bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022–10–16
forty papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes.
  2. Lysosomal solute and water transport.
  3. Saposin C, Key Regulator in the Alpha-Synuclein Degradation Mediated by Lysosome.
  4. LYECs: lysosome-enhancing compounds as potential therapeutic approaches for Alzheimer disease.
  5. Mechanistic target of rapamycin signaling in human nervous system development and disease.
  6. Purification and biochemical characterization of the Rag GTPase heterodimer.
  7. Hyperactivation of mTORC1 in a double hit mutant zebrafish model of tuberous sclerosis complex causes increased seizure susceptibility and neurodevelopmental abnormalities.
  8. The Role of the miR-17-92 Cluster in Autophagy and Atherosclerosis Supports Its Link to Lysosomal Storage Diseases.
  9. MPSI Manifestations and Treatment Outcome: Skeletal Focus.
  10. TFEB; Beyond Its Role as an Autophagy and Lysosomes Regulator.
  11. Schisandrol A protects AGEs-induced neuronal cells death by allosterically targeting ATP6V0d1 subunit of V-ATPase.
  12. Effects of lysosomal low density lipoprotein oxidation by ferritin on macrophage function.
  13. GSK3β-dependent lysosome biogenesis: An effective pathway to mitigate renal fibrosis with LM49.
  14. Pathogenic Roles of Heparan Sulfate and Its Use as a Biomarker in Mucopolysaccharidoses.
  15. Phenotypic correction of murine mucopolysaccharidosis type II by engraftment of ex vivo lentiviral vector transduced hematopoietic stem and progenitor cells.
  16. An unusual mode of baseline translation adjusts cellular protein synthesis capacity to metabolic needs.
  17. Early enzyme replacement therapy prevents dental and craniofacial abnormalities in a mouse model of mucopolysaccharidosis type VI.
  18. Decreased Prosaposin and Progranulin in the Cingulate Cortex Are Associated with Schizophrenia Pathophysiology.
  19. A quick fix for lysosomes.
  20. Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death.
  21. Glycerophosphoinositol is Elevated in Blood Samples From CLN3 Δex7-8 pigs, Cln3 Δex7-8 Mice, and CLN3-Affected Individuals.
  22. A two-tiered system for selective receptor and transporter protein degradation.
  23. The GARP complex prevents sterol accumulation at the trans-Golgi network during dendrite remodeling.
  24. Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.
  25. mTORC1-Dependent and GSDMD-Mediated Pyroptosis in Developmental Sevoflurane Neurotoxicity.
  26. An AP-3 dependent pathway directs phagosome fusion with Rab8 and Rab11 vesicles involved in TLR2 signaling.
  27. Potential Role of Sphingolipidoses-Associated Lysosphingolipids in Cancer.
  28. The aldolase inhibitor aldometanib mimics glucose starvation to activate lysosomal AMPK.
  29. Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.
  30. mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.
  31. Systematic review of mTOR inhibitor treatment, biomarkers and prophylaxis for tuberous sclerosis complex-associated seizures.
  32. Axenic Culture of Caenorhabditis elegans Alters Lysosomal/Proteasomal Balance and Increases Neuropeptide Expression.
  33. GRN-/- iPSC-derived cortical neurons recapitulate the pathological findings of both frontotemporal lobar degeneration and neuronal ceroidolipofuscinosis.
  34. Regulation of membrane phospholipid biosynthesis in mammalian cells.
  35. Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.
  36. The entry of unclosed autophagosomes into vacuoles and its physiological relevance.
  37. Lipid and protein content profiling of isolated native autophagic vesicles.
  38. Emerging degrader technologies engaging lysosomal pathways.
  39. Reducing Akt2 in retinal pigment epithelial cells causes a compensatory increase in Akt1 and attenuates diabetic retinopathy.
  40. Endocytosis at extremes: Formation and internalization of giant clathrin-coated pits under elevated membrane tension.
  1. J Cell Biol. 2023 Jan 02. pii: e202203045. [Epub ahead of print]222(1):
    Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes.
    Lingjian Zhou, Xutong Xue, Ke Yang, Zhi Feng, Min Liu, José C Pastor-Pareja.
      At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. Here, we investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. We found that Vps20, Rop, and lysosomal markers localize near the trans-Golgi. Furthermore, we document that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in our hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, we show that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. Our results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system.
    DOI:  https://doi.org/10.1083/jcb.202203045
  2. J Cell Biol. 2022 Nov 07. pii: e202109133. [Epub ahead of print]221(11):
    Lysosomal solute and water transport.
    Meiqin Hu, Nan Zhou, Weijie Cai, Haoxing Xu.
      Lysosomes mediate hydrolase-catalyzed macromolecule degradation to produce building block catabolites for reuse. Lysosome function requires an osmo-sensing machinery that regulates osmolytes (ions and organic solutes) and water flux. During hypoosmotic stress or when undigested materials accumulate, lysosomes become swollen and hypo-functional. As a membranous organelle filled with cargo macromolecules, catabolites, ions, and hydrolases, the lysosome must have mechanisms that regulate its shape and size while coordinating content exchange. In this review, we discussed the mechanisms that regulate lysosomal fusion and fission as well as swelling and condensation, with a focus on solute and water transport mechanisms across lysosomal membranes. Lysosomal H+, Na+, K+, Ca2+, and Cl- channels and transporters sense trafficking and osmotic cues to regulate both solute flux and membrane trafficking. We also provide perspectives on how lysosomes may adjust the volume of themselves, the cytosol, and the cytoplasm through the control of lysosomal solute and water transport.
    DOI:  https://doi.org/10.1083/jcb.202109133
  3. Int J Mol Sci. 2022 Oct 09. pii: 12004. [Epub ahead of print]23(19):
    Saposin C, Key Regulator in the Alpha-Synuclein Degradation Mediated by Lysosome.
    Clara Ruz, Francisco J Barrero, Javier Pelegrina, Sara Bandrés-Ciga, Francisco Vives, Raquel Duran.
      Lysosomal dysfunction has been proposed as one of the most important pathogenic molecular mechanisms in Parkinson disease (PD). The most significant evidence lies in the GBA gene, which encodes for the lysosomal enzyme β-glucocerebrosidase (β-GCase), considered the main genetic risk factor for sporadic PD. The loss of β-GCase activity results in the formation of α-synuclein deposits. The present study was aimed to determine the activity of the main lysosomal enzymes and the cofactors Prosaposin (PSAP) and Saposin C in PD and healthy controls, and their contribution to α-synuclein (α-Syn) aggregation. 42 PD patients and 37 age-matched healthy controls were included in the study. We first analyzed the β-GCase, β-galactosidase (β-gal), β-hexosaminidase (Hex B) and Cathepsin D (CatD) activities in white blood cells. We also measured the GBA, β-GAL, β-HEX, CTSD, PSAP, Saposin C and α-Syn protein levels by Western-blot. We found a 20% reduced β-GCase and β-gal activities in PD patients compared to controls. PSAP and Saposin C protein levels were significantly lower in PD patients and correlated with increased levels of α-synuclein. CatD, in contrast, showed significantly increased activity and protein levels in PD patients compared to controls. Increased CTSD protein levels in PD patients correlated, intriguingly, with a higher concentration of α-Syn. Our findings suggest that lysosomal dysfunction in sporadic PD is due, at least in part, to an alteration in Saposin C derived from reduced PSAP levels. That would lead to a significant decrease in the β-GCase activity, resulting in the accumulation of α-syn. The accumulation of monohexosylceramides might act in favor of CTSD activation and, therefore, increase its enzymatic activity. The evaluation of lysosomal activity in the peripheral blood of patients is expected to be a promising approach to investigate pathological mechanisms and novel therapies aimed to restore the lysosomal function in sporadic PD.
    Keywords:  Cathepsin D; PSAP; Parkinson’s disease; lysosomal dysfunction; β-glucocerebrosidase
    DOI:  https://doi.org/10.3390/ijms231912004
  4. Autophagy. 2022 Oct 10. 1-2
    LYECs: lysosome-enhancing compounds as potential therapeutic approaches for Alzheimer disease.
    Limin Yin, Yu Zhou, Hong Liu, Yang Li.
      More than 55 million people are suffering from Alzheimer's disease (AD), but there is still no effective treatment for it. Therefore, novel therapeutic approaches and regulatory mechanisms of protein quality control need to be further evaluated and dissected. The lysosome is one of the major degradative organelles that maintain cellular homeostasis and protein quality control. In our recent study, we have identified a group of LYsosome-Enhancing Compounds (LYECs), which significantly promote the activation of TFEB (transcription factor EB) and lysosome biogenesis via inhibiting dopamine transporters (DAT). Injection of LH2-051, a member of the LYECs identified in this study, significantly improves learning, memory, and cognitive function of APP-PSEN1 mice, in which the enhanced capability of lysosomal degradation promotes the clearance of amyloid protein aggregates. In summary, this study reports novel mechanisms of neurotransporter-mediated lysosome biogenesis and shows that DAT inhibition can alleviate the pathogenesis of Alzheimer's disease.
    Keywords:  Alzheimer‘s disease; TFEB; lysosome biogenesis; lysosome-enhancing compounds
    DOI:  https://doi.org/10.1080/15548627.2022.2131247
  5. Front Mol Neurosci. 2022 ;15 1005631
    Mechanistic target of rapamycin signaling in human nervous system development and disease.
    Marie Girodengo, Sila K Ultanir, Joseph M Bateman.
      Mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that regulates fundamental cellular processes including growth control, autophagy and metabolism. mTOR has key functions in nervous system development and mis-regulation of mTOR signaling causes aberrant neurodevelopment and neurological diseases, collectively called mTORopathies. In this mini review we discuss recent studies that have deepened our understanding of the key roles of the mTOR pathway in human nervous system development and disease. Recent advances in single-cell transcriptomics have been exploited to reveal specific roles for mTOR signaling in human cortical development that may have contributed to the evolutionary divergence from our primate ancestors. Cerebral organoid technology has been utilized to show that mTOR signaling is active in and regulates outer radial glial cells (RGCs), a population of neural stem cells that distinguish the human developing cortex. mTOR signaling has a well-established role in hamartoma syndromes such as tuberous sclerosis complex (TSC) and other mTORopathies. New ultra-sensitive techniques for identification of somatic mTOR pathway mutations have shed light on the neurodevelopmental origin and phenotypic heterogeneity seen in mTORopathy patients. These emerging studies suggest that mTOR signaling may facilitate developmental processes specific to human cortical development but also, when mis-regulated, cause cortical malformations and neurological disease.
    Keywords:  cortex; mTOR; mTORopathy; neuron; organoid; tuberous sclerosis
    DOI:  https://doi.org/10.3389/fnmol.2022.1005631
  6. Methods Enzymol. 2022 ;pii: S0076-6879(22)00245-2. [Epub ahead of print]675 131-158
    Purification and biochemical characterization of the Rag GTPase heterodimer.
    Dylan D Doxsey, Kuang Shen.
      The mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient levels in the cell and based on the availability, regulates cellular growth and proliferation. Its activity is tightly modulated by two GTPase units, the Rag GTPases and the Rheb GTPase. The Rag GTPases are the central hub of amino acid sensing as they summarize the amino acid signals from upstream regulators and control the subcellular localization of mTORC1. Unique from canonical signaling GTPases, the Rag GTPases are obligatory heterodimers, and the two subunits coordinate their nucleotide loading states to regulate their functional states. Robust biochemical analysis is indispensable to understanding the molecular mechanism governing the GTPase cycle. This chapter discusses protocols for purifying and biochemically characterizing the Rag GTPase heterodimer. We described two purification protocols to recombinantly produce the Rag GTPase heterodimer in large quantities. We then described assays to quantitatively measure the nucleotide binding and hydrolysis by the Rag GTPases. These assays allow for a thorough investigation of this unique heterodimeric GTPase, and they could be applicable to investigations of other noncanonical GTPases.
    Keywords:  Cooperativity; Enzymatic mechanism; GTP hydrolysis; Kinetics; Rag GTPase; mTORC1
    DOI:  https://doi.org/10.1016/bs.mie.2022.07.007
  7. Front Cell Dev Biol. 2022 ;10 952832
    Hyperactivation of mTORC1 in a double hit mutant zebrafish model of tuberous sclerosis complex causes increased seizure susceptibility and neurodevelopmental abnormalities.
    Ann-Sofie De Meulemeester, Lise Heylen, Aleksandra Siekierska, James D Mills, Alessia Romagnolo, Nicole N Van Der Wel, Eleonora Aronica, Peter A M de Witte.
      Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by pathogenic variants in TSC1 and TSC2 genes. TSC patients present with seizures and brain abnormalities such as tubers and subependymal giant cells astrocytoma (SEGA). Despite common molecular and clinical features, the severity of the disease varies greatly, even intrafamilially. The second hit hypothesis suggests that an additional, inactivating mutation in the remaining functional allele causes a more severe phenotype and therefore explains the phenotypic variability. Recently, second hit mutations have been detected frequently in mTORopathies. To investigate the pathophysiological effects of second hit mutations, several mouse models have been developed. Here, we opted for a double mutant zebrafish model that carries a LOF mutation both in the tsc2 and the depdc5 gene. To the best of our knowledge, this is the first time a second-hit model has been studied in zebrafish. Significantly, the DEP domain-containing protein 5 (DEPDC5) gene has an important role in the regulation of mTORC1, and the combination of a germline TSC2 and somatic DEPDC5 mutation has been described in a TSC patient with intractable epilepsy. Our depdc5 -/- x tsc2 -/- double mutant zebrafish line displayed greatly increased levels of mammalian target of rapamycin (mTORC1) activity, augmented seizure susceptibility, and early lethality which could be rescued by rapamycin. Histological analysis of the brain revealed ventricular dilatation in the tsc2 and double homozygotes. RNA-sequencing showed a linear relation between the number of differentially expressed genes (DEGs) and the degree of mTORC1 hyperactivity. Enrichment analysis of their transcriptomes revealed that many genes associated with neurological developmental processes were downregulated and mitochondrial genes were upregulated. In particular, the transcriptome of human SEGA lesions overlapped strongly with the double homozygous zebrafish larvae. The data highlight the clinical relevance of the depdc5 -/- x tsc2 -/- double mutant zebrafish larvae that showed a more severe phenotype compared to the single mutants. Finally, analysis of gene-drug interactions identified interesting pharmacological targets for SEGA, underscoring the value of our small zebrafish vertebrate model for future drug discovery efforts.
    Keywords:  RNA-sequencing; SEGA; epilepsy; mTOR; neurodevelopment; tuberous sclerosis complex (TSC); zebrafish
    DOI:  https://doi.org/10.3389/fcell.2022.952832
  8. Cells. 2022 Sep 26. pii: 2991. [Epub ahead of print]11(19):
    The Role of the miR-17-92 Cluster in Autophagy and Atherosclerosis Supports Its Link to Lysosomal Storage Diseases.
    Daniel Ortuño-Sahagún, Julia Enterría-Rosales, Vanesa Izquierdo, Christian Griñán-Ferré, Mercè Pallàs, Celia González-Castillo.
      Establishing the role of non-coding RNA (ncRNA), especially microRNAs (miRNAs), in the regulation of cell function constitutes a current research challenge. Two to six miRNAs can act in clusters; particularly, the miR-17-92 family, composed of miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a is well-characterized. This cluster functions during embryonic development in cell differentiation, growth, development, and morphogenesis and is an established oncogenic cluster. However, its role in the regulation of cellular metabolism, mainly in lipid metabolism and autophagy, has received less attention. Here, we argue that the miR-17-92 cluster is highly relevant for these two processes, and thus, could be involved in the study of pathologies derived from lysosomal deficiencies. Lysosomes are related to both processes, as they control cholesterol flux and regulate autophagy. Accordingly, we compiled, analyzed, and discussed current evidence that highlights the cluster's fundamental role in regulating cellular energetic metabolism (mainly lipid and cholesterol flux) and atherosclerosis, as well as its critical participation in autophagy regulation. Because these processes are closely related to lysosomes, we also provide experimental data from the literature to support our proposal that the miR-17-92 cluster could be involved in the pathogenesis and effects of lysosomal storage diseases (LSD).
    Keywords:  autophagy; cholesterol; enzyme deficiency; lysosomal storage diseases; metabolism; vesicle trafficking
    DOI:  https://doi.org/10.3390/cells11192991
  9. Int J Mol Sci. 2022 Sep 22. pii: 11168. [Epub ahead of print]23(19):
    MPSI Manifestations and Treatment Outcome: Skeletal Focus.
    Giada De Ponti, Samantha Donsante, Marta Frigeni, Alice Pievani, Alessandro Corsi, Maria Ester Bernardo, Mara Riminucci, Marta Serafini.
      Mucopolysaccharidosis type I (MPSI) (OMIM #252800) is an autosomal recessive disorder caused by pathogenic variants in the IDUA gene encoding for the lysosomal alpha-L-iduronidase enzyme. The deficiency of this enzyme causes systemic accumulation of glycosaminoglycans (GAGs). Although disease manifestations are typically not apparent at birth, they can present early in life, are progressive, and include a wide spectrum of phenotypic findings. Among these, the storage of GAGs within the lysosomes disrupts cell function and metabolism in the cartilage, thus impairing normal bone development and ossification. Skeletal manifestations of MPSI are often refractory to treatment and severely affect patients' quality of life. This review discusses the pathological and molecular processes leading to impaired endochondral ossification in MPSI patients and the limitations of current therapeutic approaches. Understanding the underlying mechanisms responsible for the skeletal phenotype in MPSI patients is crucial, as it could lead to the development of new therapeutic strategies targeting the skeletal abnormalities of MPSI in the early stages of the disease.
    Keywords:  endochondral bone formation; glycosaminoglycans; lysosomal alpha-L-iduronidase; lysosomal storage disease; mucopolysaccharidoses; mucopolysaccharidosis type I
    DOI:  https://doi.org/10.3390/ijms231911168
  10. Cells. 2022 Oct 07. pii: 3153. [Epub ahead of print]11(19):
    TFEB; Beyond Its Role as an Autophagy and Lysosomes Regulator.
    Berenice Franco-Juárez, Cristina Coronel-Cruz, Beatriz Hernández-Ochoa, Saúl Gómez-Manzo, Noemi Cárdenas-Rodríguez, Roberto Arreguin-Espinosa, Cindy Bandala, Luis Miguel Canseco-Ávila, Daniel Ortega-Cuellar.
      Transcription factor EB (TFEB) is considered the master transcriptional regulator of autophagy and lysosomal biogenesis, which regulates target gene expression through binding to CLEAR motifs. TFEB dysregulation has been linked to the development of numerous pathological conditions; however, several other lines of evidence show that TFEB might be a point of convergence of diverse signaling pathways and might therefore modulate other important biological processes such as cellular senescence, DNA repair, ER stress, carbohydrates, and lipid metabolism and WNT signaling-related processes. The regulation of TFEB occurs predominantly at the post-translational level, including phosphorylation, acetylation, SUMOylating, PARsylation, and glycosylation. It is noteworthy that TFEB activation is context-dependent; therefore, its regulation is subjected to coordinated mechanisms that respond not only to nutrient fluctuations but also to stress cell programs to ensure proper cell homeostasis and organismal health. In this review, we provide updated insights into novel post-translational modifications that regulate TFEB activity and give an overview of TFEB beyond its widely known role in autophagy and the lysosomal pathway, thus opening the possibility of considering TFEB as a potential therapeutic target.
    Keywords:  DNA damage repair and cell cycle; WNT signaling; carbohydrate; cellular senescence; endoplasmic reticulum stress; lipids; metabolism; transcriptional factor EB (TFEB)
    DOI:  https://doi.org/10.3390/cells11193153
  11. Acta Pharm Sin B. 2022 Oct;12(10): 3843-3860
    Schisandrol A protects AGEs-induced neuronal cells death by allosterically targeting ATP6V0d1 subunit of V-ATPase.
    Xiaoqing Zhou, Shaoyang Zhao, Tingting Liu, Lu Yao, Meimei Zhao, Xiaoming Ye, Xiaowen Zhang, Qiang Guo, Pengfei Tu, Kewu Zeng.
      Diabetes have been shown to cause progressive neuronal injury with pain and numbness via advanced glycation end-products (AGEs)-induced neuronal cell apoptosis; however, the valuable drug targets for diabetic neuropathy have been poorly reported so far. In this study, we discovered a natural small-molecule schisandrol A (SolA) with significant protective effect against AGEs-induced neuronal cell apoptosis. ATP6V0D1, a major subunit of vacuolar-type ATPase (V-ATPase) in lysosome was identified as a crucial cellular target of SolA. Moreover, SolA allosterically mediated ATP6V0D1 conformation via targeting a unique cysteine 335 residue to activate V-ATPase-dependent lysosomal acidification. Interestingly, SolA-induced lysosome pH downregulation resulted in a mitochondrial-lysosomal crosstalk by selectively promoting mitochondrial BH3-only protein BIM degradation, thereby preserving mitochondrial homeostasis and neuronal cells survival. Collectively, our findings reveal ATP6V0D1 is a valuable pharmacological target for diabetes-associated neuronal injury via controlling lysosomal acidification, and also provide the first small-molecule template allosterically activating V-ATPase for preventing diabetic neuropathy.
    Keywords:  AGEs; ATP6V0D1; Advanced glycation end-products; Allosteric regulation; Diabetic neuropathy; Lysosomal acidification; Schisandrol A; Targets; V-ATPase
    DOI:  https://doi.org/10.1016/j.apsb.2022.06.013
  12. Free Radic Res. 2022 Oct 11. 1-21
    Effects of lysosomal low density lipoprotein oxidation by ferritin on macrophage function.
    Oluwatosin O Ojo, David S Leake.
      We have previously demonstrated that low density lipoprotein (LDL) can be oxidised by iron in the lysosomes of macrophages. Some of the iron content of lysosomes might be delivered through autophagy of ferritin (the main iron-storage protein in the body). We have now investigated the effects of ferritin-mediated LDL oxidation on macrophage function. The addition of ferritin to human THP-1 cells and human monocyte-derived macrophages increased lysosomal lipid peroxidation, as shown by LPO-Foam, a fluorescent probe targetted to lysosomes. Incubating THP-1 cells with ferritin and native LDL or LDL aggregated by sphingomyelinase, to allow their endocytosis and delivery to lysosomes, led to the formation of lysosomal ceroid (an advanced lipid oxidation product), indicative of lysosomal LDL oxidation. Incubating THP-1 cells with ferritin and LDL caused metabolic activation of the cells, as shown by increased extracellular acidification and oxygen consumption measured by a Seahorse analyser. LDL oxidised by ferritin in lysosomes might be released from macrophages when the cells die and lyse and affect neighbouring cells in atherosclerotic lesions. Adding LDL oxidised by ferritin at lysosomal pH (pH 4.5) to macrophages increased their intracellular reactive oxygen species formation, shown using dihydroethidium, and increased apoptosis. Ferritin might therefore contribute to LDL oxidation in the lysosomes of macrophages and have atherogenic effects.
    Keywords:  LDL oxidation; atherosclerosis; ferritin; lysosome; macrophage
    DOI:  https://doi.org/10.1080/10715762.2022.2133703
  13. Front Pharmacol. 2022 ;13 925489
    GSK3β-dependent lysosome biogenesis: An effective pathway to mitigate renal fibrosis with LM49.
    Jinhong Ren, Huizhi Wei, Jian Sun, Xiue Feng, Yuanlin Zhang, Hongxia Yuan, Junqiu Miao, Xiaoming Qi, Yuanbiao Qiao, Baoguo Xiao, Qingshan Li.
      Renal fibrosis is an incurable disorder characterised by an imbalance of the extracellular matrix (ECM) favouring excess production over degradation. The identification of actionable pathways and agents that promote ECM degradation to restore ECM homeostasis may help mitigate renal fibrosis. In this study, we identified 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49), a compound we previously synthesised, as a small-molecule inducer of ECM degradation. LM49 administration efficiently reduced ECM deposition in renal tissue of diabetic nephropathy rats and in transforming growth factor β-treated renal fibroblast cells. LM49 promoted the cytosol-to-nucleus translocation of transcription factor EB (TFEB) to increase lysosome biogenesis, leading to lysosome-based degradation of the ECM. TFEB-mediated lysosome biogenesis was induced by LM49 directly inhibiting the activity of glycogen synthase kinase 3β (GSK3β) rather than mammalian target of rapamycin complex 1. LM49 inhibited GSK3β kinase activity concentration-dependently via competing with ATP. Direct binding between LM49 and GSK3β was confirmed by the bio-layer interferometry assay, cellular thermal shift assay, and drug affinity responsive target stability. A molecular docking and molecular dynamic simulation revealed that LM49 occupied the ATP pocket of GSK3β, which was consistent with the kinase activity assay. In summary, LM49 enhances TFEB-mediated lysosome biogenesis by directly inhibiting GSK3β, leading to the degradation of the ECM by lysosomes. The enhancement of GSK3β-dependent lysosome biogenesis to rebalance the ECM may be a novel strategy to counteract renal fibrosis, and LM49 may be a viable clinical candidate for treating this disorder.
    Keywords:  LM49; extracellular matrix; glycogen synthase kinase 3β; lysosome biogenesis; renal fibrosis
    DOI:  https://doi.org/10.3389/fphar.2022.925489
  14. Int J Mol Sci. 2022 Oct 03. pii: 11724. [Epub ahead of print]23(19):
    Pathogenic Roles of Heparan Sulfate and Its Use as a Biomarker in Mucopolysaccharidoses.
    Kohtaro Minami, Hideto Morimoto, Hiroki Morioka, Atsushi Imakiire, Masafumi Kinoshita, Ryuji Yamamoto, Tohru Hirato, Hiroyuki Sonoda.
      Heparan sulfate (HS) is an essential glycosaminoglycan (GAG) as a component of proteoglycans, which are present on the cell surface and in the extracellular matrix. HS-containing proteoglycans not only function as structural constituents of the basal lamina but also play versatile roles in various physiological processes, including cell signaling and organ development. Thus, inherited mutations of genes associated with the biosynthesis or degradation of HS can cause various diseases, particularly those involving the bones and central nervous system (CNS). Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders involving GAG accumulation throughout the body caused by a deficiency of GAG-degrading enzymes. GAGs are stored differently in different types of MPSs. Particularly, HS deposition is observed in patients with MPS types I, II, III, and VII, all which involve progressive neuropathy with multiple CNS system symptoms. While therapies are available for certain symptoms in some types of MPSs, significant unmet medical needs remain, such as neurocognitive impairment. This review presents recent knowledge on the pathophysiological roles of HS focusing on the pathogenesis of MPSs. We also discuss the possible use and significance of HS as a biomarker for disease severity and therapeutic response in MPSs.
    Keywords:  biomarker; blood-brain barrier; cerebrospinal fluid; heparan sulfate; lysosomal storage disorders; mucopolysaccharidoses
    DOI:  https://doi.org/10.3390/ijms231911724
  15. Hum Gene Ther. 2022 Oct 13.
    Phenotypic correction of murine mucopolysaccharidosis type II by engraftment of ex vivo lentiviral vector transduced hematopoietic stem and progenitor cells.
    Miles C Smith, Lalitha R Belur, Andrea D Karlen, Olivia Erlanson, Kelly M Podetz-Pedersen, Jessica McKenzie, Jenn Detellis, Khatuna Gagnidze, Geoffrey Parsons, Nicholas Robinson, Shelby Labarre, Saumil Shah, Justin Furcich, Troy Lund, Hsing-Chen Tsai, R S Mc Ivor, Melissa Bonner.
      Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X- linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). Absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase. However, ERT is demanding for the patient and does not ameliorate neurological manifestations of the disease. Using an IDS deficient mouse model that phenocopies the human disease, we evaluated hematopoietic stem and progenitor cells (HSPCs) transduced with a lentiviral vector (LVV) carrying a codon optimized human IDS coding sequence regulated by a ubiquitous MNDU3 promoter (MNDU3-IDS). Mice treated with MNDU3-IDS LVV transduced cells showed supraphysiologic levels of IDS enzyme activity in plasma, peripheral blood mononuclear cells (PBMCs), and in most analyzed tissues. These enzyme levels were sufficient to normalize GAG storage in analyzed tissues. Importantly, IDS levels in the brains of MNDU3-IDS engrafted animals were restored to 10-20% that of wild-type mice, sufficient to normalize GAG content and prevent emergence of cognitive deficit as evaluated by neurobehavioral testing. These results demonstrate the potential effectiveness of ex vivo MNDU3-IDS LVV transduced HSPCs for treatment of MPS II.
    DOI:  https://doi.org/10.1089/hum.2022.141
  16. Cell Rep. 2022 Oct 11. pii: S2211-1247(22)01317-1. [Epub ahead of print]41(2): 111467
    An unusual mode of baseline translation adjusts cellular protein synthesis capacity to metabolic needs.
    Cornelius Schneider, Florian Erhard, Beyenech Binotti, Alexander Buchberger, Jörg Vogel, Utz Fischer.
      In all domains of life, mechanisms exist that adjust translational capacity to nutrient restriction and other growth constraints. The mammalian target of rapamycin (mTOR) regulates the synthesis of ribosomal proteins and translation factors in mammalian cells via phosphorylation of the La-related protein 1 (LARP1). In the present model of starvation-induced translational silencing, LARP1 targets mRNAs carrying a 5' terminal oligopyrimidine (5'TOP) motif to shift these into subpolysomal ribonucleoprotein particles. However, how these mRNAs would be protected from degradation and rapidly made available to restore translation capacity when needed remained enigmatic. Here, to address this, we employ gradient profiling by sequencing (Grad-seq) and monosome footprinting. Challenging the above model, we find that 5'TOP mRNAs, instead of being translationally silenced during starvation, undergo low baseline translation with reduced initiation rates. This mode of regulation ensures a stable 5'TOP mRNA population under starvation and allows fast reversibility of the translational repression.
    Keywords:  5'TOP; CP: Molecular biology; LARP1; TOP mRNAs; TOP response; baseline translation; mRNA; mTORC1; starvation; translation initiation; translation regulation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111467
  17. Front Physiol. 2022 ;13 998039
    Early enzyme replacement therapy prevents dental and craniofacial abnormalities in a mouse model of mucopolysaccharidosis type VI.
    Rohit Nagpal, Gina Georgi, Sarah Knauth, Carmen Schmid-Herrmann, Nicole Muschol, Thomas Braulke, Bärbel Kahl-Nieke, Michael Amling, Thorsten Schinke, Till Koehne, Julian Petersen.
      Mucopolysaccharidosis VI (MPS VI) is a hereditary lysosomal storage disease caused by the absence of the enzyme arylsulfatase B (ARSB). Craniofacial defects are common in MPS VI patients and manifest as abnormalities of the facial bones, teeth, and temporomandibular joints. Although enzyme replacement therapy (ERT) is the treatment of choice for MPS VI, the effects on the craniofacial and dental structures are still poorly understood. In this study, we used an Arsb-deficient mouse model (Arsb m/m ) that mimics MPS VI to investigate the effects of ERT on dental and craniofacial structures and compared these results with clinical and radiological observations from three MPS VI patients. Using micro-computed tomography, we found that the craniofacial phenotype of the Arsb m/m mice was characterized by bone exostoses at the insertion points of the masseter muscles and an overall increased volume of the jaw bone. An early start of ERT (at 4 weeks of age for 20 weeks) resulted in a moderate improvement of these jaw anomalies, while a late start of ERT (at 12 weeks of age for 12 weeks) showed no effect on the craniofacial skeleton. While teeth typically developed in Arsb m/m mice, we observed a pronounced loss of tooth-bearing alveolar bone. This alveolar bone loss, which has not been described before in MPS VI, was also observed in one of the MPS VI patients. Interestingly, only an early start of ERT led to a complete normalization of the alveolar bone in Arsb m/m mice. The temporomandibular joints in Arsb m/m mice were deformed and had a porous articular surface. Histological analysis revealed a loss of physiological cartilage layering, which was also reflected in an altered proteoglycan content in the cartilage of Arsb m/m mice. These abnormalities could only be partially corrected by an early start of ERT. In conclusion, our results show that an early start of ERT in Arsb m/m mice achieves the best therapeutic effects for tooth, bone, and temporomandibular joint development. As the MPS VI mouse model in this study resembles the clinical findings in MPS VI patients, our results suggest enzyme replacement therapy should be started as early as possible.
    Keywords:  Arsbm/m mice; MPS VI; alveolar bone loss; condyle; craniofacial malformations
    DOI:  https://doi.org/10.3389/fphys.2022.998039
  18. Int J Mol Sci. 2022 Oct 10. pii: 12056. [Epub ahead of print]23(19):
    Decreased Prosaposin and Progranulin in the Cingulate Cortex Are Associated with Schizophrenia Pathophysiology.
    Yachao He, Xiaoqun Zhang, Ivana Flais, Per Svenningsson.
      Prosaposin (PSAP) and progranulin (PGRN) are two lysosomal proteins that interact and modulate the metabolism of lipids, particularly sphingolipids. Alterations in sphingolipid metabolism have been found in schizophrenia. Genetic associations of PSAP and PGRN with schizophrenia have been reported. To further clarify the role of PSAP and PGRN in schizophrenia, we examined PSAP and PGRN levels in postmortem cingulate cortex tissue from healthy controls along with patients who had suffered from schizophrenia, bipolar disorder, or major depressive disorder. We found that PSAP and PGRN levels are reduced specifically in schizophrenia patients. To understand the role of PSAP in the cingulate cortex, we used an AAV strategy to knock down PSAP in neurons located in this region. Neuronal PSAP knockdown led to the downregulation of neuronal PGRN levels and behavioral abnormalities. Cingulate-PSAP-deficient mice exhibited increased anxiety-like behavior and impaired prepulse inhibition, as well as intact locomotion, working memory, and a depression-like state. The behavioral changes were accompanied by increased early growth response protein 1 (EGR-1) and activity-dependent cytoskeleton-associated protein (ARC) levels in the sensorimotor cortex and hippocampus, regions implicated in circuitry dysfunction in schizophrenia. In conclusion, PSAP and PGRN downregulation in the cingulate cortex is associated with schizophrenia pathophysiology.
    Keywords:  cingulate cortex; progranulin; prosaposin; schizophrenia; sphingolipids
    DOI:  https://doi.org/10.3390/ijms231912056
  19. Nat Struct Mol Biol. 2022 Oct;29(10): 955
    A quick fix for lysosomes.
    Katharine H Wrighton.
      
    DOI:  https://doi.org/10.1038/s41594-022-00847-y
  20. J Biol Chem. 2022 Oct 08. pii: S0021-9258(22)01024-9. [Epub ahead of print] 102580
    Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death.
    Shikshya Shrestha, Elio Adib, Jewel Imani, Dean J Aguiar, Anthony M Lamattina, Dereje D Tassew, Elizabeth P Henske, Mark A Perrella, Carmen Priolo, Souheil El-Chemaly.
      Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth-muscle-like "LAM" cells in the lungs. LAM cells bear loss-of-function mutations in TSC1 and/or TSC2 genes resulting in hyperactivation of the proliferation promoting mTORC1 pathway. In addition, an active renin-angiotensin system specific to LAM has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show treatment of these cells with AGTR1 inhibitor losartan, or silencing of the Agtr1 gene leads to increased cell death in vitro and attenuates tumor progression in vivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increase in expression of klotho, a transmembrane protein with a cleavable extracellular domain. In TSC2-deficient cells, we showe overexpression of Klotho or treatment with recombinant (soluble) Klotho (sKlotho) mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1-inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the renin-angiotensin system in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.
    Keywords:  Agtr1; Angiotensin II; Klotho; LAM; cell death; losartan; receptor; signaling; tuberin
    DOI:  https://doi.org/10.1016/j.jbc.2022.102580
  21. Biomark Insights. 2022 ;17 11772719221107765
    Glycerophosphoinositol is Elevated in Blood Samples From CLN3 Δex7-8 pigs, Cln3 Δex7-8 Mice, and CLN3-Affected Individuals.
    Jon J Brudvig, Vicki J Swier, Tyler B Johnson, Jacob C Cain, Melissa Pratt, Mitch Rechtzigel, Hannah Leppert, An N Dang Do, Forbes D Porter, Jill M Weimer.
       Introduction: CLN3 Batten disease is a rare pediatric neurodegenerative lysosomal disorder caused by biallelic disease-associated variants in CLN3. Despite decades of intense research, specific biofluid biomarkers of disease status have not been reported, hindering clinical development of therapies. Thus, we sought to determine whether individuals with CLN3 Batten disease have elevated levels of specific metabolites in blood.
    Methods: We performed an exhaustive metabolomic screen using serum samples from a novel minipig model of CLN3 Batten disease and validated findings in CLN3 pig serum and CSF and Cln3 mouse serum. We further validate biomarker candidates with a retrospective analysis of plasma and CSF samples collected from participants in a natural history study. Plasma samples were evaluated from 22 phenotyped individuals with CLN3 disease, 15 heterozygous carriers, and 6 non-affected non-carriers (NANC).
    Results: CLN3 pig serum samples from 4 ages exhibited large elevations in 4 glycerophosphodiester species: glycerophosphoinositol (GPI), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), and glycerophosphoserine (GPS). GPI and GPE exhibited the largest elevations, with similar elevations found in CLN3 pig CSF and Cln3 mouse serum. In plasma samples from individuals with CLN3 disease, glycerophosphoethanolamine and glycerophosphoinositol were significantly elevated with glycerophosphoinositol exhibiting the clearest separation (mean 0.1338 vs 0.04401 nmol/mL for non-affected non-carriers). Glycerophosphoinositol demonstrated excellent sensitivity and specificity as a biomarker, with a receiver operating characteristic area under the curve of 0.9848 (P = .0003).
    Conclusions: GPE and GPI could have utility as biomarkers of CLN3 disease status. GPI, in particular, shows consistent elevations across a diverse cohort of individuals with CLN3. This raises the potential to use these biomarkers as a blood-based diagnostic test or as an efficacy measure for disease-modifying therapies.
    Keywords:  Biomarkers; lipid metabolism; metabolomics; neuronal ceroid-lipofuscinoses
    DOI:  https://doi.org/10.1177/11772719221107765
  22. PLoS Genet. 2022 Oct 10. 18(10): e1010446
    A two-tiered system for selective receptor and transporter protein degradation.
    Charlotte Kathleen Golden, Thomas David Daniel Kazmirchuk, Erin Kate McNally, Mariyam El Eissawi, Zeynep Derin Gokbayrak, Joël Denis Richard, Christopher Leonard Brett.
      Diverse physiology relies on receptor and transporter protein down-regulation and degradation mediated by ESCRTs. Loss-of-function mutations in human ESCRT genes linked to cancers and neurological disorders are thought to block this process. However, when homologous mutations are introduced into model organisms, cells thrive and degradation persists, suggesting other mechanisms compensate. To better understand this secondary process, we studied degradation of transporter (Mup1) or receptor (Ste3) proteins when ESCRT genes (VPS27, VPS36) are deleted in Saccharomyces cerevisiae using live-cell imaging and organelle biochemistry. We find that endocytosis remains intact, but internalized proteins aberrantly accumulate on vacuolar lysosome membranes within cells. Here they are sorted for degradation by the intralumenal fragment (ILF) pathway, constitutively or when triggered by substrates, misfolding or TOR activation in vivo and in vitro. Thus, the ILF pathway functions as fail-safe layer of defense when ESCRTs disregard their clients, representing a two-tiered system that ensures degradation of surface polytopic proteins.
    DOI:  https://doi.org/10.1371/journal.pgen.1010446
  23. J Cell Biol. 2023 Jan 02. pii: e202112108. [Epub ahead of print]222(1):
    The GARP complex prevents sterol accumulation at the trans-Golgi network during dendrite remodeling.
    Caitlin E O'Brien, Susan H Younger, Lily Yeh Jan, Yuh Nung Jan.
      Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, we find that both complexes are required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, we investigated genes that regulate sterols and related lipids at the TGN. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.
    DOI:  https://doi.org/10.1083/jcb.202112108
  24. J Inherit Metab Dis. 2022 Oct 11.
    Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.
    Takashi Miyajima, Ryo Saito, Hiroko Yanagisawa, Miki Igarashi, Chen Wu, Takeo Iwamoto, Yoshikatsu Eto.
      Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features. This article is protected by copyright. All rights reserved.
    Keywords:  Fabry disease; Globotriaosylceramide; Induced pluripotent stem cell; Lysosomal storage disorder; Neuronal cells
    DOI:  https://doi.org/10.1002/jimd.12567
  25. Mol Neurobiol. 2022 Oct 13.
    mTORC1-Dependent and GSDMD-Mediated Pyroptosis in Developmental Sevoflurane Neurotoxicity.
    Wang Wen-Yuan, Yi Wan-Qing, Hu Qi-Yun, Liu Yu-Si, Qian Shao-Jie, Liu Jin-Tao, Mao Hui, Cai Fang, Yang Hui-Ling.
      Developmental sevoflurane exposure leads to neuronal cell death, and subsequent learning and memory cognitive defects. The underlyi\ng mechanism remains to be elucidated. Gasdermin D (GSDMD)-mediated pyroptosis is a form of inflammatory cell death and participates in a variety of neurodegenerative diseases. Several studies illustrated that dysregulation of mTOR activity is involved in pyroptotic cell death. The current study was designed to interrogate the role of GSDMD-mediated pyroptosis and mTOR activity in developmental sevoflurane exposure. We found that inhibition of GSDMD pore formation with Disulfiram (DSF) or Necrosulfonamide (NSA) significantly attenuated sevoflurane neurotoxicity in vitro. In addition, treatment with DSF or NSA also mitigated damage-associated molecular patterns (DAMPs) release and subsequent plasma membrane rupture (PMR) induced by sevoflurane challenge. Further investigation showed that the overactivation of mTOR signaling is involved in sevoflurane induced pyroptosis both in vivo and in vitro. Intriguingly, we found that the DAMPs release and subsequent PMR triggered by developmental sevoflurane priming were compromised by knocking down the expression of mTORC1 component Raptor, but not mTORC2 component Rictor. Moreover, sevoflurane induced pyroptosis could also be restored by suppressing mTOR activity or knocking down the expressions of Ras-related small GTPases RagA or RagC. Finally, administration of DSF or NSA dramatically improved the spatial and emotional cognitive disorders without alternation of locomotor activity. Taken together, these results indicate that mTORC1-dependent and GSDMD-mediated pyroptosis contributes to the developmental sevoflurane neurotoxicity. Characterizing these processes may provide experimental evidence for the possible prevention of developmental sevoflurane neurotoxicity.
    Keywords:  Cognitive function; Developing brain; GSDMD; Pyroptosis; Sevoflurane; mTOR signaling
    DOI:  https://doi.org/10.1007/s12035-022-03070-4
  26. Traffic. 2022 Oct 12.
    An AP-3 dependent pathway directs phagosome fusion with Rab8 and Rab11 vesicles involved in TLR2 signaling.
    Tanja Petnicki-Ocwieja, Bijaya Sharma, Urmila Powale, Devesh Pathak, Shumin Tan, Linden T Hu.
      Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The Toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule Adaptor Protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors such as MyD88, TRAM and TAK1, in an AP-3 dependent manner. Our data supports a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.
    Keywords:  Adaptor Protein 3; Borrelia burgdorferi; Inflammation; Lyme disease; MyD88; Phagosomal trafficking; Rab11; Rab8; TAK1; TRAM; Toll-like receptor 2
    DOI:  https://doi.org/10.1111/tra.12870
  27. Cancers (Basel). 2022 Oct 05. pii: 4858. [Epub ahead of print]14(19):
    Potential Role of Sphingolipidoses-Associated Lysosphingolipids in Cancer.
    Patricia Dubot, Leonardo Astudillo, Nicole Therville, Lorry Carrié, Magali Pettazzoni, David Cheillan, Jérôme Stirnemann, Thierry Levade, Nathalie Andrieu-Abadie, Frédérique Sabourdy.
      Sphingolipids play a key structural role in cellular membranes and/or act as signaling molecules. Inherited defects of their catabolism lead to lysosomal storage diseases called sphingolipidoses. Although progress has been made toward a better understanding of their pathophysiology, several issues still remain unsolved. In particular, whether lysosphingolipids, the deacylated form of sphingolipids, both of which accumulate in these diseases, are simple biomarkers or play an instrumental role is unclear. In the meanwhile, evidence has been provided for a high risk of developing malignancies in patients affected with Gaucher disease, the most common sphingolipidosis. This article aims at analyzing the potential involvement of lysosphingolipids in cancer. Knowledge about lysosphingolipids in the context of lysosomal storage diseases is summarized. Available data on the nature and prevalence of cancers in patients affected with sphingolipidoses are also reviewed. Then, studies investigating the biological effects of lysosphingolipids toward pro or antitumor pathways are discussed. Finally, original findings exploring the role of glucosylsphingosine in the development of melanoma are presented. While this lysosphingolipid may behave like a protumorigenic agent, further investigations in appropriate models are needed to elucidate the role of these peculiar lipids, not only in sphingolipidoses but also in malignant diseases in general.
    Keywords:  Gaucher disease; cancer; glucosylsphingosine; lysoGb3; lysosulfatide; melanoma; psychosine; sphingolipid; sphingosylphosphocholine
    DOI:  https://doi.org/10.3390/cancers14194858
  28. Nat Metab. 2022 Oct 10.
    The aldolase inhibitor aldometanib mimics glucose starvation to activate lysosomal AMPK.
    Chen-Song Zhang, Mengqi Li, Yu Wang, Xiaoyang Li, Yue Zong, Shating Long, Mingliang Zhang, Jin-Wei Feng, Xiaoyan Wei, Yan-Hui Liu, Baoding Zhang, Jianfeng Wu, Cixiong Zhang, Wenhua Lian, Teng Ma, Xiao Tian, Qi Qu, Yaxin Yu, Jinye Xiong, Dong-Tai Liu, Zhenhua Wu, Mingxia Zhu, Changchuan Xie, Yaying Wu, Zheni Xu, Chunyan Yang, Junjie Chen, Guohong Huang, Qingxia He, Xi Huang, Lei Zhang, Xiufeng Sun, Qingfeng Liu, Abdul Ghafoor, Fu Gui, Kaili Zheng, Wen Wang, Zhi-Chao Wang, Yong Yu, Qingliang Zhao, Shu-Yong Lin, Zhi-Xin Wang, Hai-Long Piao, Xianming Deng, Sheng-Cai Lin.
      The activity of 5'-adenosine monophosphate-activated protein kinase (AMPK) is inversely correlated with the cellular availability of glucose. When glucose levels are low, the glycolytic enzyme aldolase is not bound to fructose-1,6-bisphosphate (FBP) and, instead, signals to activate lysosomal AMPK. Here, we show that blocking FBP binding to aldolase with the small molecule aldometanib selectively activates the lysosomal pool of AMPK and has beneficial metabolic effects in rodents. We identify aldometanib in a screen for aldolase inhibitors and show that it prevents FBP from binding to v-ATPase-associated aldolase and activates lysosomal AMPK, thereby mimicking a cellular state of glucose starvation. In male mice, aldometanib elicits an insulin-independent glucose-lowering effect, without causing hypoglycaemia. Aldometanib also alleviates fatty liver and nonalcoholic steatohepatitis in obese male rodents. Moreover, aldometanib extends lifespan and healthspan in both Caenorhabditis elegans and mice. Taken together, aldometanib mimics and adopts the lysosomal AMPK activation pathway associated with glucose starvation to exert physiological roles, and might have potential as a therapeutic for metabolic disorders in humans.
    DOI:  https://doi.org/10.1038/s42255-022-00640-7
  29. Nat Commun. 2022 Oct 12. 13(1): 6023
    Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.
    Joshua J Rennick, Cameron J Nowell, Colin W Pouton, Angus P R Johnston.
      Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in individual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.
    DOI:  https://doi.org/10.1038/s41467-022-33348-z
  30. Cell Death Dis. 2022 Oct 11. 13(10): 862
    mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.
    Shuo Wan, Yadong Sun, Jiamin Fu, Hongrui Song, Zhiqiang Xiao, Quanli Yang, Sanfeng Wang, Gongwang Yu, Peiran Feng, Wenkai Lv, Liang Luo, Zerong Guan, Feng Liu, Qinghua Zhou, Zhinan Yin, Meixiang Yang.
      The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen. Estrogen acts as an activator of mTOR signaling but its role in vaginal epithelial homeostasis is unknown. We analyzed reproductive tract-specific Rptor or Rictor conditional knockout mice to reveal the role of mTOR signaling in estrogen-dependent vaginal epithelial cell proliferation and differentiation. Loss of Rptor but not Rictor in the vagina resulted in an aberrant proliferation of epithelial cells and failure of keratinized differentiation. As gene expression analysis indicated, several estrogen-mediated genes, including Pgr and Ereg (EGF-like growth factor) were not induced by estrogen in Rptor cKO mouse vagina. Moreover, supplementation of EREG could activate the proliferation and survival of vaginal epithelial cells through YAP1 in the absence of Rptor. Thus, mTORC1 signaling integrates estrogen and growth factor signaling to mediate vaginal epithelial cell proliferation and differentiation, providing new insights into vaginal atrophy treatment for post-menopausal women.
    DOI:  https://doi.org/10.1038/s41419-022-05293-8
  31. Clin Med (Lond). 2022 Jul;22(Suppl 4): 48-49
    Systematic review of mTOR inhibitor treatment, biomarkers and prophylaxis for tuberous sclerosis complex-associated seizures.
    Jingwen Zhang.
      
    DOI:  https://doi.org/10.7861/clinmed.22-4-s48
  32. Int J Mol Sci. 2022 Sep 29. pii: 11517. [Epub ahead of print]23(19):
    Axenic Culture of Caenorhabditis elegans Alters Lysosomal/Proteasomal Balance and Increases Neuropeptide Expression.
    Huaihan Cai, Ping Wu, Lieselot Vandemeulebroucke, Ineke Dhondt, Madina Rasulova, Andy Vierstraete, Bart P Braeckman.
      Axenically cultured C. elegans show many characteristic traits of worms subjected to dietary restriction, such as slowed development, reduced fertility, and increased stress resistance. Hence, the term axenic dietary restriction (ADR) is often applied. ADR dramatically extends the worm lifespan compared to other DR regimens such as bacterial dilution. However, the underlying molecular mechanisms still remain unclear. The primary goal of this study is to comprehensively investigate transcriptional alterations that occur when worms are subjected to ADR and to estimate the molecular and physiological changes that may underlie ADR-induced longevity. One of the most enriched clusters of up-regulated genes under ADR conditions is linked to lysosomal activity, while proteasomal genes are significantly down-regulated. The up-regulation of genes specifically involved in amino acid metabolism is likely a response to the high peptide levels found in axenic culture medium. Genes related to the integrity and function of muscles and the extracellular matrix are also up-regulated. Consistent down-regulation of genes involved in DNA replication and repair may reflect the reduced fertility phenotype of ADR worms. Neuropeptide genes are found to be largely up-regulated, suggesting a possible involvement of neuroendocrinal signaling in ADR-induced longevity. In conclusion, axenically cultured worms seem to rely on increased amino acid catabolism, relocate protein breakdown from the cytosol to the lysosomes, and do not invest in DNA maintenance but rather retain muscle integrity and the extracellular matrix. All these changes may be coordinated by peptidergic signaling.
    Keywords:  C. elegans; axenic dietary restriction; lifespan extension; transcriptomics
    DOI:  https://doi.org/10.3390/ijms231911517
  33. Neurobiol Dis. 2022 Oct 08. pii: S0969-9961(22)00283-2. [Epub ahead of print] 105891
    GRN-/- iPSC-derived cortical neurons recapitulate the pathological findings of both frontotemporal lobar degeneration and neuronal ceroidolipofuscinosis.
    Patrizia Bossolasco, Sara Cimini, Emanuela Maderna, Donatella Bardelli, Laura Canafoglia, Tiziana Cavallaro, Martina Ricci, Vincenzo Silani, Gianluca Marucci, Giacomina Rossi.
      Heterozygous mutations in the gene coding for progranulin (GRN) cause frontotemporal lobar degeneration (FTLD) while homozygous mutations are linked to neuronal ceroidolipofuscinosis (NCL). While both FTLD/NCL pathological hallmarks were mostly investigated in heterozygous GRN+/- brain tissue or iPSC-derived neurons, data from homozygous GRN-/- condition are scarce, being limited to a postmortem brain tissue from a single case. Indeed, homozygous GRN-/- is an extremely rare condition reported in very few cases. Our aim was to investigate pathological phenotypes associated to both FTLD and NCL in iPSC-derived cortical neurons from a GRN-/- patient affected by NCL. iPSCs were generated from peripheral blood of a GRN wt healthy donor and a GRN-/- patient and subsequently differentiated into cortical neurons. Several pathological changes were investigated, by means of immunocytochemical, biochemical and ultrastructural analyses. GRN-/- patient-derived cortical neurons displayed both TDP-43 and phospho-TDP-43 mislocalization, enlarged autofluorescent lysosomes and electron-dense vesicles containing storage material with granular, curvilinear and fingerprints profiles. In addition, different patterns in the expression of TDP-43, caspase 3 and cleaved caspase 3 were observed by biochemical analysis at different time points of cortical differentiation. At variance with previous findings, the present data highlight the existence of both FTLD- and NCL-linked pathological features in GRN-/- iPSC-derived cortical neurons from a NCL patient. They also suggest an evolution in the appearance of these features: firstly, FTLD-related TDP-43 alterations and initial NCL storage materials were detected; afterwards, mainly well-shaped NCL storage materials were present, while some FTLD features were not observed anymore.
    Keywords:  Cortical neurons; Fingerprints; Frontotemporal lobar degeneration; Induced pluripotent stem cells; Lysosomes; Neuronal ceroidolipofuscinosis; Progranulin; TDP-43
    DOI:  https://doi.org/10.1016/j.nbd.2022.105891
  34. Biochem Pharmacol. 2022 Oct 11. pii: S0006-2952(22)00390-2. [Epub ahead of print] 115296
    Regulation of membrane phospholipid biosynthesis in mammalian cells.
    Shin-Ya Morita, Yoshito Ikeda.
      In mammalian cells, phospholipids and cholesterol are assembled into bilayer membranes forming the plasma membrane, nuclear envelope, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and endosomes. Phospholipids are divided into classes based on the molecular structures, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, cardiolipin, and sphingomyelin. In addition to their structural roles, phospholipids play important roles in many cellular processes, such as membrane protein regulation, membrane trafficking, cell growth, apoptosis, and intracellular signaling. Thus, abnormal phospholipid metabolism is associated with various diseases. In mammalian cells, phospholipid classes are generated through several enzymatic steps, predominantly in the endoplasmic reticulum, mitochondria, and Golgi apparatus. In recent years, various enzymes involved in the biosynthesis of phospholipid classes have been identified. However, little is known about the regulatory mechanisms underlying the biosynthesis of phospholipid classes. Using our recently developed enzymatic fluorometric assays for all major phospholipid classes, we have demonstrated changes in phospholipid composition in intracellular organelles during cell growth. In this review, we summarize the current understanding of the properties and functions of phospholipid biosynthesis enzymes, and discuss their regulatory mechanisms.
    Keywords:  Cell membrane; Enzyme; Intracellular organelle; Phospholipid biosynthesis
    DOI:  https://doi.org/10.1016/j.bcp.2022.115296
  35. Autophagy. 2022 Oct 10. 1-15
    Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.
    Kihyoun Park, Seong Keun Sonn, Seungwoon Seo, Jinyoung Kim, Kyu Yeon Hur, Goo Taeg Oh, Myung-Shik Lee.
      Macroautophagy/autophagy or mitophagy plays crucial roles in the maintenance of pancreatic β-cell function. PPP3/calcineurin can modulate the activity of TFEB, a master regulator of lysosomal biogenesis and autophagy gene expression, through dephosphorylation. We studied whether PPP3/calcineurin inhibitors can affect the mitophagy of pancreatic β-cells and pancreatic β-cell function employing FK506, an immunosuppressive drug against graft rejection. FK506 suppressed rotenone- or oligomycin+antimycin-A-induced mitophagy measured by Mito-Keima localization in acidic lysosomes or RFP-LC3 puncta colocalized with TOMM20 in INS-1 insulinoma cells. FK506 diminished nuclear translocation of TFEB after treatment with rotenone or oligomycin+antimycin A. Forced TFEB nuclear translocation by a constitutively active TFEB mutant transfection restored impaired mitophagy by FK506, suggesting the role of decreased TFEB nuclear translocation in FK506-mediated mitophagy impairment. Probably due to reduced mitophagy, recovery of mitochondrial potential or quenching of mitochondrial ROS after removal of rotenone or oligomycin+antimycin A was delayed by FK506. Mitochondrial oxygen consumption was reduced by FK506, indicating reduced mitochondrial function by FK506. Likely due to mitochondrial dysfunction, insulin release from INS-1 cells was reduced by FK506 in vitro. FK506 treatment also reduced insulin release and impaired glucose tolerance in vivo, which was associated with decreased mitophagy and mitochondrial COX activity in pancreatic islets. FK506-induced mitochondrial dysfunction and glucose intolerance were ameliorated by an autophagy enhancer activating TFEB. These results suggest that diminished mitophagy and consequent mitochondrial dysfunction of pancreatic β-cells contribute to FK506-induced β-cell dysfunction or glucose intolerance, and autophagy enhancement could be a therapeutic modality against post-transplantation diabetes mellitus caused by PPP3/calcineurin inhibitors.
    Keywords:  Calcineurin; TFEB; mitophagy; pancreatic β-cell; post-transplantation diabetes mellitus
    DOI:  https://doi.org/10.1080/15548627.2022.2132686
  36. PLoS Genet. 2022 Oct;18(10): e1010431
    The entry of unclosed autophagosomes into vacuoles and its physiological relevance.
    Zulin Wu, Haiqian Xu, Pei Wang, Ling Liu, Juan Cai, Yun Chen, Xiaomin Zhao, Xia You, Junze Liu, Xiangrui Guo, Tingting Xie, Jiajie Feng, Fan Zhou, Rui Li, Zhiping Xie, Yanhong Xue, Chuanhai Fu, Yongheng Liang.
      It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.
    DOI:  https://doi.org/10.1371/journal.pgen.1010431
  37. EMBO Rep. 2022 Oct 10. e53065
    Lipid and protein content profiling of isolated native autophagic vesicles.
    Daniel Schmitt, Süleyman Bozkurt, Pascale Henning-Domres, Heike Huesmann, Stefan Eimer, Laura Bindila, Christian Behrends, Emily Boyle, Florian Wilfling, Georg Tascher, Christian Münch, Christian Behl, Andreas Kern.
      Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.
    Keywords:  autophagic vesicles; autophagy; cargo profiling; lipid profiling; vesicle isolation
    DOI:  https://doi.org/10.15252/embr.202153065
  38. Chem Soc Rev. 2022 Oct 11.
    Emerging degrader technologies engaging lysosomal pathways.
    Yu Ding, Dong Xing, Yiyan Fei, Boxun Lu.
      Targeted protein degradation (TPD) provides unprecedented opportunities for drug discovery. While the proteolysis-targeting chimera (PROTAC) technology has already entered clinical trials and changed the landscape of small-molecule drugs, new degrader technologies harnessing alternative degradation machineries, especially lysosomal pathways, have emerged and broadened the spectrum of degradable targets. We have recently proposed the concept of autophagy-tethering compounds (ATTECs) that hijack the autophagy protein microtubule-associated protein 1A/1B light chain 3 (LC3) for targeted degradation. Other groups also reported degrader technologies engaging lysosomal pathways through different mechanisms including AUTACs, AUTOTACs, LYTACs and MoDE-As. In this review, we analyse and discuss ATTECs along with other lysosomal-relevant degrader technologies. Finally, we will briefly summarize the current status of these degrader technologies and envision possible future studies.
    DOI:  https://doi.org/10.1039/d2cs00624c
  39. Nat Commun. 2022 Oct 13. 13(1): 6045
    Reducing Akt2 in retinal pigment epithelial cells causes a compensatory increase in Akt1 and attenuates diabetic retinopathy.
    Haitao Liu, Nadezda A Stepicheva, Sayan Ghosh, Peng Shang, Olivia Chowdhury, Rachel A Daley, Meysam Yazdankhah, Urvi Gupta, Stacey L Hose, Mallika Valapala, Christopher Scott Fitting, Anastasia Strizhakova, Yang Shan, Derrick Feenstra, José-Alain Sahel, Ashwath Jayagopal, James T Handa, J Samuel Zigler, Patrice E Fort, Akrit Sodhi, Debasish Sinha.
      The retinal pigment epithelium (RPE) plays an important role in the development of diabetic retinopathy (DR), a leading cause of blindness worldwide. Here we set out to explore the role of Akt2 signaling-integral to both RPE homeostasis and glucose metabolism-to DR. Using human tissue and genetically manipulated mice (including RPE-specific conditional knockout (cKO) and knock-in (KI) mice), we investigate whether Akts in the RPE influences DR in models of diabetic eye disease. We found that Akt1 and Akt2 activities were reciprocally regulated in the RPE of DR donor tissue and diabetic mice. Akt2 cKO attenuated diabetes-induced retinal abnormalities through a compensatory upregulation of phospho-Akt1 leading to an inhibition of vascular injury, inflammatory cytokine release, and infiltration of immune cells mediated by the GSK3β/NF-κB signaling pathway; overexpression of Akt2 has no effect. We propose that targeting Akt1 activity in the RPE may be a novel therapy for treating DR.
    DOI:  https://doi.org/10.1038/s41467-022-33773-0
  40. Front Mol Biosci. 2022 ;9 959737
    Endocytosis at extremes: Formation and internalization of giant clathrin-coated pits under elevated membrane tension.
    Ahmet Ata Akatay, Tianyao Wu, Umidahan Djakbarova, Cristopher Thompson, Emanuele Cocucci, Roya Zandi, Joseph Rudnick, Comert Kural.
      Internalization of clathrin-coated vesicles from the plasma membrane constitutes the major endocytic route for receptors and their ligands. Dynamic and structural properties of endocytic clathrin coats are regulated by the mechanical properties of the plasma membrane. Here, we used conventional fluorescence imaging and multiple modes of structured illumination microscopy (SIM) to image formation of endocytic clathrin coats within live cells and tissues of developing fruit fly embryos. High resolution in both spatial and temporal domains allowed us to detect and characterize distinct classes of clathrin-coated structures. Aside from the clathrin pits and plaques detected in distinct embryonic tissues, we report, for the first time, formation of giant coated pits (GCPs) that can be up to two orders of magnitude larger than the canonical pits. In cultured cells, we show that GCP formation is induced by increased membrane tension. GCPs take longer to grow but their mechanism of curvature generation is the same as the canonical pits. We also demonstrate that GCPs split into smaller fragments during internalization. Considering the supporting roles played by actin filament dynamics under mechanically stringent conditions that slow down completion of clathrin coats, we suggest that local changes in the coat curvature driven by actin machinery can drive splitting and internalization of GCPs.
    Keywords:  adhesion; clathrin; embryogenesis; endocytosis; mechanobiolgy; superresolution microscopy
    DOI:  https://doi.org/10.3389/fmolb.2022.959737
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