bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022‒08‒28
forty-two papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.
  2. Identification of mEAK-7 as a human V-ATPase regulator via cryo-EM data mining.
  3. Long non-coding RNA SNHG6 couples cholesterol sensing with mTORC1 activation in hepatocellular carcinoma.
  4. Lysosomal exocytosis releases pathogenic α-synuclein species from neurons in synucleinopathy models.
  5. In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair.
  6. Rescue of lysosomal function as therapeutic strategy for SPG15 hereditary spastic paraplegia.
  7. Neuronopathic Gaucher disease: Beyond lysosomal dysfunction.
  8. Disease correction in Mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.
  9. Invading Bacterial Pathogens Activate Transcription Factor EB in Epithelial Cells through the Amino Acid Starvation Pathway of mTORC1 Inhibition.
  10. Mitochondria-lysosome crosstalk in GBA1-associated Parkinson's disease.
  11. Potassium-chelating drug sodium polystyrene sulfonate enhances lysosomal function and suppresses proteotoxicity.
  12. Preclinical Enzyme Replacement Therapy with a Recombinant β-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis.
  13. Activating transcription factor 4-dependent hsa-miR-663a transcription mediates mTORC1/p70S6K1 signaling underleucine deprivation.
  14. LIMP2 gene, evolutionarily conserved regulation by TFE3, relieves lysosomal stress induced by cholesterol.
  15. Analysis of Brain Lipids in the Early-Onset Tay-Sachs Disease Mouse Model With the Combined Deficiency of β-Hexosaminidase A and Neuraminidase 3.
  16. Lysosome exocytosis is required for mitosis in mammalian cells.
  17. Conserved Pib2 regions have distinct roles in TORC1 regulation at the vacuole.
  18. Detection of Structural Variants by NGS: Revealing Missing Alleles in Lysosomal Storage Diseases.
  19. In-Depth Characterization of Endo-Lysosomal Aβ in Intact Neurons.
  20. Cell cycle disturbances in mucopolysaccharidoses: Transcriptomic and experimental studies on cellular models.
  21. β-Galactosylceramidase Deficiency Causes Upregulation of Long Pentraxin-3 in the Central Nervous System of Krabbe Patients and Twitcher Mice.
  22. Midkine expression by stem-like tumor cells drives persistence to mTOR inhibition and an immune-suppressive microenvironment.
  23. RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1.
  24. Autophagy Guided Interventions to Modify the Cardiac Phenotype of Danon Disease.
  25. Overview of Research into mTOR Inhibitors.
  26. Jagged-1 is induced by mTOR inhibitors in renal cancer cells through an Akt/ALK5/Smad4-dependent mechanism.
  27. Targeting Akt/PKB in pediatric tumors: A review from preclinical to clinical trials.
  28. Metabolic Fingerprinting of Fabry Disease: Diagnostic and Prognostic Aspects.
  29. IL11 stimulates ERK/P90RSK to inhibit LKB1/AMPK and activate mTOR initiating a mesenchymal program in stromal, epithelial, and cancer cells.
  30. CaMK4 overexpression in polycystic kidney disease promotes mTOR-mediated cell proliferation.
  31. miRNAs and isomiRs: Serum-Based Biomarkers for the Development of Intellectual Disability and Autism Spectrum Disorder in Tuberous Sclerosis Complex.
  32. Isoform a4 of the vacuolar ATPase a subunit promotes 4T1-12B breast cancer cell-dependent tumor growth and metastasis in vivo.
  33. Evaluation of oxidative stress and mitochondrial function in a type II mucopolysaccharidosis cellular model: in vitro effects of genistein and coenzyme Q10.
  34. Spatial localization of cathepsins: Implications in immune activation and resolution during infections.
  35. Adipose mTORC2 is essential for sensory innervation in white adipose tissue and whole-body energy homeostasis.
  36. The Role of Rab Proteins in Parkinson's Disease Synaptopathy.
  37. AMPK suppresses Th2 cell responses by repressing mTORC2.
  38. The Key Role of Lysosomal Protease Cathepsins in Viral Infections.
  39. GPRC6A Mediates Glucose and Amino Acid Homeostasis in Mice.
  40. Biological Variation in Peripheral Inflammation and Oxidative Stress Biomarkers in Individuals with Gaucher Disease.
  41. Ammonia promotes the proliferation of bone marrow-derived mesenchymal stem cells by regulating the Akt/mTOR/S6k pathway.
  42. mTOR regulation of metabolism limits LPS-induced monocyte inflammatory and procoagulant responses.
  1. Science. 2022 Aug 25. eabg6621
    Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.
    Hijai R Shin, Y Rose Citron, Lei Wang, Laura Tribouillard, Claire S Goul, Robin Stipp, Yusuke Sugasawa, Aakriti Jain, Nolwenn Samson, Chun-Yan Lim, Oliver B Davis, David Castaneda-Carpio, Mingxing Qian, Daniel K Nomura, Rushika M Perera, Eunyong Park, Douglas F Covey, Mathieu Laplante, Alex S Evers, Roberto Zoncu.
      Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified LYsosomal CHOlesterol Signaling (LYCHOS, previously annotated as G-protein coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1). Cholesterol bound to the N-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a GTPase-activating protein for the Rag guanosine triphosphatases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing, and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.
    DOI:  https://doi.org/10.1126/science.abg6621
  2. Proc Natl Acad Sci U S A. 2022 Aug 30. 119(35): e2203742119
    Identification of mEAK-7 as a human V-ATPase regulator via cryo-EM data mining.
    Longfei Wang, Di Wu, Carol V Robinson, Tian-Min Fu.
      Vacuolar-type adenosine triphosphatases (V-ATPases) not only function as rotary proton pumps in cellular organelles but also serve as signaling hubs. To identify the endogenous binding partners of V-ATPase, we collected a large dataset of human V-ATPases and did extensive classification and focused refinement of human V-ATPases. Unexpectedly, about 17% of particles in state 2 of human V-ATPases display additional density with an overall resolution of 3.3 Å. Structural analysis combined with artificial intelligence modeling enables us to identify this additional density as mEAK-7, a protein involved in mechanistic target of rapamycin (mTOR) signaling in mammals. Our structure shows that mEAK-7 interacts with subunits A, B, D, and E of V-ATPases in state 2. Thus, we propose that mEAK-7 may regulate V-ATPase function through binding to V-ATPases in state 2 as well as mediate mTOR signaling.
    Keywords:  V-ATPase; lysosomal signaling; mEAK-7; mTOR
    DOI:  https://doi.org/10.1073/pnas.2203742119
  3. Nat Metab. 2022 Aug;4(8): 1022-1040
    Long non-coding RNA SNHG6 couples cholesterol sensing with mTORC1 activation in hepatocellular carcinoma.
    Fangzhou Liu, Tian Tian, Zhen Zhang, Shanshan Xie, Jiecheng Yang, Linyu Zhu, Wen Wang, Chengyu Shi, Lingjie Sang, Kaiqiang Guo, Zuozhen Yang, Lei Qu, Xiangrui Liu, Jian Liu, Qingfeng Yan, Huai-Qiang Ju, Wenqi Wang, Hai-Long Piao, Jianzhong Shao, Tianhua Zhou, Aifu Lin.
      Cholesterol contributes to the structural basis of biological membranes and functions as a signaling molecule, whose dysregulation has been associated with various human diseases. Here, we report that the long non-coding RNA (lncRNA) SNHG6 increases progression from non-alcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) by modulating cholesterol-induced mTORC1 activation. Mechanistically, cholesterol binds ER-anchored FAF2 protein to promote the formation of a SNHG6-FAF2-mTOR complex. As a putative cholesterol effector, SNHG6 enhances cholesterol-dependent mTORC1 lysosomal recruitment and activation via enhancing FAF2-mTOR interaction at ER-lysosome contacts, thereby coordinating mTORC1 kinase cascade activation with cellular cholesterol biosynthesis in a self-amplified cycle to accelerate cholesterol-driven NAFLD-HCC development. Notably, loss of SNHG6 inhibits mTORC1 signaling and impairs growth of patient-derived xenograft liver cancer tumors, identifyifng SNHG6 as a potential target for liver cancer treatment. Together, our findings illustrate the crucial role of organelle-associated lncRNA in organelle communication, nutrient sensing, and kinase cascades.
    DOI:  https://doi.org/10.1038/s42255-022-00616-7
  4. Nat Commun. 2022 Aug 22. 13(1): 4918
    Lysosomal exocytosis releases pathogenic α-synuclein species from neurons in synucleinopathy models.
    Ying Xue Xie, Nima N Naseri, Jasmine Fels, Parinati Kharel, Yoonmi Na, Diane Lane, Jacqueline Burré, Manu Sharma.
      Considerable evidence supports the release of pathogenic aggregates of the neuronal protein α-Synuclein (αSyn) into the extracellular space. While this release is proposed to instigate the neuron-to-neuron transmission and spread of αSyn pathology in synucleinopathies including Parkinson's disease, the molecular-cellular mechanism(s) remain unclear. To study this, we generated a new mouse model to specifically immunoisolate neuronal lysosomes, and established a long-term culture model where αSyn aggregates are produced within neurons without the addition of exogenous fibrils. We show that neuronally generated pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons. We then find that neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis. The released aggregates are non-membrane enveloped and seeding-competent. Additionally, we find that this release is dependent on neuronal activity and cytosolic Ca2+. These results propose lysosomal exocytosis as a central mechanism for the release of aggregated and degradation-resistant proteins from neurons.
    DOI:  https://doi.org/10.1038/s41467-022-32625-1
  5. Proc Natl Acad Sci U S A. 2022 Aug 30. 119(35): e2205590119
    In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair.
    Sankalp Shukla, Kevin P Larsen, Chenxi Ou, Kevin Rose, James H Hurley.
      The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.
    Keywords:  in vitro reconstitution; membrane biology; membrane remodeling; membrane repair; neurodegeneration
    DOI:  https://doi.org/10.1073/pnas.2205590119
  6. Brain. 2022 Aug 27. pii: awac308. [Epub ahead of print]
    Rescue of lysosomal function as therapeutic strategy for SPG15 hereditary spastic paraplegia.
    Chiara Vantaggiato, Genny Orso, Giulia Guarato, Francesca Brivio, Barbara Napoli, Elena Panzeri, Simona Masotti, Filippo Maria Santorelli, Maria Lamprou, Sentiljana Gumeni, Emilio Clementi, Maria Teresa Bassi.
      SPG15 is a hereditary spastic paraplegia (HSP) subtype caused by mutations in Spastizin, a protein encoded by the ZFYVE26 gene. Spastizin is involved in autophagosome maturation and autophagic lysosome reformation (ALR) and SPG15-related mutations lead to ALR defects with lysosome enlargement, free lysosomes depletion and autophagosome accumulation. Symptomatic and rehabilitative treatments are the only therapy currently available for patients. Here, we targeted autophagy and lysosomes in SPG15 patient-derived cells by using a library of autophagy-modulating compounds. We identified a rose of compounds, affecting intracellular calcium levels, the calcium-calpain pathway, or lysosomal functions, that reduced autophagosome accumulation. The six most effective compounds were tested in vivo in a new SPG15 loss of function Drosophila model that mimicked the reported SPG15 phenotype, with autophagosome accumulation, enlarged lysosomes, reduced free lysosomes, ALR defects and locomotor deficit. These compounds, namely verapamil, Bay K8644, 2',5'-dideoxyadenosine, trehalose, Small Molecule Enhancer of Rapamycin 28 (SMER28) and trifluoperazine, improved lysosome biogenesis and function in vivo, demonstrating that lysosomes are a key pharmacological target to rescue SPG15 phenotype. Among the others, the small molecule enhancer of autophagy SMER28 was the most effective, rescuing both ALR defects and locomotor deficit, and could be considered as a potential therapeutic compound for this HSP subtype.
    Keywords:  ALR; SMER28; SPG15; autophagy; lysosomes
    DOI:  https://doi.org/10.1093/brain/awac308
  7. Front Mol Neurosci. 2022 ;15 934820
    Neuronopathic Gaucher disease: Beyond lysosomal dysfunction.
    Nohela B Arévalo, Cristian M Lamaizon, Viviana A Cavieres, Patricia V Burgos, Alejandra R Álvarez, María J Yañez, Silvana Zanlungo.
      Gaucher disease (GD) is an inherited disorder caused by recessive mutations in the GBA1 gene that encodes the lysosomal enzyme β-glucocerebrosidase (β-GC). β-GC hydrolyzes glucosylceramide (GluCer) into glucose and ceramide in the lysosome, and the loss of its activity leads to GluCer accumulation in different tissues. In severe cases, enzymatic deficiency triggers inflammation, organomegaly, bone disease, and neurodegeneration. Neuronopathic Gaucher disease (nGD) encompasses two different forms of the disease, characterized by chronic or acute damage to the central nervous system (CNS). The cellular and molecular studies that uncover the pathological mechanisms of nGD mainly focus on lysosomal dysfunction since the lysosome is the key organelle affected in GD. However, new studies show alterations in other organelles that contribute to nGD pathology. For instance, abnormal accumulation of GluCer in lysosomes due to the loss of β-GC activity leads to excessive calcium release from the endoplasmic reticulum (ER), activating the ER-associated degradation pathway and the unfolded protein response. Recent evidence indicates mitophagy is altered in nGD, resulting in the accumulation of dysfunctional mitochondria, a critical factor in disease progression. Additionally, nGD patients present alterations in mitochondrial morphology, membrane potential, ATP production, and increased reactive oxygen species (ROS) levels. Little is known about potential dysfunction in other organelles of the secretory pathway, such as the Golgi apparatus and exosomes. This review focuses on collecting evidence regarding organelle dysfunction beyond lysosomes in nGD. We briefly describe cellular and animal models and signaling pathways relevant to uncovering the pathological mechanisms and new therapeutic targets in GD.
    Keywords:  Golgi apparatus; Neuronopathic Gaucher disease (nGD); Parkinson's disease (PD); autophagy; endoplasmic reticulum; lysosomal storage disorders (LSD); lysosome; mitochondria
    DOI:  https://doi.org/10.3389/fnmol.2022.934820
  8. Hum Mol Genet. 2022 Aug 23. pii: ddac209. [Epub ahead of print]
    Disease correction in Mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.
    Courtney J Rouse, Kimberley Hawkins, Nadia Kabbej, Justin Dalugdug, Aishwarya Kunta, Mi-Jung Kim, Shinichi Someya, Zachary Herbst, Michael Gelb, Isabella Dinelli, Elizabeth Butterworth, Darin J Falk, Erinn Rosenkrantz, Hamza Elmohd, Hamid Khaledi, Samar Mowafy, Frederick Ashby, Coy D Heldermon.
      Mucopolysaccharidoses IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared to AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site (IC6) or intracisterna magna (ICM) injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing, and survival. Histologic and enzymatic assessments both show each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB.
    DOI:  https://doi.org/10.1093/hmg/ddac209
  9. Mol Cell Biol. 2022 Aug 25. e0024122
    Invading Bacterial Pathogens Activate Transcription Factor EB in Epithelial Cells through the Amino Acid Starvation Pathway of mTORC1 Inhibition.
    Liliane Cabral-Fernandes, Shawn Goyal, Armin Farahvash, Jessica Tsalikis, Dana J Philpott, Stephen E Girardin.
      Upon pathogen infection, intricate innate signaling cascades are induced to initiate the transcription of immune effectors, including cytokines and chemokines. Transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy genes, was found recently to be a novel regulator of innate immunity in both Caenorhabditis elegans and mammals. Despite TFEB participating in critical mechanisms of pathogen recognition and in the transcriptional response to infection in mammalian macrophages, little is known about its roles in the infected epithelium or infected nonimmune cells in general. Here, we demonstrate that TFEB is activated in nonimmune cells upon infection with bacterial pathogens through a pathway dependent on mTORC1 inhibition and RAG-GTPase activity, reflecting the importance of membrane damage and amino acid starvation responses during infection. Additionally, we present data demonstrating that although TFEB does not affect bacterial killing or load in nonimmune cells, it alters the host transcriptome upon infection, thus promoting an antibacterial transcriptomic landscape. Elucidating the roles of TFEB in infected nonimmune cells and the upstream signaling cascade provides critical insight into understanding how cells recognize and respond to bacterial pathogens.
    Keywords:  Shigella; TFEB; amino acid starvation; host-microbe interactions; mTOR
    DOI:  https://doi.org/10.1128/mcb.00241-22
  10. 3 Biotech. 2022 Sep;12(9): 230
    Mitochondria-lysosome crosstalk in GBA1-associated Parkinson's disease.
    M Sahyadri, Abhishek P R Nadiga, Seema Mehdi, K Mruthunjaya, Pawan G Nayak, Vipan K Parihar, S N Manjula.
      Organelle crosstalk is significant in regulating their respective functions and subsequent cell fate. Mitochondria and lysosomes are amongst the essential organelles in maintaining cellular homeostasis. Mitochondria-lysosome connections, which may develop dynamically in the human neurons, have been identified as sites of bidirectional communication. Aberrancies are often associated with neurodegenerative disorders like Parkinson's disease (PD), suggesting the physical and functional link between these two organelles. PD is often linked with genetic mutations of several mutations discovered in the familial forms of the disease; some are considered risk factors. Many of these genes are either associated with mitochondrial function or belong to endo-lysosomal pathways. The recent investigations have indicated that neurons with mutant glucosylceramidase beta (GBA1) exhibit extended mitochondria-lysosome connections in individuals with PD. This may be due to impaired control of the untethering protein, which aids in the hydrolysis of Rab7 GTP required for contact untethering. A GCase modulator may be used to augment the reduced GBA1 lysosomal enzyme activity in the neurons of PD patients. This review focuses on how GBA1 mutation in PD is interlinked with mitochondria-lysosome (ML) crosstalk, exploring the pathways governing these interactions and mechanistically comprehending the mitochondrial and lysosomal miscommunication in the pathophysiology of PD. This review is based on the limited literature available on the topic and hence may be subject to bias in its views. Our estimates may be conservative and limited due to the lack of studies under the said discipline due to its inherent complex nature. The current association of GBA1 to PD pathogenesis is based on the limited scope of study and further research is necessary to explore the risk factors further and identify the relationship with more detail.
    Keywords:  Glucosylceramidase Beta (GBA); Lysosome; ML crosstalk; Mitochondria; Neuronal dysfunction; Parkinson's disease
    DOI:  https://doi.org/10.1007/s13205-022-03261-9
  11. Geroscience. 2022 Aug 26.
    Potassium-chelating drug sodium polystyrene sulfonate enhances lysosomal function and suppresses proteotoxicity.
    Cyrene Arputhasamy, Anna C Foulger, Mark Lucanic, Anand Rane, Minna Schmidt, Theo Garrett, Michael Broussalian, Elena Battistoni, Rachel B Brem, Gordon J Lithgow, Manish Chamoli, Julie K Andersen.
      Lysosomes are crucial for degradation and recycling of damaged proteins and cellular components. Therapeutic strategies enhancing lysosomal function are a promising approach for aging and age-related neurodegenerative diseases. Here, we show that an FDA approved drug sodium polystyrene sulfonate (SPS), used to reduce high blood potassium in humans, enhances lysosomal function both in C. elegans and in human neuronal cells. Enhanced lysosomal function following SPS treatment is accompanied by the suppression of proteotoxicity caused by expression of the neurotoxic peptides Aβ and TAU. Additionally, treatment with SPS imparts health benefits as it significantly increases lifespan in C. elegans. Overall our work supports the potential use of SPS as a prospective geroprotective intervention.
    Keywords:  Amyloid-β; Autophagy; C. elegans; Lifespan; Lysosome; Neurodegeneration; Potassium restriction; Proteotoxicity; SH-SY5Y neuronal cells
    DOI:  https://doi.org/10.1007/s11357-022-00647-8
  12. Cells. 2022 Aug 19. pii: 2579. [Epub ahead of print]11(16):
    Preclinical Enzyme Replacement Therapy with a Recombinant β-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis.
    Jason Andrew Weesner, Ida Annunziata, Tianhong Yang, Walter Acosta, Elida Gomero, Huimin Hu, Diantha van de Vlekkert, Jorge Ayala, Xiaohui Qiu, Leigh Ellen Fremuth, David N Radin, Carole L Cramer, Alessandra d'Azzo.
      GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase (β-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine β-Gal fused to the plant lectin subunit B of ricin (mβ-Gal:RTB). We show that long-term, bi-weekly systemic injection of mβ-Gal:RTB in the β-Gal-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, β-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the β-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.
    Keywords:  CNS; ERT; GM1; lysosomal storage disease; mβ-Gal:RTB
    DOI:  https://doi.org/10.3390/cells11162579
  13. Front Nutr. 2022 ;9 965771
    Activating transcription factor 4-dependent hsa-miR-663a transcription mediates mTORC1/p70S6K1 signaling underleucine deprivation.
    Junki Yamamura, Sihui Ma, Huijuan Jia, Hisanori Kato.
      The mechanistic target of rapamycin complex 1 (mTORC1) is involved in nutrient-induced signaling and is a master regulator of cell growth and metabolism. Amino acid-deficient conditions affect mTORC1 activity; however, its upstream regulators warrant further investigation. MicroRNAs are key regulators of nutrient-related responses; therefore, the present study aimed to assess the leucine starvation-induced microRNA profile and its impact on mTORC1 activity. Transcriptome analysis of human hepatocellular carcinoma cells (HepG2) under leucine deprivation revealed that hsa-miR-663a and hsa-miR-1469 were altered in a transcription factor 4-dependent manner. Overexpression of these microRNAs induced phosphorylation of the ribosomal protein S6 kinase beta-1, a mTORC1 downstream target. Furthermore, hsa-miR-663a downregulated proline-rich Akt1 substrate of 40 kDa (PRAS40), one of the mTORC1 components. In summary, this study provides new insights into the regulatory role of microRNAs in amino acid metabolism and demonstrates alterations in microRNA profile under leucine deprivation in human hepatocytes.
    Keywords:  ATF4; hsa-miR-663a; leucine deprivation; mTORC1; microRNAs
    DOI:  https://doi.org/10.3389/fnut.2022.965771
  14. Life Sci. 2022 Aug 17. pii: S0024-3205(22)00588-4. [Epub ahead of print]307 120888
    LIMP2 gene, evolutionarily conserved regulation by TFE3, relieves lysosomal stress induced by cholesterol.
    Hanze Guo, Yingying Zhu, Jiarui Li, Qipeng Zhang, Yan Chi.
      AIM: Excess cholesterol deposition in lysosomes may result in lysosomal stress and dysfunction. Here, we focus on the role of lysosome membrane protein 2 (LIMP2) in relieving the lysosomal stress caused by excess cholesterol and the mechanism that regulate its expression.MATERIAL AND METHODS: Cholesterol enrichment in lamprey liver tissue was evaluated by RNA transcriptome data analysis, RT-qPCR, H&E, and Oil Red O staining. Gene markers of autophagy and cholesterol synthesis were determined by western blot or RT-qPCR. Lysosomal morphology and pH value was measured by confocal observation or flow cytometry. Dual-Luciferase reporter assay was performed to test the expression regulation relationship.
    KEY FINDINGS: We report that lamprey limp2 (L-limp2) is evolutionarily highly conserved with human LIMP2 (H-LIMP2). The biological function of L-limp2, consistent with H-LIMP2, includes maintaining lysosomal morphology, modulating autophagy, and aiding cholesterol efflux from lysosomes. Furthermore, we find that both L-limp2 and H-limp2 can restore cholesterol-induced elevation of lysosomal pH and impaired autophagic flux. We demonstrate that lamprey transcription factor binding to IGHM enhancer 3 (L-TFE3) can bind with coordinated lysosomal expression and regulation (CLEAR) elements on the L-limp2 promoter and regulate its expression. Moreover, this regulatory relationship is also available in humans. Taken together, the present study demonstrates that the evolutionarily conserved TFE3-LIMP2 axis may have a protective role against the impaired lysosomal function caused by excess cholesterol.
    SIGNIFICANCE: The protective effect of TFE3-LIMP2 axis against cholesterol-triggered lysosomal stress may provide a new target for the treatment of diseases caused by excessive cholesterol accumulation in lysosomes.
    Keywords:  LIMP2; Lamprey; Lysosomal stress; TFE3
    DOI:  https://doi.org/10.1016/j.lfs.2022.120888
  15. Front Mol Biosci. 2022 ;9 892248
    Analysis of Brain Lipids in the Early-Onset Tay-Sachs Disease Mouse Model With the Combined Deficiency of β-Hexosaminidase A and Neuraminidase 3.
    Melike Can, Tugce Sengül, Secil Akyildiz Demir, Orhan K İnci, Hande Basırlı, Volkan Seyrantepe.
      Introduction: Tay-Sachs disease is an autosomal recessively inherited lysosomal storage disease that results from loss-of-function mutations in the HEXA gene coding β-hexosaminidase A. HEXA gene deficiency affects the central nervous system owing to GM2 ganglioside accumulation in lysosomes resulting in progressive neurodegeneration in patients. We recently generated a novel mice model with a combined deficiency of β-hexosaminidase A and neuraminidase 3 (Hexa-/-Neu3-/-) that mimics both the neuropathological and clinical abnormalities of early-onset Tay-Sachs disease. Here, we aimed to explore the secondary accumulation of lipids in the brain of Hexa-/-Neu3-/- mice. Materials and Methods: In the cortex and hippocampus of five-month-old WT, Hexa-/-, Neu3-/-, and Hexa-/-Neu3-/- mice, lipid levels belonging to glycerolipids, glycerophospholipids, and sterol lipids were evaluated using a shotgun lipidomics approach. The levels of myelin were also assessed by luxol fast blue staining and immunohistochemistry using antibodies against myelin basic protein. We further examined glycoconjugate and cholesterol levels by periodic acid-Schiff and filipin staining, respectively. Toluidine blue staining was also performed to display axonal degeneration. Results: Among glycerophospholipids, we demonstrated elevated levels of phosphatidylcholine-ether and lysophosphatidylcholine while decreased levels of phosphatidylcholine and phosphatidylserine in both cortex and hippocampus of Hexa-/-Neu3-/- mice. In the glycerolipid class, we showed an alleviated level of sphingomyelin in both cortex and hippocampus, but the higher levels of diacylglycerol and triacylglycerol were detected in only the hippocampus of Hexa-/-Neu3-/- mice. The lower level of sterol was also detected in the cortex of Hexa-/-Neu3-/- mice but not in the hippocampus. Histochemical studies showed a decrease in the myelin level and axonal degeneration indicating neuronal pathology in the brain of Hexa-/-Neu3-/- mice. Although glycoconjugate accumulation was evident both in the cortex and hippocampus, we did not detect any changes in the level of cholesterol. Conclusion: Our results indicate that alterations in lipid metabolism and neuropathology, such as demyelination and axonal degeneration, might be related to the dysfunctionality of lipid-related cellular pathways like autophagy. Understanding of brain-specific lipid alterations contributes to evaluating the effectiveness of treatments in Hexa-/-Neu3-/- mice in future studies.
    Keywords:  Tay–Sachs disease; brain; gangliosides; lipidomics; mouse model
    DOI:  https://doi.org/10.3389/fmolb.2022.892248
  16. Biochem Biophys Res Commun. 2022 Aug 16. pii: S0006-291X(22)01137-8. [Epub ahead of print]626 211-219
    Lysosome exocytosis is required for mitosis in mammalian cells.
    Charlotte Nugues, Dayani Rajamanoharan, Robert D Burgoyne, Lee P Haynes, Nordine Helassa.
      Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. In this paper we demonstrate how lysosomes dynamically redistribute during mitosis and exocytose. Antagonism of lysosomal exocytosis by pharmacological and genetic approaches causes mitosis failure in a significant proportion of cells. We speculate that either lysosomal membrane or luminal content release, possibly both, are therefore required for normal mitosis progression. These findings are important as they reveal a new process required for successful cell division.
    Keywords:  Exocytosis; Lysosome; Mitosis; Phosphoinositide; TIRFM
    DOI:  https://doi.org/10.1016/j.bbrc.2022.08.024
  17. J Cell Sci. 2022 Aug 24. pii: jcs.259994. [Epub ahead of print]
    Conserved Pib2 regions have distinct roles in TORC1 regulation at the vacuole.
    Kayla K Troutman, Natalia V Varlakhanova, Bryan A Tornabene, Rajesh Ramachandran, Marijn G J Ford.
      TORC1 is a critical controller of cell growth in eukaryotes. In yeast, the presence of nutrients is signaled to TORC1 by several upstream regulatory sensors that together coordinate TORC1 activity. TORC1 localizes to both vacuolar and endosomal membranes, where differential signaling occurs. This localization is mimicked by Pib2, a key upstream TORC1 regulator that is essential for TORC1 reactivation after nutrient starvation or pharmacological inhibition. Pib2 has both positive and negative effects on TORC1 activity, but the mechanisms remain poorly understood. Here, we pinpoint the Pib2 inhibitory function on TORC1 to residues within short, conserved N-terminal regions. We also show that Pib2 C-terminal regions, helical region E and tail, are essential for TORC1 reactivation. Further, the Pib2 FYVE domain plays a role in vacuolar localization but it is surprisingly unnecessary for recovery from rapamycin exposure. Using chimeric Pib2 targeting constructs, we show that endosomal localization is not necessary for TORC1 reactivation and cell growth after rapamycin treatment. Thus, a comprehensive molecular dissection of Pib2 demonstrates that each of its conserved regions differentially contribute to Pib2 regulation of TORC1 activity.
    Keywords:  FYVE; Pib2; Rapamycin; TORC1
    DOI:  https://doi.org/10.1242/jcs.259994
  18. Biomedicines. 2022 Jul 29. pii: 1836. [Epub ahead of print]10(8):
    Detection of Structural Variants by NGS: Revealing Missing Alleles in Lysosomal Storage Diseases.
    Valentina La Cognata, Sebastiano Cavallaro.
      Lysosomal storage diseases (LSDs) are a heterogeneous group of rare multisystem metabolic disorders occurring mostly in infancy and childhood, characterized by a gradual accumulation of non-degraded substrates inside the cells. Although biochemical enzymatic assays are considered the gold standard for diagnosis of symptomatic patients, genotyping is a requirement for inclusion in enzyme replacement programs and is a prerequisite for carrier tests in relatives and DNA-based prenatal diagnosis. The emerging next-generation sequencing (NGS) technologies are now offering a powerful diagnostic tool for genotyping LSDs patients by providing faster, cheaper, and higher-resolution testing options, and are allowing to unravel, in a single integrated workflow SNVs, small insertions and deletions (indels), as well as major structural variations (SVs) responsible for the pathology. Here, we summarize the current knowledge about the most recurrent and private SVs involving LSDs-related genes, review advantages and drawbacks related to the use of the NGS in the SVs detection, and discuss the challenges to bring this type of analysis in clinical diagnostics.
    Keywords:  CNVs; diagnosis; lysosomal storage diseases; structural variants; tNGS
    DOI:  https://doi.org/10.3390/biomedicines10081836
  19. Biosensors (Basel). 2022 Aug 20. pii: 663. [Epub ahead of print]12(8):
    In-Depth Characterization of Endo-Lysosomal Aβ in Intact Neurons.
    Alec K McKendell, Mei C Q Houser, Shane P C Mitchell, Michael S Wolfe, Oksana Berezovska, Masato Maesako.
      Amyloid-beta (Aβ) peptides are produced within neurons. Some peptides are released into the brain parenchyma, while others are retained inside the neurons. However, the detection of intracellular Aβ remains a challenge since antibodies against Aβ capture Aβ and its precursor proteins (i.e., APP and C99). To overcome this drawback, we recently developed 1) the C99 720-670 biosensor for recording γ-secretase activity and 2) a unique multiplexed immunostaining platform that enables the selective detection of intracellular Aβ with subcellular resolution. Using these new assays, we showed that C99 is predominantly processed by γ-secretase in late endosomes and lysosomes, and intracellular Aβ is enriched in the same subcellular loci in intact neurons. However, the detailed properties of Aβ in the acidic compartments remain unclear. Here, we report using fluorescent lifetime imaging microscopy (FLIM) that intracellular Aβ includes both long Aβ intermediates bound to γ-secretase and short peptides dissociated from the protease complex. Surprisingly, our results also suggest that the dissociated Aβ is bound to the glycoproteins on the inner membrane of lysosomes. Furthermore, we show striking cell-to-cell heterogeneity in intracellular Aβ levels in primary neurons and APP transgenic mouse brains. These findings provide a basis for the further investigation of the role(s) of intracellular Aβ and its relevance to Alzheimer's disease (AD).
    Keywords:  Alzheimer’s disease; fluorescence resonance energy transfer (FRET); glycoconjugates; intracellular amyloid-beta (Aβ); lysosomes; γ-secretase
    DOI:  https://doi.org/10.3390/bios12080663
  20. Exp Biol Med (Maywood). 2022 Aug 23. 15353702221114872
    Cell cycle disturbances in mucopolysaccharidoses: Transcriptomic and experimental studies on cellular models.
    Joanna Brokowska, Lidia Gaffke, Karolina Pierzynowska, Zuzanna Cyske, Grzegorz Węgrzyn.
      Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by defects in genes coding for proteins involved in degradation of glycosaminoglycans (GAGs). These complex carbohydrates accumulate in cells causing their serious dysfunctions. Apart from the physical GAG storage, secondary and tertiary changes may contribute significantly to the pathomechanism of the disease. Among processes which were not systematically investigated in MPS cells to date there is the cell cycle. Here, we studied perturbances in this crucial cellular process in majority of MPS types. Transcriptomic analyses indicated that expression of many genes coding for proteins involved in the cell cycle is dysregulated in all tested MPS cells. Importantly, levels of transcripts of particular genes were changed in the same manner (i.e. either up- or down-regulated) in most or all types of the disease, indicating a common mechanism of the dysregulation. Flow cytometric studies demonstrated that the cell cycle is disturbed in all MPS types, with increased fractions of cells in the G0/G1 phase in most types and decreased fractions of cells in the G2/M phase in all types. We found that increased levels of cyclin D1 and disturbed timing of its appearance during the cell cycle may contribute to the mechanism of dysregulation of this process in MPS. Reduction of GAG levels by either a specific enzyme or genistein-mediated inhibition of synthesis of these compounds improved, but not fully corrected, the cell cycle in MPS fibroblasts. Therefore, it is suggested that combination of the therapeutic approaches devoted to reduction of GAG levels with cyclin D1 inhibitors might be considered in further works on developing effective treatment procedures for MPS.
    Keywords:  Mucopolysaccharidosis; cell cycle; cyclins; enzyme replacement therapy; pathomechanism; substrate reduction therapy
    DOI:  https://doi.org/10.1177/15353702221114872
  21. Int J Mol Sci. 2022 Aug 21. pii: 9436. [Epub ahead of print]23(16):
    β-Galactosylceramidase Deficiency Causes Upregulation of Long Pentraxin-3 in the Central Nervous System of Krabbe Patients and Twitcher Mice.
    Daniela Coltrini, Adwaid Manu Krishna Chandran, Mirella Belleri, Pietro L Poliani, Manuela Cominelli, Francesca Pagani, Miriam Capra, Stefano Calza, Simona Prioni, Laura Mauri, Alessandro Prinetti, Julia K Kofler, Maria L Escolar, Marco Presta.
      Globoid cell leukodystrophy (GLD), or Krabbe disease, is a neurodegenerative sphingolipidosis caused by genetic deficiency of lysosomal β-galactosylceramidase (GALC), characterized by neuroinflammation and demyelination of the central (CNS) and peripheral nervous system. The acute phase protein long pentraxin-3 (PTX3) is a soluble pattern recognition receptor and a regulator of innate immunity. Growing evidence points to the involvement of PTX3 in neurodegeneration. However, the expression and role of PTX3 in the neurodegenerative/neuroinflammatory processes that characterize GLD remain unexplored. Here, immunohistochemical analysis of brain samples from Krabbe patients showed that macrophages and globoid cells are intensely immunoreactive for PTX3. Accordingly, Ptx3 expression increases throughout the course of the disease in the cerebrum, cerebellum, and spinal cord of GALC-deficient twitcher (Galctwi/twi) mice, an authentic animal model of GLD. This was paralleled by the upregulation of proinflammatory genes and M1-polarized macrophage/microglia markers and of the levels of PTX3 protein in CNS and plasma of twitcher animals. Crossing of Galctwi/twi mice with transgenic PTX3 overexpressing animals (hPTX3 mice) demonstrated that constitutive PTX3 overexpression reduced the severity of clinical signs and the upregulation of proinflammatory genes in the spinal cord of P35 hPTX3/Galctwi/twi mice when compared to Galctwi/twi littermates, leading to a limited increase of their life span. However, this occurred in the absence of a significant impact on the histopathological findings and on the accumulation of the neurotoxic metabolite psychosine when evaluated at this late time point of the disease. In conclusion, our results provide the first evidence that PTX3 is produced in the CNS of GALC-deficient Krabbe patients and twitcher mice. PTX3 may exert a protective role by reducing the neuroinflammatory response that occurs in the spinal cord of GALC-deficient animals.
    Keywords:  CNS; GALC; Krabbe disease; PTX3; neuroinflammation; sphingolipidosis
    DOI:  https://doi.org/10.3390/ijms23169436
  22. Nat Commun. 2022 Aug 26. 13(1): 5018
    Midkine expression by stem-like tumor cells drives persistence to mTOR inhibition and an immune-suppressive microenvironment.
    Yan Tang, David J Kwiatkowski, Elizabeth P Henske.
      mTORC1 is hyperactive in multiple cancer types1,2. Here, we performed integrative analysis of single cell transcriptomic profiling, paired T cell receptor (TCR) sequencing, and spatial transcriptomic profiling on Tuberous Sclerosis Complex (TSC) associated tumors with mTORC1 hyperactivity, and identified a stem-like tumor cell state (SLS) linked to T cell dysfunction via tumor-modulated immunosuppressive macrophages. Rapamycin and its derivatives (rapalogs) are the primary treatments for TSC tumors, and the stem-like tumor cells showed rapamycin resistance in vitro, reminiscent of the cytostatic effects of these drugs in patients. The pro-angiogenic factor midkine (MDK) was highly expressed by the SLS population, and associated with enrichment of endothelial cells in SLS-dominant samples. Inhibition of MDK showed synergistic benefit with rapamycin in reducing the growth of TSC cell lines in vitro and in vivo. In aggregate, this study suggests an autocrine rapamycin resistance mechanism and a paracrine tumor survival mechanism via immune suppression adopted by the stem-like state tumor cells with mTORC1 hyperactivity.
    DOI:  https://doi.org/10.1038/s41467-022-32673-7
  23. Autophagy. 2022 Aug 21. 1-17
    RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1.
    Yifei Pei, Shuning Lv, Yong Shi, Jingwen Jia, Mengru Ma, Hailong Han, Rongying Zhang, Jieqiong Tan, Xinjun Zhang.
      The endosomal system maintains cellular homeostasis by coordinating multiple vesicular trafficking events, and the retromer complex plays a critical role in endosomal cargo recognition and sorting. Here, we demonstrate an essential role for the small GTPase RAB21 in regulating retromer-mediated recycling of the glucose transporter SLC2A1/GLUT1 and macroautophagy/autophagy. RAB21 depletion mis-sorts SLC2A1 to lysosomes and affects glucose uptake, thereby activating the AMPK-ULK1 pathway to increase autophagic flux. RAB21 depletion also increases lysosome function. Notably, RAB21 depletion does not overtly affect retrograde transport of IGF2R/CI-M6PR or WLS from endosomes to the trans-Golgi network. We speculate that RAB21 regulates fission of retromer-decorated endosomal tubules, as RAB21 depletion causes accumulation of the SNX27-containing retromer complex on enlarged endosomes at the perinuclear region. Functionally, RAB21 depletion sensitizes cancer cells to energy stress and inhibits tumor growth in vivo, suggesting an oncogenic role for RAB21. Overall, our study illuminates the role of RAB21 in regulating endosomal dynamics and maintaining cellular energy homeostasis and suggests RAB21 as a potential metabolic target for cancer therapy.
    Keywords:  Autophagic flux; glucose uptake; membrane trafficking; retromer complex; small GTPases
    DOI:  https://doi.org/10.1080/15548627.2022.2114271
  24. Biochem Pharmacol. 2022 Aug 23. pii: S0006-2952(22)00323-9. [Epub ahead of print] 115229
    Autophagy Guided Interventions to Modify the Cardiac Phenotype of Danon Disease.
    Dor Yadin, Zachary Petrover, Asher Shainberg, Ronny Alcalai, Maayan Waldman, Jon Seidman, Christine E Seidman, Nader G Abraham, Edith Hochhauser, Michael Arad.
      Danon disease is a lethal X-linked genetic syndrome resulting from radical mutations in the LAMP2 gene. LAMP2 protein deficiency results in defective lysosomal function, autophagy arrest and a multisystem disorder primarily involving the heart, skeletal muscle and the central nervous system. Cardiomyopathy is the main cause of morbidity and mortality. To investigate the mechanisms of and develop therapies for cardiac Danon disease we engineered a mouse model carrying an exon 6 deletion human mutation in LAMP2, which recapitulates the human cardiac disease phenotype. Mice develop cardiac hypertrophy followed by left ventricular dilatation and systolic dysfunction, in association with progressive fibrosis, oxidative stress, accumulation of autophagosomes and activation of proteasome. Stimulation of autophagy in Danon mice (by exercise training, caloric restriction, and rapamycin) aggravate the disease phenotype by promoting dilated cardiomyopathy. Inhibiting autophagy (by high fat diet or hydroxychloroquine) is better tolerated by Danon mice compared to wild type but is not curative. Inhibiting proteasome by Velcade was found to be highly toxic to Danon mice, suggesting that proteasome is activated to compensate for defective autophagy. In conclusion, activation of autophagy should be avoided in Danon patients. Since Danon's is a lifelong disease, we suggest that lifestyle interventions to decrease cardiac stress may be useful to slow progression of Danon's cardiomyopathy. While Danon mice better tolerate high fat diet and sedentary lifestyle, the benefit regarding cardiomyopathy in humans needs to be balanced against other health consequences of such interventions.
    Keywords:  Cardiomyopathy; LAMP2; animal model of human disease; autophagy; diet; proteasome
    DOI:  https://doi.org/10.1016/j.bcp.2022.115229
  25. Molecules. 2022 Aug 19. pii: 5295. [Epub ahead of print]27(16):
    Overview of Research into mTOR Inhibitors.
    Beibei Mao, Qi Zhang, Li Ma, Dong-Sheng Zhao, Pan Zhao, Peizheng Yan.
      The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) family. The kinase exists in the forms of two complexes, mTORC1 and mTORC2, and it participates in cell growth, proliferation, metabolism, and survival. The kinase activity is closely related to the occurrence and development of multiple human diseases. Inhibitors of mTOR block critical pathways to produce antiviral, anti-inflammatory, antiproliferative and other effects, and they have been applied to research in cancer, inflammation, central nervous system diseases and viral infections. Existing mTOR inhibitors are commonly divided into mTOR allosteric inhibitors, ATP-competitive inhibitors and dual binding site inhibitors, according to their sites of action. In addition, there exist several dual-target mTOR inhibitors that target PI3K, histone deacetylases (HDAC) or ataxia telangiectasia mutated and Rad-3 related (ATR) kinases. This review focuses on the structure of mTOR protein and related signaling pathways as well as the structure and characteristics of various mTOR inhibitors. Non-rapalog allosteric inhibitors will open new directions for the development of new therapeutics specifically targeting mTORC1. The applications of ATP-competitive inhibitors in central nervous system diseases, viral infections and inflammation have laid the foundation for expanding the indications of mTOR inhibitors. Both dual-binding site inhibitors and dual-target inhibitors are beneficial in overcoming mTOR inhibitor resistance.
    Keywords:  dual inhibitors; human diseases; mTOR; mTOR inhibitors
    DOI:  https://doi.org/10.3390/molecules27165295
  26. Curr Res Pharmacol Drug Discov. 2022 ;3 100117
    Jagged-1 is induced by mTOR inhibitors in renal cancer cells through an Akt/ALK5/Smad4-dependent mechanism.
    David Danielpour, Sarah Corum, Patrick Leahy, Anusha Bangalore.
      The mammalian target of rapamycin (mTOR) plays an important role in the aggressiveness and therapeutic resistance of many cancers. Targeting mTOR continues to be under clinical investigation for cancer therapy. Despite the notable clinical success of mTOR inhibitors in extending the overall survival of patients with certain malignancies including metastatic renal cell carcinomas (RCCs), the overall impact of mTOR inhibitors on cancers has been generally disappointing and attributed to various compensatory responses. Here we provide the first report that expression of the Notch ligand Jagged-1 (JAG1), which is associated with aggressiveness of RCCs, is induced by several inhibitors of mTOR (rapamycin (Rap), BEZ235, KU-0063794) in human clear cell RCC (ccRCC) cells. Using both molecular and chemical inhibitors of PI3K, Akt, and TGF-β signaling, we provide evidence that the induction of JAG1 expression by mTOR inhibitors in ccRCC cells depends on the activation of Akt and occurs through an ALK5 kinase/Smad4-dependent mechanism. Furthermore, we show that mTOR inhibitors activate Notch1 and induce the expression of drivers of epithelial-mesenchymal transition, notably Hic-5 and Slug. Silencing JAG1 with selective shRNAs blocked the ability of KU-0063794 and Rap to induce Hic-5 in ccRCC cells. Moreover, Rap enhanced TGF-β-induced expression of Hic-5 and Slug, both of which were repressed in JAG1-silenced ccRCC cells. Silencing JAG1 selectively decreased the motility of ccRCC cells treated with Rap or TGF-β1. Moreover, inhibition of Notch signaling with γ-secretase inhibitors enhanced or permitted mTOR inhibitors to suppress the motility of ccRCC cells. We suggest targeting JAG1 may enhance therapeutic responses to mTOR inhibitors in ccRCCs.
    Keywords:  ALK5, Activin-like kinase 5 (TGF-β type I receptor); ANOVA, Analysis of variance; Akt; BEZ235; BSA, Bovine serum albumin; EDTA, Ethylenediaminetetraacetic acid; EMT; FBS, Fetal bovine serum; Hic-5; Hic-5, Hydrogen peroxide-inducible clone 5, also known as transforming growth factor beta induced transcript; IRS-1, Insulin receptor substrate-1; JAG1, Jagged-1; KU-0063794; MAML-1, Mastermind-like protein-1; Myr, Myristoylated; PI3K; PI3K, Phosphatidylinositol 3-kinase; RCC, RCC; Rap, Rapamycin; Rapamycin; Renal cancer; Rheb, Ras homologue enriched in brain; SE, Standard error; Slug; Slug, Snail family of transcription factors encoded by the SNAI2 gene; Smad, Mothers against decapentaplegic homolog; Smad4; TGF-beta; TGF-β, Transforming growth factor-beta; TSC, Tuberous Sclerosis Complex; TβRI, Transforming growth factor β receptor type 1; TβRII, Transforming growth factor β receptor type 2; ccRCC, Clear cell renal cell carcinoma; mRCC, Metastatic renal cell carcinoma; mTOR; mTORC1, Mammalian target of rapamycin complex 1; mTORC2, Mammalian target of rapamycin complex 2
    DOI:  https://doi.org/10.1016/j.crphar.2022.100117
  27. Pharmacol Res. 2022 Aug 18. pii: S1043-6618(22)00348-6. [Epub ahead of print]183 106403
    Targeting Akt/PKB in pediatric tumors: A review from preclinical to clinical trials.
    Bruno Toson, Isadora S Fortes, Rafael Roesler, Saulo F Andrade.
      The serine/threonine kinase Akt is a major player in the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and its modulation impacts multiple cellular processes such as growth, proliferation, and survival. Several abnormalities in this pathway have been documented over the years, and these alterations were shown to have great implications in tumorigenesis and resistance to chemotherapy. Thus, multiple Akt inhibitors have been developed and tested in adult tumors, and some of them are currently undergoing phase I, II, and III clinical trials for distinct cancers that arise during adulthood. Despite that, the impact of these inhibitors is still not fully understood in pediatric tumors, and Akt-specific targeting seems to be a promising approach to treat children affected by cancers. This review summarizes recent available evidence of Akt inhibitors in pediatric cancers, from both preclinical and clinical studies. In short, we demonstrate the impact that Akt inhibition provides in tumorigenesis, and we suggest targeting the PI3K/Akt/mTOR signaling pathway, alone or in combination with other inhibitors, is a feasible tool to achieve better outcomes in pediatric tumors.
    Keywords:  AZD5363 (PubChem CID 25227436); Akt inhibitor; CCT128930 (PubChem CID17751819); GDC-0068 (PubChem CID 24788740); GSK2110183 (PubChem CID 92044396); GSK2141795 (PubChem CID 51042438); GSK690693 (PubChem CID 16725726); MK-2206 (PubChem CID 24964624); PI3K/Akt/mTOR pathway; Pediatric cancer; Perifosine (PubChem CID 148177); TAS-117 (PubChem CID 66555816); Targeted therapy; Triciribine (PubChem CID 65399)
    DOI:  https://doi.org/10.1016/j.phrs.2022.106403
  28. Metabolites. 2022 Jul 28. pii: 703. [Epub ahead of print]12(8):
    Metabolic Fingerprinting of Fabry Disease: Diagnostic and Prognostic Aspects.
    Maria Teresa Rocchetti, Federica Spadaccino, Valeria Catalano, Gianluigi Zaza, Giovanni Stallone, Daniela Fiocco, Giuseppe Stefano Netti, Elena Ranieri.
      Fabry disease (FD) is an X-linked lysosomal disease due to a deficiency in the activity of the lysosomal-galactosidase A (GalA), a key enzyme in the glycosphingolipid degradation pathway. FD is a complex disease with a poor genotype-phenotype correlation. In the early stages, FD could involve the peripheral nervous system (acroparesthesias and dysautonomia) and the ski (angiokeratoma), but later kidney, heart or central nervous system impairment may significantly decrease life expectancy. The advent of omics technologies offers the possibility of a global, integrated and systemic approach well-suited for the exploration of this complex disease. In this narrative review, we will focus on the main metabolomic studies, which have underscored the importance of detecting biomarkers for a diagnostic and prognostic purpose in FD. These investigations are potentially useful to explain the wide clinical, biochemical and molecular heterogeneity found in FD patients. Moreover, the quantitative mass spectrometry methods developed to evaluate concentrations of these biomarkers in urine and plasma will be described. Finally, the complex metabolic biomarker profile depicted in FD patients will be reported, which varies according to gender, types of mutations, and therapeutic treatment.
    Keywords:  Fabry disease; LysoGb3; lysosomal storage diseases; metabolomics; systems biology
    DOI:  https://doi.org/10.3390/metabo12080703
  29. iScience. 2022 Aug 19. 25(8): 104806
    IL11 stimulates ERK/P90RSK to inhibit LKB1/AMPK and activate mTOR initiating a mesenchymal program in stromal, epithelial, and cancer cells.
    Anissa A Widjaja, Sivakumar Viswanathan, Joyce Goh Wei Ting, Jessie Tan, Shamini G Shekeran, David Carling, Wei-Wen Lim, Stuart A Cook.
      IL11 initiates fibroblast activation but also causes epithelial cell dysfunction. The mechanisms underlying these processes are not known. We report that IL11-stimulated ERK/P90RSK activity causes the phosphorylation of LKB1 at S325 and S428, leading to its inactivation. This inhibits AMPK and activates mTOR across cell types. In stromal cells, IL11-stimulated ERK activity inhibits LKB1/AMPK which is associated with mTOR activation, ⍺SMA expression, and myofibroblast transformation. In hepatocytes and epithelial cells, IL11/ERK activity inhibits LKB1/AMPK leading to mTOR activation, SNAI1 expression, and cell dysfunction. Across cells, IL11-induced phenotypes were inhibited by metformin stimulated AMPK activation. In mice, genetic or pharmacologic manipulation of IL11 activity revealed a critical role of IL11/ERK signaling for LKB1/AMPK inhibition and mTOR activation in fatty liver disease. These data identify the IL11/mTOR axis as a signaling commonality in stromal, epithelial, and cancer cells and reveal a shared IL11-driven mesenchymal program across cell types.
    Keywords:  Cell biology; Functional aspects of cell biology
    DOI:  https://doi.org/10.1016/j.isci.2022.104806
  30. J Mol Cell Biol. 2022 Aug 24. pii: mjac050. [Epub ahead of print]
    CaMK4 overexpression in polycystic kidney disease promotes mTOR-mediated cell proliferation.
    Yan Zhang, Emily A Daniel, July Metcalf, Yuqiao Dai, Gail A Reif, Darren P Wallace.
      Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of fluid-filled cysts causing nephron loss and a decline in renal function. Mammalian target of rapamycin (mTOR) is overactive in cyst-lining cells and contributes to abnormal cell proliferation and cyst enlargement; however, the mechanism for mTOR stimulation remains unclear. We discovered that calcium/calmodulin-dependent kinase IV (CaMK4), a multifunctional kinase, is overexpressed in the kidneys of ADPKD patients and polycystic kidney disease (PKD) mouse models. In human ADPKD cells, CaMK4 knockdown reduced mTOR abundance and the phosphorylation of ribosomal protein S6 kinase (S6K), a downstream target of mTOR. Pharmacologic inhibition of CaMK4 with KN-93 reduced phosphorylated S6K and S6 levels and inhibited cell proliferation and in vitro cyst formation of ADPKD cells. Moreover, inhibition of calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) and calmodulin (CaM), two key upstream regulators of CaMK4, also decreased mTOR signaling. The effects of KN-93 were independent of the LKB1-AMPK pathway and the combination of KN-93 and metformin, an AMPK activator, had additive inhibitory effects on mTOR signaling and in vitro cyst growth. Our data suggest that increased CaMK4 expression and activity contribute to mTOR signaling and proliferation of cystic cells of ADPKD kidneys.
    Keywords:  ADPKD; AMP kinase; CaMK4; CaMKKβ; LKB1; calmodulin
    DOI:  https://doi.org/10.1093/jmcb/mjac050
  31. Biomedicines. 2022 Jul 29. pii: 1838. [Epub ahead of print]10(8):
    miRNAs and isomiRs: Serum-Based Biomarkers for the Development of Intellectual Disability and Autism Spectrum Disorder in Tuberous Sclerosis Complex.
    Mirte Scheper, Alessia Romagnolo, Zein Mersini Besharat, Anand M Iyer, Romina Moavero, Christoph Hertzberg, Bernhard Weschke, Kate Riney, Martha Feucht, Theresa Scholl, Borivoj Petrak, Alice Maulisova, Rima Nabbout, Anna C Jansen, Floor E Jansen, Lieven Lagae, Malgorzata Urbanska, Elisabetta Ferretti, Aleksandra Tempes, Magdalena Blazejczyk, Jacek Jaworski, David J Kwiatkowski, Sergiusz Jozwiak, Katarzyna Kotulska, Krzysztof Sadowski, Julita Borkowska, Paolo Curatolo, James D Mills, Eleonora Aronica, Epistop Consortium Members.
      Tuberous sclerosis complex (TSC) is a rare multi-system genetic disorder characterized by a high incidence of epilepsy and neuropsychiatric manifestations known as tuberous-sclerosis-associated neuropsychiatric disorders (TANDs), including autism spectrum disorder (ASD) and intellectual disability (ID). MicroRNAs (miRNAs) are small regulatory non-coding RNAs that regulate the expression of more than 60% of all protein-coding genes in humans and have been reported to be dysregulated in several diseases, including TSC. In the current study, RNA sequencing analysis was performed to define the miRNA and isoform (isomiR) expression patterns in serum. A Receiver Operating Characteristic (ROC) curve analysis was used to identify circulating molecular biomarkers, miRNAs, and isomiRs, able to discriminate the development of neuropsychiatric comorbidity, either ASD, ID, or ASD + ID, in patients with TSC. Part of our bioinformatics predictions was verified with RT-qPCR performed on RNA isolated from patients' serum. Our results support the notion that circulating miRNAs and isomiRs have the potential to aid standard clinical testing in the early risk assessment of ASD and ID development in TSC patients.
    Keywords:  autism spectrum disorder; biomarkers; epilepsy; intellectual disability; serum; tuberous sclerosis complex
    DOI:  https://doi.org/10.3390/biomedicines10081838
  32. J Biol Chem. 2022 Aug 18. pii: S0021-9258(22)00838-9. [Epub ahead of print] 102395
    Isoform a4 of the vacuolar ATPase a subunit promotes 4T1-12B breast cancer cell-dependent tumor growth and metastasis in vivo.
    Kevin Su, Michael P Collins, Christina M McGuire, Mohammed A Alshagawi, Mariam K Alamoudi, Zhen Li, Michael Forgac.
      The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that governs the pH of various intracellular compartments and also functions at the plasma membrane in certain cell types, including cancer cells. Membrane targeting of the V-ATPase is controlled by isoforms of subunit a, and we have previously shown that isoforms a3 and a4 are important for the migration and invasion of several breast cancer cell lines in vitro. Using CRISPR-mediated genome editing to selectively disrupt each of the four a subunit isoforms, we also recently showed that a4 is critical to plasma membrane V-ATPase localization, as well as in vitro migration and invasion of 4T1-12B murine breast cancer cells. We now report that a4 is important for the growth of 4T1-12B tumors in vivo. We found that BALB/c mice bearing a4-/- 4T1-12B allografts had significantly smaller tumors than mice in the control group. In addition, we determined that a4-/- allografts showed dramatically reduced metastases to the lung and reduced luminescence intensity of metastases to bone relative to the control group. Taken together, these results suggest that the a4 isoform of the V-ATPase represents a novel potential therapeutic target to limit breast cancer growth and metastasis.
    Keywords:  breast cancer; in vivo imaging; tumor growth; tumor metastasis; vacuolar ATPase
    DOI:  https://doi.org/10.1016/j.jbc.2022.102395
  33. Metab Brain Dis. 2022 Aug 27.
    Evaluation of oxidative stress and mitochondrial function in a type II mucopolysaccharidosis cellular model: in vitro effects of genistein and coenzyme Q10.
    Carlos Eduardo Diaz Jacques, Franciele Fátima Lopes, Edina Poletto, Luisa Natalia Pimentel Vera, Priscila Vianna, Luiza Steffens Reinhardt, Guilherme Baldo, Carmen Regla Vargas.
      Mucopolysaccharidosis type II (MPS II or Hunter Syndrome) is a lysosomal disease caused by deficient degradation of glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate due to the deficiency of the enzyme iduronate-2-sulfatase. The main treatment for MPS II is the administration of the recombinant form of the enzyme, in a process known as enzyme replacement therapy (ERT). Oxidative damage can contribute to the pathophysiology of MPS II and treatment with ERT can reduce the effects of oxidative stress. For a better understanding of pathophysiology of MPS II, we evaluated biomarkers of mitochondrial dysfunction, DNA (Deoxyribonucleic acid) damage, antioxidant defenses, reactive species production and lysosomal size in IDS-deficient HEK 293 cells and investigate the in vitro effect of genistein and coenzyme Q10 (CoQ) on these biomarkers. An increase in the production of reactive species was demonstrated, as well as an increase in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Also, an increase in lysosomal volume and oxidative damage to DNA were verified. There was no evidence of a change in mitochondrial function in this cell model. In the HEK 293 (human embryonic kidney 293) knockout (KO) HP10 cell model we found that genistein at concentrations of 25 and 50 μm decreased in vitro the production of reactive species and the activity of the SOD enzyme, showing an antioxidant protective effect. Still, in these cells we verified that the coenzyme Q10 in the concentrations of 5 and 10 μm decreased in vitro the activity of the SOD enzyme and in the concentration of 10 μm decreased in vitro the DNA damage, also demonstrating antioxidant protection. In conclusion, MPS II knockout cells demonstrated oxidative stress and DNA damage and genistein, as well as coenzyme Q10, have been shown to have an important protective effect in vitro against these oxidative damages.
    Keywords:  Coenzyme Q10; Genistein; HEK 293 cells; Mucopolysaccharidosis type II; Oxidative stress
    DOI:  https://doi.org/10.1007/s11011-022-01062-w
  34. Front Immunol. 2022 ;13 955407
    Spatial localization of cathepsins: Implications in immune activation and resolution during infections.
    Elsa Anes, David Pires, Manoj Mandal, José Miguel Azevedo-Pereira.
      Cathepsins were first described, as endolysosomal proteolytic enzymes in reference to the organelles where they degrade the bulk of endogenous and exogenous substrates in a slightly acidic environment. These substrates include pathogens internalized via endocytosis and/or marked for destruction by autophagy. However, the role of cathepsins during infection far exceeds that of direct digestion of the pathogen. Cathepsins have been extensively investigated in the context of tumour associated immune cells and chronic inflammation. Several cathepsin-dependent immune responses develop in the endocytic pathway while others take place in the cytosol, the nucleus, or in the extracellular space. In this review we highlight the spatial localization of cathepsins and their implications in immune activation and resolution pathways during infection.
    Keywords:  adaptive immunity; cathepsins; immune resolution; infection; inflammation; innate immunity
    DOI:  https://doi.org/10.3389/fimmu.2022.955407
  35. Mol Metab. 2022 Aug 23. pii: S2212-8778(22)00149-1. [Epub ahead of print] 101580
    Adipose mTORC2 is essential for sensory innervation in white adipose tissue and whole-body energy homeostasis.
    Irina C Frei, Diana Weissenberger, Danilo Ritz, Wolf Heusermann, Marco Colombi, Mitsugu Shimobayashi, Michael N Hall.
      OBJECTIVE: Adipose tissue, via sympathetic and possibly sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue are poorly characterized.METHODS AND RESULTS: Taking advantage of recent progress in whole-mount three-dimensional imaging, we identified a network of calcitonin gene-related protein (CGRP)-positive sensory neurons in murine white adipose tissue (WAT). We found that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, is required for arborization of sensory, but not of sympathetic neurons. Time course experiments revealed that adipose mTORC2 is required for maintenance of sensory neurons. Furthermore, loss of sensory innervation in WAT coincided with systemic insulin resistance. Finally, we established that neuronal protein growth-associated protein 43 (GAP43) is a marker for sensory neurons in adipose tissue.
    CONCLUSION: Our findings indicate that adipose mTORC2 is necessary for sensory innervation in WAT. In addition, our results also suggest that WAT may affect whole-body energy homeostasis via sensory neurons.
    Keywords:  CGRP; adipose tissue; diabetes; mTORC2; neuropathy; sensory nervous system; whole-body energy homeostasis
    DOI:  https://doi.org/10.1016/j.molmet.2022.101580
  36. Biomedicines. 2022 Aug 10. pii: 1941. [Epub ahead of print]10(8):
    The Role of Rab Proteins in Parkinson's Disease Synaptopathy.
    Arianna Bellucci, Francesca Longhena, Maria Grazia Spillantini.
      In patients affected by Parkinson's disease (PD), the most common neurodegenerative movement disorder, the brain is characterized by the loss of dopaminergic neurons in the nigrostriatal system, leading to dyshomeostasis of the basal ganglia network activity that is linked to motility dysfunction. PD mostly arises as an age-associated sporadic disease, but several genetic forms also exist. Compelling evidence supports that synaptic damage and dysfunction characterize the very early phases of either sporadic or genetic forms of PD and that this early PD synaptopathy drives retrograde terminal-to-cell body degeneration, culminating in neuronal loss. The Ras-associated binding protein (Rab) family of small GTPases, which is involved in the maintenance of neuronal vesicular trafficking, synaptic architecture and function in the central nervous system, has recently emerged among the major players in PD synaptopathy. In this manuscript, we provide an overview of the main findings supporting the involvement of Rabs in either sporadic or genetic PD pathophysiology, and we highlight how Rab alterations participate in the onset of early synaptic damage and dysfunction.
    Keywords:  GBA1; LRRK2; Parkinson’s disease; Rab proteins; alpha-synuclein; autophagy; synaptopathy
    DOI:  https://doi.org/10.3390/biomedicines10081941
  37. Exp Mol Med. 2022 Aug 23.
    AMPK suppresses Th2 cell responses by repressing mTORC2.
    Mahesh Pandit, Maheshwor Timilshina, Ye Gu, Suman Acharya, Yeonseok Chung, Sang-Uk Seo, Jae-Hoon Chang.
      Allergic inflammation is a T helper 2 (Th2) cell-driven pathophysiological phenomenon, but the mechanism by which the metabolic cascade affects Th2 cell differentiation remains unclear. In this study, we investigated the roles of AMP-activated protein kinase (AMPK) and intracellular energy sensors in Th2 cell differentiation and the pathogenesis of allergic inflammation. Accordingly, T-cell-specific AMPK or Sirtuin 1 (Sirt1)-knockout mice were subjected to allergic inflammation, and their Th2 cell responses were investigated. The results demonstrated that inducing allergic inflammation in AMPK- and Sirt1-knockout mice increased Th2 cell responses and exacerbated allergic phenotypes. Furthermore, treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, ameliorated allergic inflammation in mice. Mechanistically, our findings revealed that AMPK repressed mechanistic target of rapamycin complex 2 (mTORC2), which downregulated the expression of suppressor of cytokine signaling 5 (SOCS5) in CD4+ T cells. In addition, the loss of AMPK signaling reduced SOCS5 expression and increased interleukin-4-STAT6-GATA3 axis-mediated Th2 cell differentiation. Finally, the T-cell-specific deletion of Rictor, a member of mTORC2, in Sirt1T-KO mice led to the reversal of allergic exacerbation to the level in control mice. Overall, our findings suggest that AMPK in CD4+ T cells inhibits the differentiation of Th2 cells by repressing mTORC2 and thus serves as a potential target for Th2 cell-associated diseases.
    DOI:  https://doi.org/10.1038/s12276-022-00832-x
  38. Int J Mol Sci. 2022 Aug 13. pii: 9089. [Epub ahead of print]23(16):
    The Key Role of Lysosomal Protease Cathepsins in Viral Infections.
    Melania Scarcella, Danila d'Angelo, Mariangela Ciampa, Simona Tafuri, Luigi Avallone, Luigi Michele Pavone, Valeria De Pasquale.
      Cathepsins encompass a family of lysosomal proteases that mediate protein degradation and turnover. Although mainly localized in the endolysosomal compartment, cathepsins are also found in the cytoplasm, nucleus, and extracellular space, where they are involved in cell signaling, extracellular matrix assembly/disassembly, and protein processing and trafficking through the plasma and nuclear membrane and between intracellular organelles. Ubiquitously expressed in the body, cathepsins play regulatory roles in a wide range of physiological processes including coagulation, hormone secretion, immune responses, and others. A dysregulation of cathepsin expression and/or activity has been associated with many human diseases, including cancer, diabetes, obesity, cardiovascular and inflammatory diseases, kidney dysfunctions, and neurodegenerative disorders, as well as infectious diseases. In viral infections, cathepsins may promote (1) activation of the viral attachment glycoproteins and entry of the virus into target cells; (2) antigen processing and presentation, enabling the virus to replicate in infected cells; (3) up-regulation and processing of heparanase that facilitates the release of viral progeny and the spread of infection; and (4) activation of cell death that may either favor viral clearance or assist viral propagation. In this review, we report the most relevant findings on the molecular mechanisms underlying cathepsin involvement in viral infection physiopathology, and we discuss the potential of cathepsin inhibitors for therapeutical applications in viral infectious diseases.
    Keywords:  cathepsins; infection; physiopathology; therapy; viruses
    DOI:  https://doi.org/10.3390/ijms23169089
  39. Metabolites. 2022 Aug 12. pii: 740. [Epub ahead of print]12(8):
    GPRC6A Mediates Glucose and Amino Acid Homeostasis in Mice.
    Yumin He, Jingyun Su, Hongrui Gao, Jianzhong Li, Zemeng Feng, Yulong Yin.
      GPRC6A, an important member of the G-protein-coupled receptor superfamily, has been widely studied in body health maintenance and related diseases. However, it is still controversial whether GPRC6A plays a vital role in glucose homeostasis, and the role of GPRC6A on amino acid homeostasis has not been reported. In this study, GPRC6A was knocked out in C57BL6 mice, and we found that GPRC6A plays an important role in the glucose metabolism, mainly affecting the glucose clearance capacity and gluconeogenesis in mice. GPRC6A plays an important role in maintaining amino acid homeostasis under dietary restrictions, and this may be realized by participating in the regulation of autophagy. Since a large amount of amino acid is lost from urine in aged GPRC6A-/- mice, it is possible that GPRC6A regulates amino acid homeostasis by affecting the integrity of tissue structure. GPRC6A is involved in the regulation of mTORC1 activation but is not necessary for mTORC1 activation under sufficient nutritional supply. In the absence of exogenous amino acids, the loss of GPRC6A induces the GCN2 pathway activation and excessive autophagy of cells, leading to the overactivation of mTORC1, which may be detrimental to body health and cell survival. In summary, this study provides a theoretical and experimental basis for the metabolic process of GPRC6A in body growth and health.
    Keywords:  GPRC6A; amino acids; autophagy; glucose; mTORC1
    DOI:  https://doi.org/10.3390/metabo12080740
  40. Int J Mol Sci. 2022 Aug 16. pii: 9189. [Epub ahead of print]23(16):
    Biological Variation in Peripheral Inflammation and Oxidative Stress Biomarkers in Individuals with Gaucher Disease.
    Siddhee A Sahasrabudhe, Marcia R Terluk, Kyle D Rudser, James C Cloyd, Reena V Kartha.
      The lack of reliable biomarkers is a significant challenge impeding progress in orphan drug development. For appropriate interpretation of intervention-based results or for evaluating candidate biomarkers, other things being equal, lower variability in biomarker measurement would be helpful. However, variability in rare disease biomarkers is often poorly understood. Type 1 Gaucher disease (GD1) is one such rare lysosomal storage disorder. Oxidative stress and inflammation have been linked to the pathophysiology of GD1 and validated measures of these processes can provide predictive value for treatment success or disease progression. This study was undertaken to investigate and compare the extent of longitudinal biological variation over a three-month period for various blood-based oxidative stress and inflammation markers in participants with GD1 on stable standard-of-care therapy (N = 13), treatment-naïve participants with GD1 (N = 5), and in age- and gender-matched healthy volunteers (N = 18). We utilized Bland-Altman plots for visual comparison of the biological variability among the three measurements. We also report group-wise means and the percentage of coefficient of variation (%CV) for 15 biomarkers. Qualitatively, we show specific markers (IL-1Ra, IL-8, and MIP-1b) to be consistently altered in GD1, irrespective of therapy status, highlighting the need for adjunctive therapies that can target and modulate these biomarkers. This information can help guide the selection of candidate biomarkers for future intervention-based studies in GD1 patients.
    Keywords:  Bland–Altman; Gaucher disease; biomarkers; inflammation; oxidative stress; therapy; variability
    DOI:  https://doi.org/10.3390/ijms23169189
  41. Bone Res. 2022 Aug 26. 10(1): 57
    Ammonia promotes the proliferation of bone marrow-derived mesenchymal stem cells by regulating the Akt/mTOR/S6k pathway.
    Yu Liu, Xiangxian Zhang, Wei Wang, Ting Liu, Jun Ren, Siyuan Chen, Tianqi Lu, Yan Tie, Xia Yuan, Fei Mo, Jingyun Yang, Yuquan Wei, Xiawei Wei.
      Ammonia plays an important role in cellular metabolism. However, ammonia is considered a toxic product. In bone marrow-derived mesenchymal stem cells, multipotent stem cells with high expression of glutamine synthetase (GS) in bone marrow, ammonia and glutamate can be converted to glutamine via glutamine synthetase activity to support the proliferation of MSCs. As a major nutritional amino acid for biosynthesis, glutamine can activate the Akt/mTOR/S6k pathway to stimulate cell proliferation. The activation of mTOR can promote cell entry into S phase, thereby enhancing DNA synthesis and cell proliferation. Our studies demonstrated that mesenchymal stem cells can convert the toxic waste product ammonia into nutritional glutamine via GS activity. Then, the Akt/mTOR/S6k pathway is activated to promote bone marrow-derived mesenchymal stem cell proliferation. These results suggest a new therapeutic strategy and potential target for the treatment of diseases involving hyperammonemia.
    DOI:  https://doi.org/10.1038/s41413-022-00215-y
  42. Commun Biol. 2022 Aug 26. 5(1): 878
    mTOR regulation of metabolism limits LPS-induced monocyte inflammatory and procoagulant responses.
    Nina C Lund, Yetunde Kayode, Melanie R McReynolds, Deanna C Clemmer, Hannah Hudson, Isabelle Clerc, Hee-Kyung Hong, Jason M Brenchley, Joseph Bass, Richard T D'Aquila, Harry E Taylor.
      Translocated lipopolysaccharide (LPS) activates monocytes via TLR4 and is hypothesized to increase cardiovascular disease risk in persons living with HIV. We tested whether mTOR activity supports LPS-stimulated monocyte production of pro-inflammatory cytokines and tissue factor (TF), as it propels the inflammatory response in several immune cell types besides monocytes. However, multi-omics analyses here demonstrate that mTOR activates a metabolic pathway that limits abundance of these gene products in monocytes. Treatment of primary human monocytes with catalytic mTOR inhibitors (mTORi) increased LPS-induced polyfunctional responses, including production of IL-1β, IL-6, and the pro-coagulant, TF. NF-κB-driven transcriptional activity is enhanced with LPS stimulation after mTORi treatment to increase expression of F3 (TF). Moreover, intracellular NAD+ availability is restricted due to decreased salvage pathway synthesis. These results document mTOR-mediated restraint of the LPS-induced transcriptional response in monocytes and a metabolic mechanism informing strategies to reverse enhanced risk of coagulopathy in pro-inflammatory states.
    DOI:  https://doi.org/10.1038/s42003-022-03804-z
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