bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒11‒14
thirty-six papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing


  1. Nat Nanotechnol. 2021 Nov 11.
      Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.
    DOI:  https://doi.org/10.1038/s41565-021-00988-z
  2. Glycoconj J. 2021 Nov 10.
      It is well established that lysosomal glucocerebrosidase gene (GBA) variants are a risk factor for Parkinson's disease (PD), with increasing evidence suggesting a loss of function mechanism. One question raised by this genetic association is whether variants of genes involved in other aspects of sphingolipid metabolism are also associated with PD. Recent studies in sporadic PD have identified variants in multiple genes linked to diseases of glycosphingolipid (GSL) metabolism to be associated with PD. GSL biosynthesis is a complex pathway involving the coordinated action of multiple enzymes in the Golgi apparatus. GSL catabolism takes place in the lysosome and is dependent on the action of multiple acid hydrolases specific for certain substrates and glycan linkages. The finding that variants in multiple GSL catabolic genes are over-represented in PD in a heterozygous state highlights the importance of GSLs in the healthy brain and how lipid imbalances and lysosomal dysfunction are associated with normal ageing and neurodegenerative diseases. In this article we will explore the link between lysosomal storage disorders and PD, the GSL changes seen in both normal ageing, lysosomal storage disorders (LSDs) and PD and the mechanisms by which these changes can affect neurodegeneration.
    Keywords:  Ageing; GBA; Gangliosides; Glycosphingolipid (GSL); Lysosomal storage disease (LSD); Parkinson’s disease (PD)
    DOI:  https://doi.org/10.1007/s10719-021-10023-x
  3. Signal Transduct Target Ther. 2021 Nov 08. 6(1): 379
      In recent years, accumulating evidence has elucidated the role of lysosomes in dynamically regulating cellular and organismal homeostasis. Lysosomal changes and dysfunction have been correlated with the development of numerous diseases. In this review, we interpreted the key biological functions of lysosomes in four areas: cellular metabolism, cell proliferation and differentiation, immunity, and cell death. More importantly, we actively sought to determine the characteristic changes and dysfunction of lysosomes in cells affected by these diseases, the causes of these changes and dysfunction, and their significance to the development and treatment of human disease. Furthermore, we outlined currently available targeting strategies: (1) targeting lysosomal acidification; (2) targeting lysosomal cathepsins; (3) targeting lysosomal membrane permeability and integrity; (4) targeting lysosomal calcium signaling; (5) targeting mTOR signaling; and (6) emerging potential targeting strategies. Moreover, we systematically summarized the corresponding drugs and their application in clinical trials. By integrating basic research with clinical findings, we discussed the current opportunities and challenges of targeting lysosomes in human disease.
    DOI:  https://doi.org/10.1038/s41392-021-00778-y
  4. Nano Lett. 2021 Nov 08.
      Cholesterol crystals (CCs), originally accumulating in the lysosome of cholesterol-laden cells, can aggravate the progression of atherosclerosis. β-cyclodextrin (CD) is a potent cholesterol acceptor or CC solubilizer. However, the random extraction of cholesterol impedes the in vivo application of CD for removing lysosomal CCs. Here, we exploit poly-β-cyclodextrin (pCD) as a lysosomal CC solubilizer and dextran sulfate grafted with benzimidazole (BM) as a pH-sensitive switch (pBM) to self-assemble into a supramolecular nanoassembly (pCD/pBM-SNA). The CD cavity in pCD/pBM-SNA can be efficiently sealed by hydrophobic BM at pH 7.4 (OFF). After it enters the lysosome, pCD/pBM-SNA disassembles, recovers the CD cavity to dissolve CCs into free cholesterol due to the protonation of BM (ON), and reduces CCs, finally enhancing the cholesterol efflux and promoting atherosclerosis regression. Our findings provide an "OFF-ON" tactic to remove lysosomal CCs for antiatherosclerosis as well as other diseases such as Niemann-Pick type C diseases with excessive cholesterol accumulation in the lysosome.
    Keywords:  atherosclerosis; cholesterol crystals; pH sensitive; supramolecular nanoassembly; β-cyclodextrin
    DOI:  https://doi.org/10.1021/acs.nanolett.1c03664
  5. Neurooncol Adv. 2021 Jan-Dec;3(1):3(1): vdab152
      Background: Increased membrane trafficking is observed in numerous cancer types, including glioblastoma. Targeting the oncogenic driven acquired alterations in membrane trafficking by synthetic cationic amphiphilic small molecules has recently been shown to induce death of glioblastoma cells, although the molecular targets are unknown.Methods: The mechanism of action of the cationic amphiphilic drug Vacquinol-1 (Vacq1)-induced cytotoxicity was investigated using cell biology, biochemistry, functional experiments, chemical biology, unbiased antibody-based post-translation modification profiling, and mass spectrometry-based chemical proteomic analysis on patient-derived glioblastoma cells.
    Results: Vacq1 induced two types of abnormal endolysosomal vesicles, enlarged vacuoles and acidic vesicle organelles (AVOs). Mechanistically, enlarged vacuoles were formed by the impairment of lysosome reformation through the direct interaction and inhibition of calmodulin (CaM) by Vacq1, while AVO formation was induced by Vacq1 accumulation and acidification in the endosomal compartments through its activation of the v-ATPase. As a consequence of v-ATPase activation, cellular ATP consumption markedly increased, causing cellular energy shortage and cytotoxicity. This effect of Vacq1 was exacerbated by its inhibitory effects on calmodulin, causing lysosomal depletion and a failure of acidic vesicle organelle clearance.
    Conclusion: Our study identifies the targets of Vacq1 and the mechanisms underlying its selective cytotoxicity in glioblastoma cells. The dual function of Vacq1 sets in motion a glioblastoma-specific vicious cycle of ATP consumption resulting in cellular energy crisis and cell death.
    Keywords:  cationic amphiphilic drug; endolysosome; glioblastoma; nonapoptotic cell death
    DOI:  https://doi.org/10.1093/noajnl/vdab152
  6. FASEB J. 2021 Dec;35(12): e22031
      Loss of skeletal muscle mass and force is of critical importance in numerous pathologies, like age-related sarcopenia or cancer. It has been shown that the Akt-mTORC1 pathway is critical for stimulating adult muscle mass and function, however, it is unknown if mTORC1 is the only mediator downstream of Akt and which intracellular processes are required for functional muscle growth. Here, we show that loss of Raptor reduces muscle hypertrophy after Akt activation and completely prevents increases in muscle force. Interestingly, the residual hypertrophy after Raptor deletion can be completely prevented by administration of the mTORC1 inhibitor rapamycin. Using a quantitative proteomics approach we find that loss of Raptor affects the increases in mitochondrial proteins, while rapamycin mainly affects ribosomal proteins. Taken together, these results suggest that mTORC1 is the key mediator of Akt-dependent muscle growth and its regulation of the mitochondrial proteome is critical for increasing muscle force.
    Keywords:  Raptor; hypertrophy; mTOR; mitochondria; rapamycin; skeletal muscle
    DOI:  https://doi.org/10.1096/fj.202101054RR
  7. Pediatr Endocrinol Diabetes Metab. 2021 ;pii: 45203. [Epub ahead of print]27(3): 201-208
      Mucopolysaccharidoses (MPSs) are known as rare genetic diseases which are caused by mutation in the enzyme heparin sulfate, which normally leads to degradation and accumulation of glycosaminoglycans in the cells. There are 11 types of MPSs, whereby neuropathy may occur in seven of them (MPS I, II, IIIA, IIIB, IIIC, IIID and VII). Accumulation of degraded heparin sulfate in lysosomes causes cellular dysfunction and malfunction of several organs. However, the exact molecular mechanism how protein degradation and storage leads to cellular dysfunction is not understood, yet. Nonetheless, several genetic and biochemical methods for diagnosis of MPSs are available nowadays. Here we provide an overview on known molecular basis of MPS in general, including enzyme defects and symptoms of MPS; however, the main focus is on MPS type III together with potential and perspective therapy-options.
    Keywords:   clinical diagnosis.; glycosaminoglycan; lysosomal storage diseases (LSDs); therapy for mucopolysccharidosis; mucopolysaccharidosis III (MPS III)
    DOI:  https://doi.org/10.5114/pedm.2021.109270
  8. STAR Protoc. 2021 Dec 17. 2(4): 100916
      The lysosomal compartment is a key hub for cell metabolism, and morphological alterations have been described in several pathological conditions. Here, we describe the use of amino acid analogs modified by the presence of a methyl ester group that accumulates within lysosomes. This generates an intraluminal osmotic effect able to trigger a rapid and selective expansion of the lysosomal compartment within 2 h of treatment. We also present protocols to preserve lysosomal morphology, which yields a more accurate size measurement. For complete details on the use and execution of this protocol, please refer to Scerra et al. (2021).
    Keywords:  Cell Biology; Microscopy; Molecular/Chemical Probes
    DOI:  https://doi.org/10.1016/j.xpro.2021.100916
  9. Int J Mol Sci. 2021 Nov 08. pii: 12083. [Epub ahead of print]22(21):
      The autophagy-lysosome pathway is a major protein degradation pathway stimulated by multiple cellular stresses, including nutrient or growth factor deprivation, hypoxia, misfolded proteins, damaged organelles, and intracellular pathogens. Recent studies have revealed that transcription factor EB (TFEB) and transcription factor E3 (TFE3) play a pivotal role in the biogenesis and functions of autophagosome and lysosome. Here we report that three translation inhibitors (cycloheximide, lactimidomycin, and rocaglamide A) can facilitate the nuclear translocation of TFEB/TFE3 via dephosphorylation and 14-3-3 dissociation. In addition, the inhibitor-mediated TFEB/TFE3 nuclear translocation significantly increases the transcriptional expression of their downstream genes involved in the biogenesis and function of autophagosome and lysosome. Furthermore, we demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. These results highlight the previously unrecognized function of the translation inhibitors as activators of TFEB/TFE3, suggesting a novel biological role of translation inhibition in autophagy regulation.
    Keywords:  TFEB; TFEB nuclear translocation; autophagy-lysosome pathway; calcineurin; eIF4A helicase; mTOR; ribosome; translation inhibitor
    DOI:  https://doi.org/10.3390/ijms222112083
  10. Mol Cell Proteomics. 2021 Nov 08. pii: S1535-9476(21)00146-8. [Epub ahead of print] 100174
      The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis and role in the unconventional secretion of cysteine peptidases.
    Keywords:  Trichomonas vaginalis; cysteine peptidase; glycosylation; mannose-6-phosphate receptor; phagolysosome; proteome
    DOI:  https://doi.org/10.1016/j.mcpro.2021.100174
  11. Drugs. 2021 Nov 08.
      Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A (AGAL/GLA) gene. The lysosomal accumulation of the substrates globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) results in progressive renal failure, cardiomyopathy associated with cardiac arrhythmia, and recurrent strokes, significantly limiting life expectancy in affected patients. Current treatment options for FD include recombinant enzyme-replacement therapies (ERTs) with intravenous agalsidase-α (0.2 mg/kg body weight) or agalsidase-β (1 mg/kg body weight) every 2 weeks, facilitating cellular Gb3 clearance and an overall improvement of disease burden. However, ERT can lead to infusion-associated reactions, as well as the formation of neutralizing anti-drug antibodies (ADAs) in ERT-treated males, leading to an attenuation of therapy efficacy and thus disease progression. In this narrative review, we provide a brief overview of the clinical picture of FD and diagnostic confirmation. The focus is on the biochemical and clinical significance of neutralizing ADAs as a humoral response to ERT. In addition, we provide an overview of different methods for ADA measurement and characterization, as well as potential therapeutic approaches to prevent or eliminate ADAs in affected patients, which is representative for other ERT-treated lysosomal storage diseases.
    DOI:  https://doi.org/10.1007/s40265-021-01621-y
  12. J Hematol Oncol. 2021 Nov 08. 14(1): 189
      The hypoxic microenvironment is presumed to be a sanctuary for myeloid leukemia cells that causes relapse following chemotherapy, but the underlying mechanism remains elusive. Using a zebrafish xenograft model, we observed that the hypoxic hematopoietic tissue preserved most of the chemoresistant leukemic cells following the doxorubicin (Dox) treatment. And hypoxia upregulated TFEB, a master regulator of lysosomal biogenesis, and increased lysosomes in leukemic cells. Specimens from relapsed myeloid leukemia patients also harbored excessive lysosomes, which trapped Dox and prevented drug nuclear influx leading to leukemia chemoresistance. Pharmaceutical inhibition of lysosomes enhanced Dox-induced cytotoxicity against leukemic cells under hypoxia circumstance. To overcome lysosome associated chemoresistance, we developed a pH-sensitive dextran-doxorubicin nanomedicine (Dex-Dox) that efficiently released Dox from lysosomes and increased drug nuclear influx. More importantly, Dex-Dox treatment significantly improved the chemotherapy outcome in the zebrafish xenografts transplanted with cultured leukemic cells or relapsed patient specimens. Overall, we developed a novel lysosome targeting nanomedicine that is promising to overcome the myeloid leukemia chemoresistance.
    Keywords:  Chemotherapy; Doxorubicin; Hypoxia; Lysosome; Myeloid leukemia
    DOI:  https://doi.org/10.1186/s13045-021-01199-8
  13. Neurosci Lett. 2021 Nov 09. pii: S0304-3940(21)00717-5. [Epub ahead of print] 136338
      Alzheimer's disease (AD) is the most common cause of dementia affecting millions of people. Neuronal death in AD is initiated by oligomeric amyloid-β (Aβ) peptides. Recently, we proposed the amyloid degradation toxicity hypothesis, which explains multiple major observations associated with AD including autophagy failure and a decreased metabolism. According to the hypothesis, the key event in the cellular toxicity of amyloid is the formation of non-selective membrane channels in lysosomal membranes by amyloid fragments that are produced by the digestion of Aβ previously absorbed by endocytosis. Electrophysiological data suggest that amyloid-formed channels have different sizes, which can be explained by the fact that channel creating barrel-shaped amyloid aggregates can consist of different number of monomers. To estimate the ability of channels to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in non-conductive membranes that pass spheres with a density of average globular proteins. As a basis, we used the conductance distribution taken from the previously published experimental dataset, in which single channels with electrical conductance of up to one nanosiemens were registered. Our calculations show that channels with such a giant conductance can allow for passing macromolecules such as large as lysosomal cathepsins implicated in the activation of apoptosis. The formation of giant channels is disproportionally promoted in an acidic environment. Also, amyloid fragments leaking from permeabilized lysosomes can reach the internal leaflet of the plasma membrane and permeabilize it. We conclude that while dissipation of the proton gradient by any (even smallest) amyloid channels readily explains lysosomal failure, the relatively rare events of lysosomal permeabilization to large macromolecules can be an additional mechanism of cellular death induced by exposure to Aβ.
    Keywords:  Alzheimer’s disease; beta-amyloid; cell death; lysosomal permeabilization; lysosome; membrane channel
    DOI:  https://doi.org/10.1016/j.neulet.2021.136338
  14. Eur J Clin Invest. 2021 Nov 09. e13707
      BACKGROUND: Mesenchymal stem cells' (MSC) therapeutic potential has been investigated for the treatment of several neurodegenerative diseases. The fact that these cells can mediate a beneficial effect in different neurodegenerative contexts strenghens their competence to target diverse mechanisms. On the other hand, distinct disorders may share similar mechanisms despite having singular neuropathological characteristics.METHODS: We have previously shown that MSC can be beneficial for two disorders, one belonging to the groups of Lysosomal Storage Disorders (LSDs) - the Krabbe Disease or Globoid Cell Leukodystrophy, and the other to the family of Polyglutamine diseases (PolyQs) - the Machado-Joseph Disease or Spinocerebellar ataxia type-3. We gave also input into disease characterization since neuropathology and MSC's effects are intrinsically associated. This review aims at describing MSC's multimode of action in these disorders, while emphasizing to possible mechanistic alterations they must share due to the accumulation of cellular toxic products.
    RESULTS: LSDs and PolyQs have different aetiology and associated symptoms, but both result from the accumulation of undegradable products inside neuronal cells due to inefficient clearance by the endosomal/lysosomal pathway. Moreover, numerous cellular mechanisms that become compromised latter are also shared by these two disease groups.
    CONCLUSIONS: Here we emphasise MSC's effect in improving proteostasis and autophagy cycling turnover, neuronal survival, synaptic activity, and axonal transport. LSDs and PolyQs, though rare in their predominance, collectively affect many people and require our utmost dedication and efforts to get successful therapies due to their tremendous impact on patient's lives and society.
    Keywords:  Axonal survival; Endosomal/lysosomal pathway; Immunomodulation; Lysosomal storage disorders (LSD); Mesenchymal stem cells (MSC); Polyglutamine diseases (PolyQ)
    DOI:  https://doi.org/10.1111/eci.13707
  15. iScience. 2021 Nov 19. 24(11): 103274
      Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery.
    Keywords:  Biological sciences; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103274
  16. STAR Protoc. 2021 Dec 17. 2(4): 100918
      We present a protocol for measuring the activity of the mechanistic target of rapamycin (mTOR) pathway in ex vivo isolated mouse primary hepatocytes. It can be used as a tool for genetic, pharmacological, metabolomic, and signal transduction procedures. We discuss critical aspects for improving yield, viability, and modulation of the mTOR pathway. This protocol can be adapted to other signaling cascades and is compatible with multiple readouts. For complete details on the use and execution of this protocol, please refer to Ortega-Molina et al. (2021).
    Keywords:  Cell culture; Cell isolation; Cell-based Assays; Metabolism; Metabolomics; Signal Transduction
    DOI:  https://doi.org/10.1016/j.xpro.2021.100918
  17. iScience. 2021 Nov 19. 24(11): 103260
      Spermidine (Spd) is a nitrogen sink and signaling molecule that plays pivotal roles in eukaryotic cell growth and must be finetuned to meet various energy demands. In eukaryotes, target of rapamycin (TOR) is a central nutrient sensor, especially N, and a master-regulator of growth and development. Here, we discovered that Spd stimulates the growth of maize and Arabidopsis seedlings through TOR signaling. Inhibition of Spd biosynthesis led to TOR inactivation and growth defects. Furthermore, disruption of a TOR complex partner RAPTOR1B abolished seedling growth stimulation by Spd. Strikingly, TOR activated by Spd promotes translation of key metabolic enzyme upstream open reading frame (uORF)-containing mRNAs, PAO and CuAO, by facilitating translation reinitiation and providing feedback to polyamine metabolism and TOR activation. The Spd-TOR relay protected young-age seedlings of maize from expeditious stress heat shock. Our results demonstrate Spd is an upstream effector of TOR kinase in planta and provide its potential application for crop protection.
    Keywords:  cell biology; plant biology; plant physiology
    DOI:  https://doi.org/10.1016/j.isci.2021.103260
  18. Cell. 2021 Nov 04. pii: S0092-8674(21)01233-2. [Epub ahead of print]
      The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.
    Keywords:  ATG16L1; Atg8ylation; COVID-19; FIP200; Golgi; SARS-CoV-2; Syntaxin 17; autophagy; coronavirus; endosome
    DOI:  https://doi.org/10.1016/j.cell.2021.10.017
  19. Front Cell Dev Biol. 2021 ;9 716919
      Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro. ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention.
    Keywords:  ALS; FUS; lyso-pHluorin; lysosomes; optodroplets; phase separation
    DOI:  https://doi.org/10.3389/fcell.2021.716919
  20. Chem Sci. 2021 Oct 27. 12(41): 13909-13913
      Gaucher's disease, the most prevalent lysosomal storage disorder, is caused by missense mutation of the GBA gene, ultimately resulting in deficient GCase activity, hence the excessive build-up of cellular glucosylceramide. Among different therapeutic strategies, pharmacological chaperoning of mutant GCase represents an attractive approach that relies on small organic molecules acting as protein stabilizers. Herein, we expand upon a new class of transient GCase inactivators based on a reactive 2-deoxy-2-fluoro-β-d-glucoside tethered to an array of lipid-mimicking phosphorus-based aglycones, which not only improve the selectivity and inactivation efficiency, but also the stability of these compounds in aqueous media. This hypothesis was further validated with kinetic and cellular studies confirming restoration of catalytic activity in Gaucher cells after treatment with these pharmacological chaperones.
    DOI:  https://doi.org/10.1039/d1sc03831a
  21. J Cachexia Sarcopenia Muscle. 2021 Nov 09.
      BACKGROUND: Skeletomuscular diseases result in significant muscle loss and decreased performance, paralleled by a loss in mitochondrial and oxidative capacity. Insulin and insulin-like growth factor-1 (IGF-1) are two potent anabolic hormones that activate a host of signalling intermediates including the serine/threonine kinase AKT to influence skeletal muscle physiology. Defective AKT signalling is associated with muscle pathology, including cachexia, sarcopenia, and disuse; however, the mechanistic underpinnings remain unresolved.METHODS: To elucidate the role of AKT signalling in muscle mass and physiology, we generated both congenital and inducible mouse models of skeletal muscle-specific AKT deficiency. To understand the downstream mechanisms mediating AKT's effects on muscle biology, we generated mice lacking AKT1/2 and FOXO1 (M-AKTFOXO1TKO and M-indAKTFOXO1TKO) to inhibit downstream FOXO1 signalling, AKT1/2 and TSC1 (M-AKTTSCTKO and M-indAKTTSCTKO) to activate mTORC1, and AKT1/2, FOXO1, and TSC1 (M-QKO and M-indQKO) to simultaneously activate mTORC1 and inhibit FOXO1 in AKT-deficient skeletal muscle. Muscle proteostasis and physiology were assessed using multiple assays including metabolic labelling, mitochondrial function, fibre typing, ex vivo physiology, and exercise performance.
    RESULTS: Here, we show that genetic ablation of skeletal muscle AKT signalling resulted in decreased muscle mass and a loss of oxidative metabolism and muscle performance. Specifically, deletion of muscle AKT activity during development or in adult mice resulted in a significant reduction in muscle growth by 30-40% (P  < 0.0001; n = 12-20) and 15% (P < 0.01 and P < 0.0001; n = 20-30), respectively. Interestingly, this reduction in muscle mass was primarily due to an ~40% reduction in protein synthesis in both M-AKTDKO and M-indAKTDKO muscles (P < 0.05 and P < 0.01; n = 12-20) without significant changes in proteolysis or autophagy. Moreover, a significant reduction in oxidative capacity was observed in both M-AKTDKO (P < 0.05, P < 0.01 and P < 0.001; n = 5-12) and M-indAKTDKO (P < 0.05 and P < 0.01; n = 4). Mechanistically, activation and inhibition of mTORC1/FOXO1, respectively, but neither alone, were sufficient to restore protein synthesis, muscle oxidative capacity, and muscle function in the absence of AKT in vivo. In a mouse model of disuse-induced muscle loss, simultaneous activation of mTORC1 and inhibition of FOXO1 preserved muscle mass following immobilization (~5-10% reduction in casted M-indFOXO1TSCDKO muscles vs. ~30-40% casted M-indControl muscles, P < 0.05 and P < 0.0001; n = 8-16).
    CONCLUSIONS: Collectively, this study provides novel insights into the AKT-dependent mechanisms that underlie muscle protein homeostasis, function, and metabolism in both normal physiology and disuse-induced muscle wasting.
    Keywords:  AKT signalling; Disuse-induced muscle wasting; Fibre specification; Insulin action
    DOI:  https://doi.org/10.1002/jcsm.12846
  22. Mol Ther Methods Clin Dev. 2021 Dec 10. 23 370-389
      Delivery of adeno-associated viral vectors (AAVs) to cerebrospinal fluid (CSF) has emerged as a promising approach to achieve widespread transduction of the central nervous system (CNS) and peripheral nervous system (PNS), with direct applicability to the treatment of a wide range of neurological diseases, particularly lysosomal storage diseases. Although studies in small animal models have provided proof of concept and experiments in large animals demonstrated feasibility in bigger brains, there is not much information on long-term safety or durability of the effect. Here, we report a 7-year study in healthy beagle dogs after intra-CSF delivery of a single, clinically relevant dose (2 × 1013 vg/dog) of AAV9 vectors carrying the canine sulfamidase, the enzyme deficient in mucopolysaccharidosis type IIIA. Periodic monitoring of CSF and blood, clinical and neurological evaluations, and magnetic resonance and ultrasound imaging of target organs demonstrated no toxicity related to treatment. AAV9-mediated gene transfer resulted in detection of sulfamidase activity in CSF throughout the study. Analysis at tissue level showed widespread sulfamidase expression and activity in the absence of histological findings in any region of encephalon, spinal cord, or dorsal root ganglia. Altogether, these results provide proof of durability of expression and long-term safety for intra-CSF delivery of AAV-based gene transfer vectors encoding therapeutic proteins to the CNS.
    Keywords:  adeno-associated viral vector; brain; central nervous system; cerebrospinal fluid; dorsal root ganglia; durability; gene therapy; lysosomal storage disease; mucopolysaccharidosis type IIIA; safety
    DOI:  https://doi.org/10.1016/j.omtm.2021.09.017
  23. Int J Mol Sci. 2021 Oct 26. pii: 11557. [Epub ahead of print]22(21):
      Progranulin (PGRN) is a secreted glycoprotein that regulates numerous cellular processes. The role of PGRN as a regulator of lysosomes has recently received attention. The purpose of this study was to characterize the retinal phenotype in mature PGRN knockout (Grn-/-) mice. The a-wave amplitude of scotopic electroretinogram and outer nuclear thickness were significantly reduced at 6 months of age in Grn-/- mice compared to wild-type (Grn+/+) mice. In Grn-/- mice, retinal microglial cells accumulated on the retinal pigment epithelium (RPE) apical layer, and the number of infiltrated microglia and white fundus lesions between 2 and 6 months of age showed a close affinity. In Grn+/+ mice, PGRN was located in the retina, while the strongest PGRN signals were detected in the RPE-choroid. The different effects of PGRN deficiency on the expression of lysosomal proteins between the retina and RPE-choroid were demonstrated. Our data suggest that the subretinal translocation of microglia is a characteristic phenotype in the retina of mature PGRN knockout mice. The different effects of PGRN deficiency on the expression of lysosomal proteins between the retina and RPE-choroid might modulate microglial dynamics in PGRN knockout mice.
    Keywords:  lysosome; microglia; neuronal ceroid lipofuscinosis; progranulin; retina; retinal pigment epithelium
    DOI:  https://doi.org/10.3390/ijms222111557
  24. Int J Mol Sci. 2021 Oct 20. pii: 11339. [Epub ahead of print]22(21):
      Fabry disease (FD) is caused by mutations in the α-galactosidase A (GLA) gene encoding the lysosomal AGAL enzyme. Loss of enzymatic AGAL activity and cellular accumulation of sphingolipids (mainly globotriaosylcermide) may lead to podocyturia and renal loss of function with increased cardiovascular morbidity and mortality in affected patients. To identify dysregulated cellular pathways in FD, we established a stable AGAL-deficient podocyte cell line to perform a comprehensive proteome analysis. Imbalanced protein expression and function were analyzed in additional FD cell lines including endothelial, epithelial kidney, patient-derived urinary cells and kidney biopsies. AGAL-deficient podocytes showed dysregulated proteins involved in thermogenesis, lysosomal trafficking and function, metabolic activity, cell-cell interactions and cell cycle. Proteins associated with neurological diseases were upregulated in AGAL-deficient podocytes. Rescues with inducible AGAL expression only partially normalized protein expression. A disturbed protein expression was confirmed in endothelial, epithelial and patient-specific cells, pointing toward fundamental pathway disturbances rather than to cell type-specific alterations in FD. We conclude that a loss of AGAL function results in profound changes of cellular pathways, which are ubiquitously in different cell types. Due to these profound alterations, current approved FD-specific therapies may not be sufficient to completely reverse all dysregulated pathways.
    Keywords:  Fabry disease; podocytes; proteome analysis; sphingolipids; α-galactosidase A-deficiency
    DOI:  https://doi.org/10.3390/ijms222111339
  25. eNeuro. 2021 Nov 10. pii: ENEURO.0310-21.2021. [Epub ahead of print]
      The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) causes familial Parkinson's disease (PD) and is also found in a subset of idiopathic cases. Prior studies in Drosophila and human induced pluripotent stem cell (iPSC)-derived dopamine neurons uncovered a pronounced effect of G2019S LRRK2 on mRNA translation. It was previously reported that G2019S LRRK2 promotes translation of mRNAs with complex 5' untranslated region (UTR) secondary structure, resulting in increased expression of calcium channels and dysregulated calcium homeostasis in human dopamine neurons. Here, we show that dysregulated translation occurs in the brains of mammalian LRRK2 models in vivo Through ribosome profiling studies of global translation, we observe that mRNAs with complex 5'UTR structure are also preferentially translated in the G2019S LRRK2-expressing mouse brain. Reporter assays suggest that this 5'UTR preference is independent of translation initiation factors. Conversely, translation of mRNAs with complex 5'UTR secondary structure is downregulated in LRRK2 knock-out (KO) mouse brain, indicating a robust link between LRRK2 kinase activity and translation of mRNA with complex 5'UTR structure. Further, substantia nigra pars compacta (SNpc) dopamine neurons in the G2019S LRRK2-expressing brain exhibit increased calcium influx, which is consistent with the previous report from human dopamine neurons. These results collectively suggest that LRRK2 plays a mechanistic role in translational regulation, and the G2019S mutation in LRRK2 causes translational defects leading to calcium dysregulation in the mammalian brain.Significance StatementParkinson's disease (PD)-linked G2019S mutation of leucine-rich repeat kinase 2 (LRRK2) is known to cause abnormalities in mRNA translation. These translational defects were suggested to cause calcium dysregulation, thereby imposing a long-term cellular stress to dopamine neurons. While these effects of G2019S LRRK2 on mRNA translation have been seen in Drosophila brain tissues and cultured mammalian neurons, translational profiling of the mammalian brain expressing G2019S LRRK2 has not been reported. In this study, we employed ribosome profiling to survey mRNA translation in the brains of LRRK2 mouse models, thereby demonstrating that the G2019S LRRK2 mutation broadly alters mRNA translation in the mouse brain.
    Keywords:  LRRK2; RPS15; translation
    DOI:  https://doi.org/10.1523/ENEURO.0310-21.2021
  26. Front Cell Dev Biol. 2021 ;9 742714
      ATP6V1B2 encodes the V1B2 subunit in V-ATPase, a proton pump responsible for the acidification of lysosomes. Mutations in this gene cause DDOD syndrome, DOORS syndrome, and Zimmermann-Laband syndrome, which share overlapping feature of congenital sensorineural deafness, onychodystrophy, and different extents of intellectual disability without or with epilepsy. However, the underlying mechanisms remain unclear. To investigate the pathological role of mutant ATP6V1B2 in the auditory system, we evaluated auditory brainstem response, distortion product otoacoustic emissions, in a transgenic line of mice carrying c.1516 C > T (p.Arg506∗) in Atp6v1b2, Atp6v1b2 Arg506*/Arg506* . To explore the pathogenic mechanism of neurodegeneration in the auditory pathway, immunostaining, western blotting, and RNAscope analyses were performed in Atp6v1b2Arg506*/Arg506* mice. The Atp6v1b2Arg506*/Arg506* mice showed hidden hearing loss (HHL) at early stages and developed late-onset hearing loss. We observed increased transcription of Atp6v1b1 in hair cells of Atp6v1b2Arg506*/Arg506* mice and inferred that Atp6v1b1 compensated for the Atp6v1b2 dysfunction by increasing its own transcription level. Genetic compensation in hair cells explains the milder hearing impairment in Atp6v1b2Arg506*/Arg506* mice. Apoptosis activated by lysosomal dysfunction and the subsequent blockade of autophagic flux induced the degeneration of spiral ganglion neurons and further impaired the hearing. Intraperitoneal administration of the apoptosis inhibitor, BIP-V5, improved both phenotypical and pathological outcomes in two live mutant mice. Based on the pathogenesis underlying hearing loss in Atp6v1b2-related syndromes, systemic drug administration to inhibit apoptosis might be an option for restoring the function of spiral ganglion neurons and promoting hearing, which provides a direction for future treatment.
    Keywords:  Atp6v1b2; apoptosis; function compensation; lysosome; syndromic hearing loss
    DOI:  https://doi.org/10.3389/fcell.2021.742714
  27. Biomed Pharmacother. 2021 Nov 10. pii: S0753-3322(21)01029-5. [Epub ahead of print] 112245
      Cathepsin S (CTSS), a lysosomal protease, belongs to a family of cysteine cathepsin proteases that promote degradation of damaged proteins in the endolysosomal pathway. Aberrant CTSS expression and regulation are associated with the pathogenesis of several diseases, including lung diseases. CTSS overexpression causes a variety of pathological processes, including pulmonary fibrosis, with increased CTSS secretion and accelerated extracellular matrix remodeling. Compared to many other cysteine cathepsin family members, CTSS has unique features that it presents limited tissue expression and retains its enzymatic activity at a neutral pH, suggesting its decisive involvement in disease microenvironments. In this review, we investigated the role of CTSS in lung disease, exploring recent studies that have indicated that CTSS mediates fibrosis in unique ways, along with its structure, substrates, and distinct regulation. We also outlined examples of CTSS inhibitors in clinical and preclinical development and proposed CTSS as a potential therapeutic target for pulmonary fibrosis.
    Keywords:  Cathepsin S; Clinical and preclinical; Inhibitors; Pulmonary fibrosis; Therapeutic target
    DOI:  https://doi.org/10.1016/j.biopha.2021.112245
  28. Anal Chem. 2021 Nov 10.
      DNA logic nanodevices have prospects in molecular recognitions but still face challenges in achieving DNA computation-controlled regulation in specific compartments of living cells. By incorporating the i-motif sequence and ATP aptamers into a Y-shaped DNA (Y-DNA) structure, and applying gold nanoparticles (AuNPs) as the transporting carrier, herein we present a new type of DNA logic nanodevices to monitor the ATP levels in lysosomes of living cells. Triple energy transfers including dual fluorescent resonance energy transfers (FRETs) and a nanometal surface energy transfer (NSET) occurred in the DNA logic nanodevices. It was identified that the proposed nanodevices perform an AND logic operation to output FRET signals only when an endogenous proton and ATP simultaneously exist in the cellular microenvironment. Owing to the use of the i-motif sequence, the nanodevices have lysosome-recognizing capacity without causing alkalization of the acidic organelle, making DNA computation-controlled regulation at the level of cellular organelles achievable. These DNA logic nanodevices show high application prospects in lysosome-related cellular function and disease treatment.
    DOI:  https://doi.org/10.1021/acs.analchem.1c02829
  29. J Cell Sci. 2021 Nov 08. pii: jcs.259306. [Epub ahead of print]
      LncRNAs are long RNA transcripts that do not code for proteins and that have been shown to play a major role in cellular processes through diverse mechanisms. DRAIC, a lncRNA which is downregulated in castration-resistant advanced prostate cancer, inhibits the NF-kB pathway by inhibiting the IκB kinase. Decreased DRAIC expression predicted poor patient outcome in gliomas and seven other cancers. We now report that DRAIC suppresses invasion, migration, colony formation and xenograft growth of glioblastoma derived cell lines. DRAIC activates AMPK by downregulating the NF-κB target gene GLUT1, and thus represses mTOR, leading to downstream effects such as decrease in protein translation and increase in autophagy. DRAIC, therefore, has an effect on multiple signal transduction pathways that are important for oncogenesis: the NF-κB pathway and AMPK-mTOR-S6K/ULK1 pathway. The regulation of NF-κB, protein translation and autophagy by the same lncRNA explains the tumor suppressive role of DRAIC in different cancers and reinforces the importance of lncRNAs as emerging regulators of signal transduction pathways.
    Keywords:  AMPK; Autophagy; DRAIC lncRNA; MTORC1; Protein-translation
    DOI:  https://doi.org/10.1242/jcs.259306
  30. Brain Dev. 2021 Oct 27. pii: S0387-7604(21)00186-8. [Epub ahead of print]
      BACKGROUND: The role of PI3K/AKT/mTOR pathway hyperactivation in localized brain overgrowth is evolving. We describe two patients with focal cortical dysplasia (FCD) who demonstrated somatic mutations in TSC1 and TSC2 genes in the dysplastic brain tissue but not peripheral blood.METHODS: Paired whole-exome sequencing was performed on genomic DNA extracted from blood and excised brain tissue in two children with FCD who underwent excision of dysplastic tissue.
    RESULTS: Patient 1, a 14-year boy, had drug-resistant focal epilepsy with onset at 20 months. His brain MRI showed abnormalities suggestive of FCD in the left superior and middle frontal lobes. Patient 2 presented at the age of 10 years with pharmaco-resistant focal epilepsy (onset at six years). His MRI suggested FCD in the left insular lobe. Both patients underwent surgical excision of FCD, and excised tissues were pathologically confirmed to have type IIb FCD. For patient 1, a missense mutation (c.64C > T; p.Arg22Trp) was detected in the TSC1 gene in DNA of dysplastic brain tissue but not peripheral blood lymphocytes. Similarly, for patient 2, a frameshift mutation (c.4258_4261delCAGT; p.Ser1420GlyfsTer55) in the TSC2 gene was identified in the brain tissue but not blood. Both gene variants are likely pathogenic and cause mTOR pathway activation.
    CONCLUSION: Our report of TSC1/TSC2 somatic mutations in patients with non-syndromic FCD suggests that localized hyperactivation of the mTOR pathway can cause focal malformations during cortical development and presents pharmacological targets for precision therapy in FCD management.
    Keywords:  Cortical malformations; Drug-resistant epilepsy; Epilepsy; mTOR, AKT, PI3K
    DOI:  https://doi.org/10.1016/j.braindev.2021.10.002
  31. Cancers (Basel). 2021 Oct 24. pii: 5337. [Epub ahead of print]13(21):
      Glioblastoma is a tumor type of unmet need despite the development of multimodal treatment strategies. The main factors contributing to the poor prognosis of glioblastoma patients are diverse genetic and epigenetic changes driving glioblastoma persistence and recurrence. Complemented are these factors by extracellular cues mediated through cell surface receptors, which further aid in fostering pro-invasion and pro-survival signaling contributing to glioblastoma therapy resistance. The underlying mechanisms conferring this therapy resistance are poorly understood. Here, we show that the cytoskeleton regulator Lamellipodin (Lpd) mediates invasiveness, proliferation and radiosensitivity of glioblastoma cells. Phosphoproteome analysis identified the epidermal growth factor receptor (EGFR) signaling axis commonly hyperactive in glioblastoma to depend on Lpd. Mechanistically, EGFR signaling together with an interaction between Lpd and the Rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR) jointly regulate glioblastoma radiosensitivity. Collectively, our findings demonstrate an essential function of Lpd in the radiation response and invasiveness of glioblastoma cells. Thus, we uncover a novel Lpd-driven resistance mechanism, which adds an additional critical facet to the complex glioblastoma resistance network.
    Keywords:  EGFR; Lamellipodin; RICTOR; glioblastoma; invasion; radiosensitivity
    DOI:  https://doi.org/10.3390/cancers13215337
  32. J Neuroinflammation. 2021 Nov 08. 18(1): 262
      BACKGROUND: Neuronal ceroid lipofuscinoses, (NCLs or Batten disease) are a group of inherited, early onset, fatal neurodegenerative diseases associated with mutations in 13 genes. All forms of the disease are characterized by lysosomal accumulation of fluorescent storage material, as well as profound neurodegeneration, but the relationship of the various genes' function to a single biological process is not obvious. In this study, we used a well-characterized mouse model of classical late infantile NCL (cLINCL) in which the tripeptidyl peptidase 1 (Tpp1) gene is disrupted by gene targeting, resulting in loss of detectable TPP1 activity and leading to progressive neurological phenotypes including ataxia, increased motor deficiency, and early death.METHODS: In order to identify genes and pathways that may contribute to progression of the neurodegenerative process, we analyzed forebrain/midbrain and cerebellar transcriptional differences at 1, 2, 3 and 4 months of age in control and TPP1-deficient mice by global RNA-sequencing.
    RESULTS: Progressive neurodegenerative inflammatory responses involving microglia, astrocytes and endothelial cells were observed, accompanied by activation of leukocyte extravasation signals and upregulation of nitric oxide production and reactive oxygen species. Several astrocytic (i.e., Gfap, C4b, Osmr, Serpina3n) and microglial (i.e., Ctss, Itgb2, Itgax, Lyz2) genes were identified as strong markers for assessing disease progression as they showed increased levels of expression in vivo over time. Furthermore, transient increased expression of choroid plexus genes was observed at 2 months in the lateral and fourth ventricle, highlighting an early role for the choroid plexus and cerebrospinal fluid in the disease pathology. Based on these gene expression changes, we concluded that neuroinflammation starts, for the most part, after 2 months in the Tpp1-/- brain and that activation of microglia and astrocytes occur more rapidly in cerebellum than in the rest of the brain; confirming increased severity of inflammation in this region.
    CONCLUSIONS: These findings have led to a better understanding of cLINCL pathological onset and progression, which may aid in development of future therapeutic treatments for this disease.
    Keywords:  Choroid plexus; Lysosomal tripeptidyl peptidase 1; Neuroinflammation; Neuronal ceroid lipofuscinoses; Pediatric neurodegeneration; Transcriptome
    DOI:  https://doi.org/10.1186/s12974-021-02302-z
  33. Open Biol. 2021 Nov;11(11): 210194
      The membranes of endosomes, phagosomes and macropinosomes can become damaged by the physical properties of internalized cargo, by active pathogenic invasion or by cellular processes, including endocytic maturation. Loss of membrane integrity is often deleterious and is, therefore, prevented by mitigation and repair mechanisms. However, it can occasionally be beneficial and actively induced by cells. Here, we summarize the mechanisms by which cells, in particular phagocytes, try to prevent membrane damage and how, when this fails, they repair or destroy damaged endocytic organelles. We also detail how one type of phagocyte, the dendritic cell, can deliberately trigger localized damage to endocytic organelles to allow for major histocompatibility complex class I presentation of exogenous antigens and initiation of CD8+ T-cell responses to viruses and tumours. Our review highlights mechanisms for the regulation of endocytic organelle membrane integrity at the intersection of cell biology and immunology that could be co-opted for improving vaccination and intracellular drug delivery.
    Keywords:  cross-presentation; endosomal escape; endosome; membrane damage; membrane repair; phagosome
    DOI:  https://doi.org/10.1098/rsob.210194
  34. Cell Signal. 2021 Nov 06. pii: S0898-6568(21)00275-8. [Epub ahead of print] 110186
      Rare gain of function mutations in the gene encoding Dyrk1b, a key regulator of skeletal muscle differentiation, have been associated with sarcopenic obesity (SO) and metabolic syndrome (MetS) in humans. So far, the global gene networks regulated by Dyrk1b during myofiber differentiation have remained elusive. Here, we have performed untargeted proteomics to determine Dyrk1b-dependent gene-network in differentiated C2C12 myofibers. This analysis led to identification of translational inhibitor, 4e-bp1 as a post-transcriptional target of Dyrk1b in C2C12 cells. Accordingly, CRISPR/Cas9 mediated knockout of Dyrk1b in zebrafish identified 4e-bp1 as a downstream target of Dyrk1b in-vivo. The Dyrk1b knockout zebrafish embryos exhibited markedly reduced myosin heavy chain 1 expression in poorly developed myotomes and were embryonic lethal. Using knockdown and overexpression approaches in C2C12 cells, we found that 4e-bp1 enhances autophagy and mediates the effects of Dyrk1b on skeletal muscle differentiation. Dyrk1bR102C, the human sarcopenic obesity-associated mutation impaired muscle differentiation via excessive activation of 4e-bp1/autophagy axis in C2C12 cells. Strikingly, the defective muscle differentiation in Dyrk1bR102C cells was rescued by reduction of autophagic flux. The identification of Dyrk1b-4e-bp1-autophagy axis provides significant insight into pathways that are relevant to human skeletal muscle development and disorders.
    Keywords:  4e-bp1; Myogenesis; Sarcopenic obesity; dyrk1b
    DOI:  https://doi.org/10.1016/j.cellsig.2021.110186
  35. PLoS Comput Biol. 2021 Nov 08. 17(11): e1009522
      Genome-scale metabolic models (GEMs) are comprehensive knowledge bases of cellular metabolism and serve as mathematical tools for studying biological phenotypes and metabolic states or conditions in various organisms and cell types. Given the sheer size and complexity of human metabolism, selecting parameters for existing analysis methods such as metabolic objective functions and model constraints is not straightforward in human GEMs. In particular, comparing several conditions in large GEMs to identify condition- or disease-specific metabolic features is challenging. In this study, we showcase a scalable, model-driven approach for an in-depth investigation and comparison of metabolic states in large GEMs which enables identifying the underlying functional differences. Using a combination of flux space sampling and network analysis, our approach enables extraction and visualisation of metabolically distinct network modules. Importantly, it does not rely on known or assumed objective functions. We apply this novel approach to extract the biochemical differences in adipocytes arising due to unlimited vs blocked uptake of branched-chain amino acids (BCAAs, considered as biomarkers in obesity) using a human adipocyte GEM (iAdipocytes1809). The biological significance of our approach is corroborated by literature reports confirming our identified metabolic processes (TCA cycle and Fatty acid metabolism) to be functionally related to BCAA metabolism. Additionally, our analysis predicts a specific altered uptake and secretion profile indicating a compensation for the unavailability of BCAAs. Taken together, our approach facilitates determining functional differences between any metabolic conditions of interest by offering a versatile platform for analysing and comparing flux spaces of large metabolic networks.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009522