bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022‒02‒13
thirty-five papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Built to last: lysosome remodeling and repair in health and disease.
  2. Asymmetric Notch activity by differential inheritance of lysosomes in human neural stem cells.
  3. Structural mechanism of allosteric activation of TRPML1 by PI(3,5)P2 and rapamycin.
  4. Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport.
  5. Ppt1-deficiency dysregulates lysosomal Ca++ -homeostasis contributing to pathogenesis in a mouse model of CLN1 disease.
  6. Cryo-EM of the Yeast VO Complex Reveals Distinct Binding Sites for Macrolide V-ATPase Inhibitors.
  7. Mutant glucocerebrosidase impairs α-synuclein degradation by blockade of chaperone-mediated autophagy.
  8. TRPMLs and TPCs: Targets for lysosomal storage and neurodegenerative disease therapy?
  9. Lysosomal TRPML1 Channel: Implications in Cardiovascular and Kidney Diseases.
  10. Neuronal endolysosomal transport and lysosomal functionality in maintaining axonostasis.
  11. Activation of endo-lysosomal two-pore channels by NAADP and PI(3,5)P2. Five things to know.
  12. Correlation of age of onset and clinical severity in Niemann-Pick disease type C1 with lysosomal abnormalities and gene expression.
  13. Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.
  14. Multisystem involvement, defective lysosomes, and impaired autophagy in a novel rat model of Nephropathic Cystinosis.
  15. NPRL3 loss alters neuronal morphology, mTOR localization, cortical lamination, and seizure threshold.
  16. Disrupting the MYC-TFEB Circuit Impairs Amino Acid Homeostasis and Provokes Metabolic Anergy.
  17. Rap_GAP Domain of TSC2 Contributes to Tumor Suppression Through mTOR Signaling in Human Hepatocellular Carcinoma.
  18. Novel frameshift CTSF mutation causing kufs disease type B mimicking frontotemporal dementia-parkinsonism.
  19. Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease.
  20. Role of mTORC1 activity during early retinal development and lamination in human-induced pluripotent stem cell-derived retinal organoids.
  21. Genotype-phenotype spectrum of 130 unrelated Indian families with Mucopolysaccharidosis type II.
  22. In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells.
  23. Low-Density Lipoprotein Internalization, Degradation and Receptor Recycling Along Membrane Contact Sites.
  24. Exosome-mediated miR-7-5p delivery enhances the anticancer effect of Everolimus via blocking MNK/eIF4E axis in non-small cell lung cancer.
  25. Endosomes supporting fusion mediated by vesicular stomatitis virus glycoprotein have distinctive motion and acidification.
  26. mTOR inhibition by TAK-228 is effective against growth, survival and angiogenesis in preclinical retinoblastoma models.
  27. The different autophagy degradation pathways and neurodegeneration.
  28. TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells.
  29. Chaperone-mediated autophagy controls proteomic and transcriptomic pathways to maintain glioma stem cell activity.
  30. mTOR inhibition prevents angiotensin II-induced aortic rupture and pseudoaneurysm but promotes dissection in Apoe-deficient mice.
  31. Stress hormone signaling inhibits Th1 polarization in a CD4 T-cell-intrinsic manner via mTORC1 and the circadian gene PER1.
  32. GLUT4 On the move.
  33. Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates.
  34. ESCRT dysfunction compromises endoplasmic reticulum maturation and autophagosome biogenesis in Drosophila.
  35. Caloric restriction has a new player.
  1. Trends Cell Biol. 2022 Feb 02. pii: S0962-8924(22)00001-0. [Epub ahead of print]
    Built to last: lysosome remodeling and repair in health and disease.
    Roberto Zoncu, Rushika M Perera.
      Lysosomes play major roles in growth regulation and catabolism and are recognized as critical mediators of cellular remodeling. An emerging theme is how the lysosome is itself subjected to extensive remodeling in order to perform specific tasks that meet the changing demands of the cell. Accordingly, lysosomes can sustain physical damage and undergo dramatic changes in composition following pathogen infection, accumulation of protein aggregates, or cellular transformation, necessitating dedicated pathways for their repair, remodeling, and restoration. In this review, we focus on emerging molecular mechanisms for piecemeal remodeling of lysosomal components and wholesale repair and discuss their implications in physiological and pathogenic challenges such as cancer, neurodegeneration, and pathogen infection.
    Keywords:  cancer; infection; lysosome; membrane damage; neurodegeneration; repair
    DOI:  https://doi.org/10.1016/j.tcb.2021.12.009
  2. Sci Adv. 2022 Feb 11. 8(6): eabl5792
    Asymmetric Notch activity by differential inheritance of lysosomes in human neural stem cells.
    Bettina Bohl, Ammar Jabali, Julia Ladewig, Philipp Koch.
      Symmetric and asymmetric cell divisions are conserved strategies for stem cell expansion and the generation of more committed progeny, respectively. Here, we demonstrate that in human neural stem cells (NSCs), lysosomes are asymmetrically inherited during mitosis. We show that lysosomes contain Notch receptors and that Notch activation occurs the acidic lysosome environment. The lysosome asymmetry correlates with the expression of the Notch target gene HES1 and the activity of Notch signaling in the daughter cells. Furthermore, an asymmetry of lysosomes and Notch receptors was also observed in a human organoid model of brain development with mitotic figures showing preferential inheritance of lysosomes and Notch receptor in that daughter cell remaining attached to the apical membrane. Thus, this study suggests a previously unknown function of lysosomes as a signaling hub to establish a bias in Notch signaling activity between daughter cells after an asymmetric cell division of human NSCs.
    DOI:  https://doi.org/10.1126/sciadv.abl5792
  3. Proc Natl Acad Sci U S A. 2022 Feb 15. pii: e2120404119. [Epub ahead of print]119(7):
    Structural mechanism of allosteric activation of TRPML1 by PI(3,5)P2 and rapamycin.
    Ninghai Gan, Yan Han, Weizhong Zeng, Yan Wang, Jing Xue, Youxing Jiang.
      Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-permeable, nonselective cation channel ubiquitously expressed in the endolysosomes of mammalian cells and its loss-of-function mutations are the direct cause of type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease. TRPML1 is a ligand-gated channel that can be activated by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] as well as some synthetic small-molecule agonists. Recently, rapamycin has also been shown to directly bind and activate TRPML1. Interestingly, both PI(3,5)P2 and rapamycin have low efficacy in channel activation individually but together they work cooperatively and activate the channel with high potency. To reveal the structural basis underlying the synergistic activation of TRPML1 by PI(3,5)P2 and rapamycin, we determined the high-resolution cryoelectron microscopy (cryo-EM) structures of the mouse TRPML1 channel in various states, including apo closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus (a rapamycin analog)-bound open states. These structures, combined with electrophysiology, elucidate the molecular details of ligand binding and provide structural insight into how the TRPML1 channel integrates two distantly bound ligand stimuli and facilitates channel opening.
    Keywords:  PI(3,5)P2; TRPML1; lysosomal channel; rapamycin
    DOI:  https://doi.org/10.1073/pnas.2120404119
  4. J Biol Chem. 2022 Feb 08. pii: S0021-9258(22)00142-9. [Epub ahead of print] 101702
    Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport.
    Shuo Du, Guopeng Wang, Zhiying Zhang, Chengying Ma, Ning Gao, Junyu Xiao.
      GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-Acetylglucosamine-1-Phosphate Transferase Subunits Alpha And Beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase (UGGT). Comparison with UGGT also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved, and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.
    Keywords:  CRISPR/Cas; cryo‐electron microscopy; glycoprotein; lysosomal storage disease; mannose‐6‐phosphate (M6P)
    DOI:  https://doi.org/10.1016/j.jbc.2022.101702
  5. J Inherit Metab Dis. 2022 Feb 12.
    Ppt1-deficiency dysregulates lysosomal Ca++ -homeostasis contributing to pathogenesis in a mouse model of CLN1 disease.
    Avisek Mondal, Abhilash P Appu, Tamal Sadhukhan, Maria B Bagh, Rafael M Previde, Sriparna Sadhukhan, Stanko Stojilkovic, Aiyi Liu, Anil B Mukherjee.
      Inactivating mutations in the PPT1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S-palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1-deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl-protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1-deficiency may impair lysosomal degradative function leading to INCL pathogenesis. Here we report that in Ppt1-/- mice, which mimic CLN1 disease, low level of inositol 3-phosphate receptor-1 (IP3R1) that mediates Ca++ -transport from the ER to the lysosome dysregulated lysosomal Ca++ homeostasis. Intriguingly, the transcription factor NFATC4, which regulates IP3R1-expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Ppt1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1-expression, thereby, dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++ -dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Ppt1-/- mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca++ -homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease. This article is protected by copyright. All rights reserved.
    Keywords:  Batten disease; Infantile neuronal ceroid lipofuscinosis; Lysosomal storage disease; Neurodegeneration; Neuronal ceroid lipofuscinosis; Palmitoyl-protein thioesterase-1; S-palmitoylation
    DOI:  https://doi.org/10.1002/jimd.12485
  6. ACS Chem Biol. 2022 Feb 11.
    Cryo-EM of the Yeast VO Complex Reveals Distinct Binding Sites for Macrolide V-ATPase Inhibitors.
    Kristine A Keon, Samir Benlekbir, Susanne H Kirsch, Rolf Müller, John L Rubinstein.
      Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V1 region that hydrolyzes ATP and a membrane-embedded VO region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small-molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for the treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated VO complex from Saccharomyces cerevisiae bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.
    DOI:  https://doi.org/10.1021/acschembio.1c00894
  7. Sci Adv. 2022 Feb 11. 8(6): eabm6393
    Mutant glucocerebrosidase impairs α-synuclein degradation by blockade of chaperone-mediated autophagy.
    Sheng-Han Kuo, Inmaculada Tasset, Melody M Cheng, Antonio Diaz, Ming-Kai Pan, Ori J Lieberman, Samantha J Hutten, Roy N Alcalay, Sangjun Kim, Pilar Ximénez-Embún, Li Fan, Donghoon Kim, Han Seok Ko, Talene Yacoubian, Ellen Kanter, Ling Liu, Guomei Tang, Javier Muñoz, Sergio Pablo Sardi, Aiqun Li, Li Gan, Ana Maria Cuervo, David Sulzer.
      The most common genetic risk factors for Parkinson's disease (PD) are a set of heterozygous mutant (MT) alleles of the GBA1 gene that encodes β-glucocerebrosidase (GCase), an enzyme normally trafficked through the ER/Golgi apparatus to the lysosomal lumen. We found that half of the GCase in lysosomes from postmortem human GBA-PD brains was present on the lysosomal surface and that this mislocalization depends on a pentapeptide motif in GCase used to target cytosolic protein for degradation by chaperone-mediated autophagy (CMA). MT GCase at the lysosomal surface inhibits CMA, causing accumulation of CMA substrates including α-synuclein. Single-cell transcriptional analysis and proteomics of brains from GBA-PD patients confirmed reduced CMA activity and proteome changes comparable to those in CMA-deficient mouse brain. Loss of the MT GCase CMA motif rescued primary substantia nigra dopaminergic neurons from MT GCase-induced neuronal death. We conclude that MT GBA1 alleles block CMA function and produce α-synuclein accumulation.
    DOI:  https://doi.org/10.1126/sciadv.abm6393
  8. Cell Calcium. 2022 Feb 05. pii: S0143-4160(22)00028-8. [Epub ahead of print]103 102553
    TRPMLs and TPCs: Targets for lysosomal storage and neurodegenerative disease therapy?
    Einar Krogsaeter, Anna Scotto Rosato, Christian Grimm.
      Neurodegenerative diseases (ND) pose a serious health burden to society and healthcare systems alike, with increasing incidence rates especially within aging populations. Alzheimer's disease (AD) is the most prevalent type of ND or dementia, followed by Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. Progressive neurological dysfunction and regional neuronal loss constitute the common characteristics of ND. Many ND are accompanied by accumulation of protein aggregates such as extracellular amyloid-β (in AD), intraneuronal hyper-phosphorylated tau (in AD), or α-synuclein (in PD). Two main systems are responsible for the clearance of damaged, dysfunctional or senescent proteins inside cells: the autophagy-lysosomal pathway and the ubiquitin-proteasome system. The importance of lysosomes in neurodegenerative processes is further highlighted by clinical phenotypes of lysosomal storage disorders (LSDs), comprising more than 70 inheritable diseases caused by mutations in lysosomal enzymes or lysosomal membrane proteins, often resulting in severe neurodegeneration. Dysfunctional lysosomal proteins and enzymes result in the lysosomal accumulation of undigested macromolecules, e.g. lipids, glycoproteins, glycosaminoglycans, or gangliosides. Defects in intracellular transport pathways involving endosomes and lysosomes are increasingly recognized as drivers of neurodegenerative disease pathology including AD and PD. Thus, accumulation of damaged proteins and organelles (e.g. mitochondria) in neurons and glial cells overwhelms the capacity of intracellular recycling and degradation mechanisms, exacerbating disease pathology. Endolysosomal ion channels have recently been established as important regulators of lysosomal exocytosis, ion homeostasis/pH, endolysosomal trafficking, fusion and fission, and autophagy. In particular two non-selective endolysosomal cation channel families, the mucolipin/TRPML/MCOLN channels and the two-pore channels/TPCs will be discussed here as potential pharmacological targets for LSD/ND treatment.
    Keywords:  Alzheimer; Dementia; Endosome; LSD; Lysosomal storage disorder; Lysosome; Parkinson; TPC1; TPC2; TRPML; TRPML1; TRPML2; TRPML3
    DOI:  https://doi.org/10.1016/j.ceca.2022.102553
  9. Adv Exp Med Biol. 2021 ;1349 275-301
    Lysosomal TRPML1 Channel: Implications in Cardiovascular and Kidney Diseases.
    Guangbi Li, Pin-Lan Li.
      Lysosomal ion channels mediate ion flux from lysosomes and regulate membrane potential across the lysosomal membrane, which are essential for lysosome biogenesis, nutrient sensing, lysosome trafficking, lysosome enzyme activity, and cell membrane repair. As a cation channel, the transient receptor potential mucolipin 1 (TRPML1) channel is mainly expressed on lysosomes and late endosomes. Recently, the normal function of TRPML1 channels has been demonstrated to be important for the maintenance of cardiovascular and renal glomerular homeostasis and thereby involved in the pathogenesis of some cardiovascular and kidney diseases. In arterial myocytes, it has been found that Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP), an intracellular second messenger, can induce Ca2+ release through the lysosomal TRPML1 channel, leading to a global Ca2+ release response from the sarcoplasmic reticulum (SR). In podocytes, it has been demonstrated that lysosomal TRPML1 channels control lysosome trafficking and exosome release, which contribute to the maintenance of podocyte functional integrity. The defect or functional deficiency of lysosomal TRPML1 channels has been shown to critically contribute to the initiation and development of some chronic degeneration or diseases in the cardiovascular system or kidneys. Here we briefly summarize the current evidence demonstrating the regulation of lysosomal TRPML1 channel activity and related signaling mechanisms. We also provide some insights into the canonical and noncanonical roles of TRPML1 channel dysfunction as a potential pathogenic mechanism for certain cardiovascular and kidney diseases and associated therapeutic strategies.
    Keywords:  Atherosclerosis; Autophagy; Chronic kidney disease; Exosomes; Lysosome; NAADP; TRPML1 channel
    DOI:  https://doi.org/10.1007/978-981-16-4254-8_13
  10. J Cell Biol. 2022 Feb 10. pii: e202111077. [Epub ahead of print]221(3):
    Neuronal endolysosomal transport and lysosomal functionality in maintaining axonostasis.
    Joseph C Roney, Xiu-Tang Cheng, Zu-Hang Sheng.
      Lysosomes serve as degradation hubs for the turnover of endocytic and autophagic cargos, which is essential for neuron function and survival. Deficits in lysosome function result in progressive neurodegeneration in most lysosomal storage disorders and contribute to the pathogenesis of aging-related neurodegenerative diseases. Given their size and highly polarized morphology, neurons face exceptional challenges in maintaining cellular homeostasis in regions far removed from the cell body where mature lysosomes are enriched. Neurons therefore require coordinated bidirectional intracellular transport to sustain efficient clearance capacity in distal axonal regions. Emerging lines of evidence have started to uncover mechanisms and signaling pathways regulating endolysosome transport and maturation to maintain axonal homeostasis, or "axonostasis," that is relevant to a range of neurologic disorders. In this review, we discuss recent advances in how axonal endolysosomal trafficking, distribution, and lysosomal functionality support neuronal health and become disrupted in several neurodegenerative diseases.
    DOI:  https://doi.org/10.1083/jcb.202111077
  11. Cell Calcium. 2022 Jan 25. pii: S0143-4160(22)00018-5. [Epub ahead of print]103 102543
    Activation of endo-lysosomal two-pore channels by NAADP and PI(3,5)P2. Five things to know.
    Sandip Patel, Yu Yuan, Gihan S Gunaratne, Taufiq Rahman, Jonathan S Marchant.
      Two-pore channels are ancient members of the voltage-gated ion channel superfamily that are expressed predominantly on acidic organelles such as endosomes and lysosomes. Here we review recent advances in understanding how TPCs are activated by their ligands and identify five salient features: (1) TPCs are Ca2+-permeable non-selective cation channels gated by NAADP. (2) NAADP activation is indirect through associated NAADP receptors. (3) TPCs are also Na+-selective channels gated by PI(3,5)P2. (4) PI(3,5)P2 activation is direct through a structurally-resolved binding site. (5) TPCs switch their ion selectivity in an agonist-dependent manner.
    Keywords:  Ca2+; Endosomes; JPT2; LSM12; Lysosomes; TPC1; TPC2:TPC3; TPCN1; TPCN2; TPCN3
    DOI:  https://doi.org/10.1016/j.ceca.2022.102543
  12. Sci Rep. 2022 Feb 09. 12(1): 2162
    Correlation of age of onset and clinical severity in Niemann-Pick disease type C1 with lysosomal abnormalities and gene expression.
    Laura L Baxter, Dawn E Watkins-Chow, Nicholas L Johnson, Nicole Y Farhat, Frances M Platt, Ryan K Dale, Forbes D Porter, William J Pavan, Jorge L Rodriguez-Gil.
      Niemann-Pick disease type C1 (NPC1) is a rare, prematurely fatal lysosomal storage disorder which exhibits highly variable severity and disease progression as well as a wide-ranging age of onset, from perinatal stages to adulthood. This heterogeneity has made it difficult to obtain prompt diagnosis and to predict disease course. In addition, small NPC1 patient sample sizes have been a limiting factor in acquiring genome-wide transcriptome data. In this study, primary fibroblasts from an extensive cohort of 41 NPC1 patients were used to validate our previous findings that the lysosomal quantitative probe LysoTracker can be used as a predictor for age of onset and disease severity. We also examined the correlation between these clinical parameters and RNA expression data from primary fibroblasts and identified a set of genes that were significantly associated with lysosomal defects or age of onset, in particular neurological symptom onset. Hierarchical clustering showed that these genes exhibited distinct expression patterns among patient subgroups. This study is the first to collect transcriptomic data on such a large scale in correlation with clinical and cellular phenotypes, providing a rich genomic resource to address NPC1 clinical heterogeneity and discover potential biomarkers, disease modifiers, or therapeutic targets.
    DOI:  https://doi.org/10.1038/s41598-022-06112-y
  13. IBRO Neurosci Rep. 2022 Jun;12 131-141
    Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.
    Lauren S Whyte, Célia Fourrier, Sofia Hassiotis, Adeline A Lau, Paul J Trim, Leanne K Hein, Kathryn J Hattersley, Julien Bensalem, John J Hopwood, Kim M Hemsley, Timothy J Sargeant.
      Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer's disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of β-hexosaminidase) result in accumulation of amyloid-β (Aβ) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for β-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between β-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of β-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in App NL-G-F/NL-G-F (App KI/KI ) mice. App KI/KI and Hexb +/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in App KI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.
    Keywords:  AD, Alzheimer’s disease; APP, amyloid precursor protein; Alzheimer’s disease; AppNL-G-F/NL-G-F; Aβ, amyloid beta; CD68, cluster of differentiation 68; Dementia; ELISA, enzyme-linked immunosorbent assay; GFAP, glial fibrillary acidic protein; HEXB, β-hexosaminidase β subunit; Hexb; IBA1, ionised calcium binding adaptor molecule 1; IL, interleukin; Knock-in; LAMP1, lysosome associated membrane protein 1; LSDs, lysosomal storage disorders; Lysosome; PBS, phosphate buffered saline; TREM2, triggering receptor expressed on myeloid cells 2; β-hexosaminidase
    DOI:  https://doi.org/10.1016/j.ibneur.2022.01.004
  14. Hum Mol Genet. 2022 Feb 08. pii: ddac033. [Epub ahead of print]
    Multisystem involvement, defective lysosomes, and impaired autophagy in a novel rat model of Nephropathic Cystinosis.
    Patrick Krohn, Laura Rita Rega, Marianne Harvent, Beatrice Paola Festa, Anna Taranta, Alessandro Luciani, Joseph Dewulf, Alessio Cremonesi, Francesca Diomedi Camassei, James V M Hanson, Christina Gerth-Kahlert, Francesco Emma, Marine Berquez, Olivier Devuyst.
      Recessive mutations in the CTNS gene encoding the lysosomal transporter cystinosin cause cystinosis, a lysosomal storage disease leading to kidney failure and multisystem manifestations. A Ctns knock-out mouse model recapitulates features of cystinosis, but the delayed onset of kidney manifestations, phenotype variability, and strain effects limit its use for mechanistic and drug development studies. To provide a better model for cystinosis, we generated a Ctns knock-out rat model using CRISPR/Cas9 technology. The Ctns-/- rats display progressive cystine accumulation and crystal formation in multiple tissues including kidney, liver and thyroid. They show an early onset and progressive loss of urinary solutes, indicating generalized proximal tubule dysfunction, with development of typical swan-neck lesions, tubulointerstitial fibrosis and kidney failure, and decreased survival. The Ctns-/- rats also present crystals in the cornea, and bone and liver defects, like in patients. Mechanistically, the loss of cystinosin induces a phenotype switch associating abnormal proliferation and dedifferentiation, loss of apical receptors and transporters, and defective lysosomal activity and autophagy in the cells. Primary cultures of proximal tubule cells derived from the Ctns-/- rat kidneys confirmed the key changes caused by cystine overload, including reduced endocytic uptake, increased proliferation and defective lysosomal dynamics and autophagy. The novel Ctns-/- rat model and derived proximal tubule cell system provide invaluable tools to investigate the pathogenesis of cystinosis and to accelerate drug discovery.
    DOI:  https://doi.org/10.1093/hmg/ddac033
  15. Brain. 2022 Feb 08. pii: awac044. [Epub ahead of print]
    NPRL3 loss alters neuronal morphology, mTOR localization, cortical lamination, and seizure threshold.
    Philip H Iffland, Mariah E Everett, Katherine M Cobb-Pitstick, Lauren E Bowser, Allan E Barnes, Janice K Babus, Andrea J Romanowski, Marianna Baybis, Soad Elziny, Erik G Puffenberger, Claudia Gonzaga-Jauregui, Alexandros Poulopoulos, Vincent J Carson, Peter B Crino.
      Mutations in nitrogen permease regulator-like 3 (NPRL3), a component of the GATOR1 complex within the mechanistic target of rapamycin (mTOR) pathway, are associated with epilepsy and malformations of cortical development. Little is known about the effects of NPRL3 loss on neuronal mTOR signaling and morphology, or cerebral cortical development and seizure susceptibility. We report the clinical phenotypic spectrum of a founder NPRL3 pedigree (c.349delG, p.Glu117LysFS; n = 133) among Old Order Mennonites dating to 1727. Next, as a strategy to define the role of NPRL3 in cortical development, CRISPR/Cas9 Nprl3 knockout in Neuro2a cells in vitro and in fetal mouse brain in vivo was used to assess effects of Nprl3 knockout on mTOR activation, subcellular mTOR localization, nutrient signaling, cell morphology and aggregation, cerebral cortical cytoarchitecture, and network integrity. The NPRL3 pedigree exhibited an epilepsy penetrance of 28% and heterogeneous clinical phenotypes with a range of epilepsy semiologies i.e., focal or generalized onset, brain imaging abnormalities i.e., polymicrogyria, focal cortical dysplasia, or normal imaging, and EEG findings, e.g., focal, multi-focal, or generalized spikes, focal or generalized slowing. Whole exome analysis comparing a seizure-free group (n = 37) to those with epilepsy (n = 24) to search for gene modifiers for epilepsy did not identify a unique genetic modifier that explained the variability in seizure penetrance in this cohort. Nprl3 knockout in vitro caused mTOR pathway hyperactivation, cell soma enlargement, and the formation of cellular aggregates seen in time-lapse videos that were prevented with the mTOR inhibitors rapamycin or torin1. In Nprl3 KO cells, mTOR remained localized on the lysosome in a constitutively active conformation, as evidenced by phosphorylation of S6 and 4E-BP1 proteins, even under nutrient starvation (amino acid free) conditions, demonstrating that Nprl3 loss decouples mTOR activation from neuronal metabolic state. To model human malformations of cortical development associated with NPRL3 variants, we created a focal Nprl3 KO in fetal mouse cortex by in utero electroporation and found altered cortical lamination and white matter heterotopic neurons, effects which were prevented with rapamycin treatment. EEG recordings showed network hyperexcitability and reduced seizure threshold to pentylenetetrazol treatment. NPRL3 variants are linked to a highly variable clinical phenotype which we propose result from mTOR-dependent effects on cell structure, cortical development, and network organization.
    Keywords:  GATOR1; cortical malformations; epilepsy; focal cortical dysplasia; mTOR
    DOI:  https://doi.org/10.1093/brain/awac044
  16. Cancer Res. 2022 Feb 11. pii: canres.1168.2021. [Epub ahead of print]
    Disrupting the MYC-TFEB Circuit Impairs Amino Acid Homeostasis and Provokes Metabolic Anergy.
    Mario R Fernandez, Franz X Schaub, Chunying Yang, Weimin Li, Seongseok Yun, Stephanie K Schaub, Frank C Dorsey, Min Liu, Meredith A Steeves, Andrea Ballabio, Alexandar Tzankov, Zhihua Chen, John M Koomen, Anders E Berglund, John L Cleveland.
      MYC family oncoproteins are regulators of metabolic reprogramming that sustains cancer cell anabolism. Normal cells adapt to nutrient-limiting conditions by activating autophagy, which is required for amino acid (AA) homeostasis. Here we report that the autophagy pathway is suppressed by Myc in normal B cells, in premalignant and neoplastic B cells of Eμ-Myc transgenic mice, and in human MYC-driven Burkitt lymphoma. Myc suppresses autophagy by antagonizing the expression and function of transcription factor EB (TFEB), a master regulator of autophagy. Mechanisms that sustained AA pools in MYC-expressing B cells include coordinated induction of the proteasome and increases in AA transport. Reactivation of the autophagy-lysosomal pathway by TFEB disabled the malignant state by disrupting mitochondrial functions, proteasome activity, amino acid transport, and amino acid and nucleotide metabolism, leading to metabolic anergy, growth arrest and apoptosis. This phenotype provides therapeutic opportunities to disable MYC-driven malignancies, including AA restriction and treatment with proteasome inhibitors.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-1168
  17. DNA Cell Biol. 2022 Feb;41(2): 215-224
    Rap_GAP Domain of TSC2 Contributes to Tumor Suppression Through mTOR Signaling in Human Hepatocellular Carcinoma.
    Mengying Cui, Weibo Jiang, Jiyao Sheng, Jiacheng Wu, Xuewen Zhang.
      Hepatocellular carcinoma (HCC) is an aggressive disease with a high degree of tumor heterogeneity. Genetic lesions of mTOR-related genes, including TSC2 and hyperactivation of mTOR signaling, are common in HCC. However, the association of genetic alterations with hepatocarcinogenesis remains unclear. In this study, continuous truncating mutations occurred within or upstream of the TSC2 Rap_GAP domain in clinical HCC samples. To elucidate whether hyperactivation of mTOR signaling in HCC is caused by TSC2 truncating mutations, HCC cell models carrying the TSC2 deletion (CRISPR/Cas9) or the TSC2 truncating mutation (mutagenesis) were established. Our findings showed that either TSC2 deletion or TSC2 mutant could lead to TSC2 loss-of-function and hyperactivation of mTOR signaling. Furthermore, hyperactivation of mTOR signaling was relieved by rapamycin. Immunohistochemistry of clinical samples confirmed frequent TSC2 loss in HCC. Thus, our study revealed that genetic alterations cause TSC2 loss of function and result in the hyperactivation of mTOR, and high frequency of TSC2 truncating mutations around RAP_GAP domain may be one of the reasons for the hyperactivation of mTOR in HCC patients.
    Keywords:  HCC; Rap_GAP domain; TSC2; mTOR; truncating mutation
    DOI:  https://doi.org/10.1089/dna.2021.0769
  18. Neurocase. 2022 Feb 09. 1-3
    Novel frameshift CTSF mutation causing kufs disease type B mimicking frontotemporal dementia-parkinsonism.
    Murat Gultekin, Zeynep Tufekcioglu, Recep Baydemir.
      Adult-onset neuronal ceroid lipofuscinoses (ANCLs, Kufs disease-KD) are rare, inherited, progressive, neurodegenerative, lysosomal storage diseases. Mutations in cathepsin F (CTSF) were linked to KD type B. Conversely, Frontotemporal dementia (FTD) is the second most common type of presenile dementia and Parkinsonism is a mostly common accompanying feature. Due to pronounced behavioral, cognitive, and motor features in the patients with KD type B, mutations in CTSF may resemble FTD-parkinsonism. Here, we present a case of KD type B with a novel homozygous frameshift pathogenic variant (p.Gly439Alafs*36) in the Cathepsin F (CTSF) gene presenting behavioral changes, cognitive disturbances and parkinsonism with a family history mimicking FTD-parkinsonism.
    Keywords:  Kufs disease; cognition; frontotemporal dementia; lipofuscinoses; parkinsonism
    DOI:  https://doi.org/10.1080/13554794.2022.2038635
  19. Cell Syst. 2022 Feb 07. pii: S2405-4712(22)00040-0. [Epub ahead of print]
    Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease.
    Todd M Greco, Christopher Secker, Eduardo Silva Ramos, Joel D Federspiel, Jeh-Ping Liu, Alma M Perez, Ismael Al-Ramahi, Jeffrey P Cantle, Jeffrey B Carroll, Juan Botas, Scott O Zeitlin, Erich E Wanker, Ileana M Cristea.
      Huntington disease (HD) is a monogenic neurodegenerative disorder with one causative gene, huntingtin (HTT). Yet, HD pathobiology is multifactorial, suggesting that cellular factors influence disease progression. Here, we define HTT protein-protein interactions (PPIs) perturbed by the mutant protein with expanded polyglutamine in the mouse striatum, a brain region with selective HD vulnerability. Using metabolically labeled tissues and immunoaffinity purification-mass spectrometry, we establish that polyglutamine-dependent modulation of HTT PPI abundances and relative stability starts at an early stage of pathogenesis in a Q140 HD mouse model. We identify direct and indirect PPIs that are also genetic disease modifiers using in-cell two-hybrid and behavioral assays in HD human cell and Drosophila models, respectively. Validated, disease-relevant mHTT-dependent interactions encompass mediators of synaptic neurotransmission (SNAREs and glutamate receptors) and lysosomal acidification (V-ATPase). Our study provides a resource for understanding mHTT-dependent dysfunction in cortico-striatal cellular networks, partly through impaired synaptic communication and endosomal-lysosomal system. A record of this paper's Transparent Peer Review process is included in the supplemental information.
    Keywords:  AMPA receptors; Arp2/3; D. melanogaster; LuTHy; SNARE; immunoaffinity purification-mass spectrometry; label-free quantification; metabolic labeling; protein interactions; synaptic biology; vesicular trafficking
    DOI:  https://doi.org/10.1016/j.cels.2022.01.005
  20. Cell Death Discov. 2022 Feb 08. 8(1): 56
    Role of mTORC1 activity during early retinal development and lamination in human-induced pluripotent stem cell-derived retinal organoids.
    Si Hyung Lee, Jung Woo Han, Jin Young Yang, Hyoung Oh Jun, Ji Hong Bang, Heejeong Shin, Ji Hye Choi, Jongwoo Lee, Sanjar Batirovich Madrakhimov, Kyung Hwun Chung, Hun Soo Chang, Jungmook Lyu, Tae Kwann Park.
      Retinal organoids derived from human-induced pluripotent stem cells (hiPSC) are powerful tools for studying retinal development as they model spatial and temporal differentiation of retinal cell types. Vertebrate retinal development involves a delicate and coordinated process of retinal progenitor cell (RPC) differentiation, and the mammalian target of rapamycin complex 1 (mTORC1) has been reported to play a significant role in this complex process. Herein, using hiPSC-derived retinal organoids, we identify the time-dependent role of mTORC1 in retinal development, specifically in retinal ganglion cell (RGC) differentiation and the retinal lamination process, during the early stages of retinal organoid (RO) development. mTORC1 activity in ROs was the highest at 40 days of differentiation. MHY1485-induced hyperactivation of mTORC1 during this period resulted in a significant increase in the overall size of ROs compared to the untreated controls and rapamycin-treated Ros; there was also a marked increase in proliferative activity within the inner and outer layers of ROs. Moreover, the MHY1485-treated ROs showed a significant increase in the number of ectopic RGCs in the outer layers (indicating disruption of retinal laminar structure), with robust expression of HuC/D-binding proteins in the inner layers. These results demonstrate that mTORC1 plays a critical role in the development of hiPSC-derived ROs, especially during the early stages of differentiation.
    DOI:  https://doi.org/10.1038/s41420-022-00837-5
  21. Eur J Med Genet. 2022 Feb 07. pii: S1769-7212(22)00028-3. [Epub ahead of print] 104447
    Genotype-phenotype spectrum of 130 unrelated Indian families with Mucopolysaccharidosis type II.
    Neha Agrawal, Gaurav Verma, Deepti Saxena, Madhulika Kabra, Neerja Gupta, Kausik Mandal, Amita Moirangthem, Jayesh Sheth, Ratna Dua Puri, Sunita Bijarnia-Mahay, Seema Kapoor, Sumita Danda, Sankar V H, Chaitanya A Datar, Prajnya Ranganath, Anju Shukla, Ashwin Dalal, Priyanka Srivastava, Radha Rama Devi, Shubha R Phadke.
      MPS II is an X linked recessive lysosomal storage disorder with multi-system involvement and marked molecular heterogeneity. In this study, we explored the clinical and molecular spectrum of 144 Indian patients with MPS II from 130 unrelated families. Clinical information was collected on a predesigned clinical proforma. Sanger method was employed to sequence all the exons and exon/intron boundaries of the IDS gene. In cases where causative variation was not detected by Sanger sequencing, MLPA and RFLP were performed to identify large deletions/duplications and complex rearrangements. Cytogenetic microarray was done in one patient to see the breakpoints and extent of deletion. In one patient with no detectable likely pathogenic or pathogenic variation, whole-genome sequencing was also performed. Novel variants were systematically assessed by in silico prediction software and protein modelling. The pathogenicity of variants was established based on ACMG criteria. An attempt was also made to establish a genotype-phenotype correlation. Positive family history was present in 31% (41/130) of patients. Developmental delay and intellectual disability were the main reasons for referral. Macrocephaly, coarse facies and dysostosis were present in almost all patients. Hepatosplenomegaly, joint contractures and short stature were the characteristic features, seen in 87% (101/116), 67.8% (74/109) and 41.4% (41/99) patients respectively. Attenuated phenotype was seen in 32.6% (47/144) patients, while severe phenotype was seen in 63% (91/144) patients. The detection rate for likely pathogenic or pathogenic variants in our cohort is 95.5% (107/112) by Sanger sequencing, MLPA and RFLP. We also found two variants of unknown significance, one each by Sanger sequencing and WGS. Total of 71 variants were identified by Sanger sequencing and 29 of these variants were found to be novel. Amongst the novel variants, there was a considerable proportion (51%) of frameshift variants (15/29). Almost half of the causative variants were located in exon 3,8 and 9. A significant genotype-phenotype correlation was also noted for both known and novel variants. This information about the genotype spectrum and phenotype will be helpful for diagnostic and prognostic purposes.
    Keywords:  Hunter syndrome; IDS; Iduronate 2-sulfatase (I2S) enzyme; Lysosomal storage disorder; MPS II; Mucopolysaccharidoses; Novel variants
    DOI:  https://doi.org/10.1016/j.ejmg.2022.104447
  22. Nat Commun. 2022 Feb 10. 13(1): 805
    In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells.
    Bonnie Huang, James D Phelan, Silvia Preite, Julio Gomez-Rodriguez, Kristoffer H Johansen, Hirofumi Shibata, Arthur L Shaffer, Qin Xu, Brendan Jeffrey, Martha Kirby, Stacie Anderson, Yandan Yang, Selamawit Gossa, Dorian B McGavern, Louis M Staudt, Pamela L Schwartzberg.
      T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.
    DOI:  https://doi.org/10.1038/s41467-022-28378-6
  23. Front Cell Dev Biol. 2022 ;10 826379
    Low-Density Lipoprotein Internalization, Degradation and Receptor Recycling Along Membrane Contact Sites.
    Mohammad Majharul Islam, Iryna Hlushchenko, Simon G Pfisterer.
      Low-density lipoprotein (LDL) internalization, degradation, and receptor recycling is a fundamental process underlying hypercholesterolemia, a high blood cholesterol concentration, affecting more than 40% of the western population. Membrane contact sites influence endosomal dynamics, plasma membrane lipid composition, and cellular cholesterol distribution. However, if we focus on LDL-related trafficking events we mostly discuss them in an isolated fashion, without cellular context. It is our goal to change this perspective and to highlight that all steps from LDL internalization to receptor recycling are likely associated with dynamic membrane contact sites in which endosomes engage with the endoplasmic reticulum and other organelles.
    Keywords:  endosomal degradation; endosomal recycling; hypercholesterolemia; low-density lipoprotein (LDL); low-density lipoprotein receptor (LDLR); membrane contact site
    DOI:  https://doi.org/10.3389/fcell.2022.826379
  24. Cell Death Dis. 2022 Feb 08. 13(2): 129
    Exosome-mediated miR-7-5p delivery enhances the anticancer effect of Everolimus via blocking MNK/eIF4E axis in non-small cell lung cancer.
    Sile Liu, Weiyuan Wang, Yue Ning, Hongmei Zheng, Yuting Zhan, Haihua Wang, Yang Yang, Jiadi Luo, Qiuyuan Wen, Hongjing Zang, Jinwu Peng, Jian Ma, Songqing Fan.
      Everolimus is a kind of mammalian target of rapamycin (mTOR) inhibitors. Activated mitogen-activated protein kinase interacting kinases/eukaryotic translation initiation factor 4E (MNK/eIF4E) axis plays a crucial role in resistance to Everolimus in non-small cell lung cancer (NSCLC). The eIF4E phosphorylation increased by mTOR inhibitors is mainly mediated by MNKs. However, the mechanisms are poorly understood. Recently, extensive reprogramming of miRNA profiles has also been found after long-term mTOR inhibitor exposure. Our previous studies have confirmed that tumor suppressor miR-7-5p is decreased in A549 cells after treatment with Everolimus. Exactly, MNK1 is the target of miR-7-5p. In this study, we investigated the biological functions and potential molecular mechanisms of miR-7-5p in the NSCLC undergoing treatment with Everolimus. We confirmed that Everolimus targeted mTORC1 inducing NSCLC cells to secrete miR-7-5p-loaded exosomes in Rab27A and Rab27B-dependent manners. Loss of intracellular miR-7-5p induced phosphorylation of MNK/eIF4E axis, but a supplement of extra exosomal miR-7-5p could reverse it. Of note, both low expression of miR-7-5p and elevated MNK1 protein were associated with a poor prognosis of NSCLC. Both endogenous miR-7-5p and exo-miR-7-5p enhanced the therapeutic efficacy of Everolimus by inhibiting the proliferation, migration, and metastasis of NSCLC in vitro and in vivo. The combination of miR-7-5p with Everolimus induced apoptosis to exhibit a synergistic anticancer therapeutic efficacy through dual abrogation of MNK/eIF4E and mTOR in NSCLC. In conclusion, Everolimus decreases the intracellular miR-7-5p by releasing of miR-7-5p loaded exosomes from NSCLC cells in Rab27A and Rab27B dependent manners. Either endogenous miR-7-5p or exo-miR-7-5p combined with Everolimus can enhance the anticancer efficacy by targeting MNK/eIF4E axis and mTOR. Besides, both low levels of miR-7-5p and positive expression of MNK1 act as independent poor prognostic biomarkers for NSCLC. Therefore, restoring miR-7-5p carried by exosome may be a promising novel combined therapeutic strategy with Everolimus for NSCLC.
    DOI:  https://doi.org/10.1038/s41419-022-04565-7
  25. Traffic. 2022 Feb 10.
    Endosomes supporting fusion mediated by vesicular stomatitis virus glycoprotein have distinctive motion and acidification.
    Maya Cabot, Volker Kiessling, Judith M White, Lukas K Tamm.
      Most enveloped viruses infect cells by binding receptors at the cell surface and undergo trafficking through the endocytic pathway to a compartment with the requisite conditions to trigger fusion with a host endosomal membrane. Broad categories of compartments in the endocytic pathway include early and late endosomes, which can be further categorized into subpopulations with differing rates of maturation and motility characteristics. Endocytic compartments have varying protein and lipid components, luminal ionic conditions and pH that provide uniquely hospitable environments for specific viruses to fuse. In order to characterize compartments that permit fusion, we studied the trafficking and fusion of viral particles pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) on their surface and equipped with a novel pH sensor and a fluorescent content marker to measure pH, motion, and fusion at the single particle level in live cells. We found that the VSV-G particles fuse predominantly from more acidic and more motile endosomes, and that a significant fraction of particles is trafficked to more static and less acidic endosomes that do not support their fusion. Moreover, the fusion-supporting endosomes undergo directed motion. This article is protected by copyright. All rights reserved.
    Keywords:  endocytosis; endosomes; fluorescent pH sensor; organelle motility; vesicular stomatitis; viral trafficking; virus entry
    DOI:  https://doi.org/10.1111/tra.12836
  26. Pharmacol Res Perspect. 2022 Feb;10(1): e00930
    mTOR inhibition by TAK-228 is effective against growth, survival and angiogenesis in preclinical retinoblastoma models.
    Lanlan Tang, Yu Fu, Jiarun Song, Taibing Hu, Kun Li, Zhi Li.
      We and others have shown that aberrant activation of the mammalian target of rapamycin (mTOR) signalling is essential for retinoblastoma progression and has potential therapeutic value. TAK-228 is a potent inhibitor of mTOR1 and 2 with preclinical activity in a variety of cancers. In this study, we report that TAK-228 is a dual inhibitor of retinoblastoma and angiogenesis. TAK-228 inhibits growth and induces apoptosis in a panel of retinoblastoma cell lines, with IC50 at ~0.2 μM. Under the same experimental conditions, TAK-228 was less effective in inhibiting growth and survival in normal retinal and fibroblast cells than retinoblastoma cells. In addition, TAK-228 inhibited retinal endothelial cell capillary network formation, migration, growth and survival. We further demonstrate that TAK-228 inhibits retinoblastoma and retinal angiogenesis through inhibiting mTOR signalling. Rescue studies confirm that mTOR is the target of TAK-228 in both retinoblastoma and retinal endothelial cells. Finally, we confirm the inhibitory effects of TAK-228 on tumor and angiogenesis in retinoblastoma xenograft mouse model. Our findings provide a preclinical rationale to explore TAK-228 as a strategy to treat retinoblastoma and highlight the therapeutic value of targeting mTOR in retinoblastoma.
    Keywords:  TAK-228; angiogenesis; mTOR; retinoblastoma
    DOI:  https://doi.org/10.1002/prp2.930
  27. Neuron. 2022 Jan 31. pii: S0896-6273(22)00056-3. [Epub ahead of print]
    The different autophagy degradation pathways and neurodegeneration.
    Angeleen Fleming, Mathieu Bourdenx, Motoki Fujimaki, Cansu Karabiyik, Gregory J Krause, Ana Lopez, Adrián Martín-Segura, Claudia Puri, Aurora Scrivo, John Skidmore, Sung Min Son, Eleanna Stamatakou, Lidia Wrobel, Ye Zhu, Ana Maria Cuervo, David C Rubinsztein.
      The term autophagy encompasses different pathways that route cytoplasmic material to lysosomes for degradation and includes macroautophagy, chaperone-mediated autophagy, and microautophagy. Since these pathways are crucial for degradation of aggregate-prone proteins and dysfunctional organelles such as mitochondria, they help to maintain cellular homeostasis. As post-mitotic neurons cannot dilute unwanted protein and organelle accumulation by cell division, the nervous system is particularly dependent on autophagic pathways. This dependence may be a vulnerability as people age and these processes become less effective in the brain. Here, we will review how the different autophagic pathways may protect against neurodegeneration, giving examples of both polygenic and monogenic diseases. We have considered how autophagy may have roles in normal CNS functions and the relationships between these degradative pathways and different types of programmed cell death. Finally, we will provide an overview of recently described strategies for upregulating autophagic pathways for therapeutic purposes.
    DOI:  https://doi.org/10.1016/j.neuron.2022.01.017
  28. iScience. 2022 Feb 18. 25(2): 103675
    TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells.
    Chihiro Yamada, Aya Morooka, Seira Miyazaki, Masayoshi Nagai, Satoru Mase, Kenji Iemura, Most Naoshia Tasnin, Tsuneyuki Takuma, Shotaro Nakamura, Shamsul Morshed, Naoki Koike, Md Golam Mostofa, Muhammad Arifur Rahman, Tasnuva Sharmin, Haruko Katsuta, Kotaro Ohara, Kozo Tanaka, Takashi Ushimaru.
      Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.
    Keywords:  Biological sciences; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103675
  29. Cancer Res. 2022 Feb 07. pii: canres.2161.2021. [Epub ahead of print]
    Chaperone-mediated autophagy controls proteomic and transcriptomic pathways to maintain glioma stem cell activity.
    Jaione Auzmendi-Iriarte, Maddalen Otaegi-Ugartemendia, Estefania Carrasco-Garcia, Mikel Azkargorta, Antonio Diaz, Ander Saenz-Antoñanzas, Joaquin Andrés Andermatten, Mikel García-Puga, Idoia Garcia, Alejandro Elua-Pinin, Irune Ruiz, Nicolás Samprón, Felix Elortza, Ana Maria Cuervo, Ander Matheu.
      Chaperone-mediated autophagy (CMA) is a homeostatic process essential for the lysosomal degradation of a selected subset of the proteome. CMA activity directly depends on the levels of LAMP2A, a critical receptor for CMA substrate proteins at the lysosomal membrane. In glioblastoma (GBM), the most common and aggressive brain cancer in adulthood, high levels of LAMP2A in the tumor and tumor-associated pericytes have been linked to temozolomide resistance and tumor progression. However, the role of LAMP2A, and hence CMA, in any cancer stem cell type or in glioblastoma stem cells (GSC) remains unknown. In this work, we show that LAMP2A expression is enriched in patient-derived GSCs, and its depletion diminishes GSC-mediated tumorigenic activities. Conversely, overexpression of LAMP2A facilitates the acquisition of GSC properties. Proteomic and transcriptomic analysis of LAMP2A-depleted GSCs revealed reduced extracellular matrix (ECM) interaction effectors in both analyses. Moreover, pathways related to mitochondrial metabolism and the immune system were differentially deregulated at the proteome level. Furthermore, clinical samples of GBM tissue presented with overexpression of LAMP2, which correlated with advanced glioma grade and poor overall survival. In conclusion, these results identify a novel role of CMA in directly regulating GSCs activity via multiple pathways at the proteome and transcriptome levels.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-2161
  30. JCI Insight. 2022 Feb 08. pii: e155815. [Epub ahead of print]7(3):
    mTOR inhibition prevents angiotensin II-induced aortic rupture and pseudoaneurysm but promotes dissection in Apoe-deficient mice.
    Changshun He, Bo Jiang, Mo Wang, Pengwei Ren, Sae-Il Murtada, Alexander W Caulk, Guangxin Li, Lingfeng Qin, Roland Assi, Constantinos J Lovoulos, Martin A Schwartz, Jay D Humphrey, George Tellides.
      Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II-induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II-induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.
    Keywords:  Cardiovascular disease; Mouse models; Surgery; Vascular Biology
    DOI:  https://doi.org/10.1172/jci.insight.155815
  31. Immunology. 2022 Feb 10.
    Stress hormone signaling inhibits Th1 polarization in a CD4 T-cell-intrinsic manner via mTORC1 and the circadian gene PER1.
    Christophe M Capelle, Anna Chen, Ni Zeng, Alexandre Baron, Kamil Grzyb, Thais Arns, Alexander Skupin, Markus Ollert, Feng Q Hefeng.
      Stress hormones are believed to skew the CD4 T-cell differentiation toward a Th2 response via a T cell-extrinsic mechanism. Using primary human naïve and memory CD4 T cells, here we show that both adrenergic- and glucocorticoid-mediated stress signaling pathways play a CD4 naïve T cell-intrinsic role in regulating the Th1/Th2 differentiation balance. Both stress hormones reduced theTh1 program and cytokine production by inhibiting mTORC1 signaling via two parallel mechanisms. Stress hormone signaling inhibited mTORC1 in naïve CD4 T cells 1) by affecting the PI3K/1/AKT pathway and 2) via regulating the expression of the circadian rhythm gene, period circadian regulator 1 (PER1). Both stress hormones induced the expression of PER1, which inhibited mTORC1 signaling, thus reducing Th1 differentiation. This previously unrecognized cell-autonomous mechanism connects stress hormone signaling with CD4 T-cell differentiation via mTORC1 and a specific circadian clock gene, namely PER1.
    Keywords:  Adrenergic signalling; CD4 T cells; Circadian rhythm; Neuroimmunology; Stress; T cell differentiation
    DOI:  https://doi.org/10.1111/imm.13448
  32. Biochem J. 2022 Feb 11. 479(3): 445-462
    GLUT4 On the move.
    Daniel J Fazakerley, Francoise Koumanov, Geoffrey D Holman.
      Insulin rapidly stimulates GLUT4 translocation and glucose transport in fat and muscle cells. Signals from the occupied insulin receptor are translated into downstream signalling changes in serine/threonine kinases within timescales of seconds, and this is followed by delivery and accumulation of the glucose transporter GLUT4 at the plasma membrane. Kinetic studies have led to realisation that there are distinct phases of this stimulation by insulin. There is a rapid initial burst of GLUT4 delivered to the cell surface from a subcellular reservoir compartment and this is followed by a steady-state level of continuing stimulation in which GLUT4 recycles through a large itinerary of subcellular locations. Here, we provide an overview of the phases of insulin stimulation of GLUT4 translocation and the molecules that are currently considered to activate these trafficking steps. Furthermore, we suggest how use of new experimental approaches together with phospho-proteomic data may help to further identify mechanisms for activation of these trafficking processes.
    Keywords:  GLUT4; glucose transport; insulin; membrane traffic; membrane trafficking kinetics; signal transduction
    DOI:  https://doi.org/10.1042/BCJ20210073
  33. Nat Commun. 2022 Feb 11. 13(1): 844
    Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates.
    Remkes A Scheele, Laurens H Lindenburg, Maya Petek, Markus Schober, Kevin N Dalby, Florian Hollfelder.
      The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.
    DOI:  https://doi.org/10.1038/s41467-022-28396-4
  34. Curr Biol. 2022 Feb 01. pii: S0960-9822(22)00093-8. [Epub ahead of print]
    ESCRT dysfunction compromises endoplasmic reticulum maturation and autophagosome biogenesis in Drosophila.
    Ruoxi Wang, Guangyan Miao, James L Shen, Tina M Fortier, Eric H Baehrecke.
      Autophagy targets cytoplasmic materials for degradation and influences cell health. Organelle contact and trafficking systems provide membranes for autophagosome formation, but how different membrane systems are selected for use during autophagy remains unclear. Here, we report a novel function of the endosomal sorting complex required for transport (ESCRT) in the regulation of endoplasmic reticulum (ER) coat protein complex II (COPII) vesicle formation that influences autophagy. The ESCRT functions in a pathway upstream of Vps13D to influence COPII vesicle transport, ER-Golgi intermediate compartment (ERGIC) assembly, and autophagosome formation. Atg9 functions downstream of the ESCRT to facilitate ERGIC and autophagosome formation. Interestingly, cells lacking either ESCRT or Vps13D functions exhibit dilated ER structures that are similar to cranio-lenticulo-sutural dysplasia patient cells with SEC23A mutations, which encodes a component of COPII vesicles. Our data reveal a novel ESCRT-dependent pathway that influences the ERGIC and autophagosome formation.
    Keywords:  Atg9; Drosophila; ESCRT; Vps13D; autophagy
    DOI:  https://doi.org/10.1016/j.cub.2022.01.040
  35. Science. 2022 Feb 11. 375(6581): 620-621
    Caloric restriction has a new player.
    Timothy W Rhoads, Rozalyn M Anderson.
      Reverse translation of a human caloric restriction trial finds an immunometabolic regulator.
    DOI:  https://doi.org/10.1126/science.abn6576
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