Fang, Tina Giese, Do Jin Kim, Nicholas Liang, Hoang N Nguyen, Hilda Solanoy, Buyankhishig Tsogtbaatar, Julie C Ullman, Junhua Wang, Mark S Dennis, Dolores Diaz, Kannan Gunasekaran, Kirk R Henne, Joseph W Lewcock, Pascal E Sanchez, Matthew D Troyer, Jeffrey M Harris, Kimberly Scearce-Levie, Lu Shan, Ryan J Watts, Robert G Thorne, Anastasia G Henry, Mihalis S Kariolis.

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

DOI: https://doi.org/10.1084/jem.20211057
PLoS Biol. 2022 Mar;20(3): e3001548

The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast.

Sylvain Tollis, Jaspal Singh, Roger Palou, Yogitha Thattikota, Ghada Ghazal, Jasmin Coulombe-Huntington, Xiaojing Tang, Susan Moore, Deborah Blake, Eric Bonneil, Catherine A Royer, Pierre Thibault, Mike Tyers.

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions.

DOI: https://doi.org/10.1371/journal.pbio.3001548
Mol Genet Metab Rep. 2022 Jun;31 100851

Reduced α-galactosidase A activity in zebrafish (Danio rerio) mirrors distinct features of Fabry nephropathy phenotype.

Hassan O A Elsaid, Jessica Furriol, Maria Blomqvist, Mette Diswall, Sabine Leh, Naouel Gharbi, Jan Haug Anonsen, Janka Babickova, Camilla Tøndel, Einar Svarstad, Hans-Peter Marti, Maximilian Krause.

  Fabry disease (FD) is a rare genetic lysosomal storage disorder, resulting from partial or complete lack of alpha-galactosidase A (α-GAL) enzyme, leading to systemic accumulation of substrate glycosphingolipids with a broad range of tissue damage. Current in vivo models are laborious, expensive, and fail to adequately mirror the complex FD physiopathology. To address these issues, we developed an innovative FD model in zebrafish. Zebrafish GLA gene encoding α-GAL enzyme presents a high (>70%) homology with its human counterpart, and the corresponding protein has a similar tissue distribution, as evaluated by immunohistochemistry. Moreover, a similar enzymatic activity in different life stages could be demonstrated. By using CRISPR/Cas9 technology, we generated a mutant zebrafish with decreased GLA gene expression, and decreased expression of the specific gene product in the kidney. Mutant animals showed higher plasma creatinine levels and proteinuria. Transmission electron microscopy (TEM) studies documented an increased podocyte foot process width (FPW) in mutant, as compared to wild type zebrafish. This zebrafish model reliably mirrors distinct features of human FD and could be advantageously used for the identification of novel biomarkers and for an effective screening of innovative therapeutic approaches.

Keywords:  Fabry disease; GLA; Zebrafish; α-GAL activity; α-Galactosidase A

DOI:  https://doi.org/10.1016/j.ymgmr.2022.100851
Biochem Pharmacol. 2022 Feb 25. pii: S0006-2952(22)00077-6. [Epub ahead of print]198 114983

ROS-mediated activation of p38 protects hepatocellular carcinoma cells from caspase-independent death elicited by lysosomal damage.

Serena Castelli, Enrico Desideri, Maria Rosa Ciriolo.

  Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancer in the world. Despite this, few effective drugs are available for its treatment, in part due to the development of resistance, and surgical resection remains the most valuable option, when applicable. Upregulation of anti-apoptotic proteins, downregulation of pro-apoptotic factors and the acquisition of mutations in signaling pathways leading to caspase activation are a few examples of mechanisms that allow cancer cells to evade caspase-dependent apoptosis and continue to grow. The identification of drugs triggering the activation of caspase-independent death may therefore be an effective strategy to circumvent resistance and kill cancer cells. Here, we show that the lysosome damaging compound glycyl-l-phenylalanine 2-naphthylamide (GPN) induces cell death by a caspase-independent mechanism in HCC cell lines. Additionally, we identify the MAPK p38 as a novel mediator of the lysosomal stress response. Indeed, a ROS-dependent activation of p38 occurs in response to lysosomal damage, promoting the recovery of lysosomal integrity. As a consequence, pharmacological or genetic inhibition of p38 increases cell death elicited by GPN. Our findings identify p38 as a potential target to potentiate the cytotoxic effects of lysosomal damage and induce caspase-independent cell death in HCC cells, laying the ground for future evaluation of the efficacy of combination therapy.

Keywords:  Caspase-independent death; Hepatocellular carcinoma; Lysosomal stress response; P38; Reactive oxygen species

DOI:  https://doi.org/10.1016/j.bcp.2022.114983
Cell Death Discov. 2022 Feb 28. 8(1): 91

Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) expression in glioblastoma is driven by ETS1- and MYBL2-dependent transcriptional activation.

Laura Hauffe, Daniel Picard, Julian Musa, Marc Remke, Thomas G P Grünewald, Barak Rotblat, Guido Reifenberger, Gabriel Leprivier.

Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) encodes the 4EBP1 protein, a negative regulator of mRNA translation and a substrate of the mechanistic target of rapamycin (mTOR), whose function and relevance in cancer is still under debate. Here, we analyzed EIF4EBP1 expression in different glioma patient cohorts and investigated its mode of transcriptional regulation in glioblastoma cells. We verified that EIF4EBP1 mRNA is overexpressed in malignant gliomas, including isocitrate dehydrogenase (IDH)-wildtype glioblastomas, relative to non-neoplastic brain tissue in multiple publically available datasets. Our analyses revealed that EIF4EBP1 overexpression in malignant gliomas is neither due to gene amplification nor to altered DNA methylation, but rather results from aberrant transcriptional activation by distinct transcription factors. We found seven transcription factor candidates co-expressed with EIF4EBP1 in gliomas and bound to the EIF4EBP1 promoter, as revealed by chromatin immunoprecipitation (ChIP)-sequencing data. We investigated the ability of these candidates to activate the EIF4EBP1 promoter using luciferase reporter assays, which supported four transcription factors as candidate EIF4EBP1 regulators, namely MYBL2, ETS1, HIF-1A, and E2F6. Finally, by employing transient knock-down experiments to repress either of these transcription factors, we identified MYBL2 and ETS1 as the relevant transcriptional drivers of enhanced EIF4EBP1 expression in malignant glioma cells. Taken together, our findings confirm enhanced expression of EIF4EBP1 in malignant gliomas relative to non-neoplastic brain tissue and characterize the underlying molecular pathomechanisms.

DOI: https://doi.org/10.1038/s41420-022-00883-z
Curr Biol. 2022 Feb 25. pii: S0960-9822(22)00243-3. [Epub ahead of print]

Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila.

Michael J Texada, Mette Lassen, Lisa H Pedersen, Takashi Koyama, Alina Malita, Kim Rewitz.

  Nutrition is one of the most important influences on growth and the timing of maturational transitions including mammalian puberty and insect metamorphosis. Childhood obesity is associated with precocious puberty, but the assessment mechanism that links body fat to early maturation is unknown. During development, the intake of nutrients promotes signaling through insulin-like systems that govern the growth of cells and tissues and also regulates the timely production of the steroid hormones that initiate the juvenile-adult transition. We show here that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty.

Keywords:  Adipose; Developmental timing; Ecdysone; Glia; NPC1; Nutrients; Nutrition; TOR

DOI:  https://doi.org/10.1016/j.cub.2022.02.021
J Biol Chem. 2022 Feb 26. pii: S0021-9258(22)00220-4. [Epub ahead of print] 101780

In Situ Artificial Contact Sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein tethering activities.

Julie Milanini, Maud Magdeleine, Nicolas Fuggetta, Souade Ikhlef, Frédéric Brau, Sophie Abelanet, Fabien Alpy, Catherine Tomasetto, Guillaume Drin.

  Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum (ER) via the specific interaction of the FFAT motif with ER-resident vesicle-associated membrane protein-associated proteins (VAPs). This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins, and of a protein construct derived from steroidogenic acute regulatory protein-related lipid transfer domain-3 (STARD3). Collectively, these data indicate that the creation of ISACS represents an efficient approach for studying the membrane tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.

Keywords:  Cell biology; Confocal microscopy; Endoplasmic reticulum (ER); FFAT motif; Lipid transport; Liposome; Membrane contact sites; OSBP; STARD3; VAP

DOI:  https://doi.org/10.1016/j.jbc.2022.101780
Curr Opin Cell Biol. 2022 Feb 25. pii: S0955-0674(22)00007-2. [Epub ahead of print]74 104-111

Compartmentation and functions of sphingolipids.

Carolin Körner, Florian Fröhlich.

Sphingolipids (SLs) are one of the three major lipid classes in all eukaryotic cells. They function as structural molecules of membranes and can also act as highly active signaling molecules. SL biosynthesis is mainly occurring at the endoplasmic reticulum and the Golgi apparatus. However, SL intermediates are also generated at other organelles such as the plasma membrane and the lysosome. SL biosynthesis is therefore highly compartmentalized. Maintaining SL levels is necessary for the function of multiple trafficking pathways. One major challenge is to decipher the complex regulatory networks controlling SL biosynthesis, the coordination of vesicular and non-vesicular SL transport as well as their role in trafficking. Recent investigations have shed new light on the regulation of SL biosynthesis. Here, we review how SL biosynthesis is coordinated, how SLs are transported and how their levels affect trafficking pathways. Finally, we discuss recently developed methods to study SL metabolism with spatio-temporal resolution.

DOI: https://doi.org/10.1016/j.ceb.2022.01.006
Cell Calcium. 2022 Feb 18. pii: S0143-4160(22)00039-2. [Epub ahead of print]103 102564

Dynamin 1 controls vesicle size and endocytosis at hippocampal synapses.

Bo Shi, Ying-Hui Jin, Ling-Gang Wu.

  Following calcium-triggered vesicle exocytosis, endocytosis regenerates vesicles to maintain exocytosis and thus synaptic transmission, which underlies neuronal circuit activities. Although most molecules involved in endocytosis have been identified, it remains rather poorly understood how endocytic machinery regulates vesicle size. Vesicle size, together with the transmitter concentration inside the vesicle, determines the amount of transmitter the vesicle can release, the quantal size, that may control the strength of synaptic transmission. Here, we report that, surprisingly, knockout of the GTPase dynamin 1, the most abundant brain dynamin isoform known to catalyze fission of the membrane pit's neck (the last step of endocytosis), not only significantly slowed endocytosis but also increased the synaptic vesicle diameter by as much as ∼40-64% at cultured hippocampal synapses. Furthermore, dynamin 1 knockout increased the size of membrane pits, the precursor for endocytic vesicle formation. These results suggest an important function of dynamin other than its well-known fission function - control of vesicle size at the pit formation stage.

Keywords:  Dynamin; Endocytosis; Hippocampus; Synaptic transmission; Vesicle recycling; Vesicle size

DOI:  https://doi.org/10.1016/j.ceca.2022.102564
Mol Cell Proteomics. 2022 Feb 25. pii: S1535-9476(22)00029-9. [Epub ahead of print] 100221

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases.

Hanna G Budayeva, Arundhati Sengupta-Ghosh, Lilian Phu, John G Moffat, Gai Ayalon, Donald S Kirkpatrick.

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed two decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its co-receptor Lrp4 to delay the onset of muscle denervation in mouse models of amyotrophic lateral sclerosis (ALS). Here we performed dose response and time course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK kinase inhibition. Importantly, mutation of this site in Rab10 disrupts association with adaptor molecules Mical1/3. Together these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.

DOI: https://doi.org/10.1016/j.mcpro.2022.100221
Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2110415119

Amino acid sensor conserved from bacteria to humans.

Vadim M Gumerov, Ekaterina P Andrianova, Miguel A Matilla, Karen M Page, Elizabet Monteagudo-Cascales, Annette C Dolphin, Tino Krell, Igor B Zhulin.

  SignificanceAmino acids are the building blocks of life and important signaling molecules. Despite their common structure, no universal mechanism for amino acid recognition by cellular receptors is currently known. We discovered a simple motif, which binds amino acids in various receptor proteins from all major life-forms. In humans, this motif is found in subunits of calcium channels that are implicated in pain and neurodevelopmental disorders. Our findings suggest that γ-aminobutyric acid-derived drugs bind to the same motif in human proteins that binds natural ligands in bacterial receptors, thus enabling future improvement of important drugs.

Keywords:  evolution; gabapentin; ion channels; serine/threonine kinases; signal transduction

DOI:  https://doi.org/10.1073/pnas.2110415119