bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒07‒19
38 papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

  1. J Cell Biol. 2020 Aug 03. pii: e201911036. [Epub ahead of print]219(8):
    Yin Q, Jian Y, Xu M, Huang X, Wang N, Liu Z, Li Q, Li J, Zhou H, Xu L, Wang Y, Yang C.
      Lysosomes are degradation and signaling organelles that adapt their biogenesis to meet many different cellular demands; however, it is unknown how lysosomes change their numbers for cell division. Here, we report that the cyclin-dependent kinases CDK4/6 regulate lysosome biogenesis during the cell cycle. Chemical or genetic inactivation of CDK4/6 increases lysosomal numbers by activating the lysosome and autophagy transcription factors TFEB and TFE3. CDK4/6 interact with and phosphorylate TFEB/TFE3 in the nucleus, thereby inactivating them by promoting their shuttling to the cytoplasm. During the cell cycle, lysosome numbers increase in S and G2/M phases when cyclin D turnover diminishes CDK4/6 activity. These findings not only uncover the molecular events that direct the nuclear export of TFEB/TFE3, but also suggest a mechanism that controls lysosome biogenesis in the cell cycle. CDK4/6 inhibitors promote autophagy and lysosome-dependent degradation, which has important implications for the therapy of cancer and lysosome-related disorders.
  2. Cell Rep. 2020 Jul 14. pii: S2211-1247(20)30876-7. [Epub ahead of print]32(2): 107895
    Zhu X, Zhang H, Mendell JT.
      Nonsense-mediated decay (NMD) is a pathway that degrades mRNAs containing premature termination codons. Here we describe a genome-wide screen for NMD factors that uncovers an unexpected mechanism that broadly governs 3' untranslated region (UTR)-directed regulation. The screen reveals that NMD requires lysosomal acidification, which allows transferrin-mediated iron uptake, which, in turn, is necessary for iron-sulfur (Fe-S) cluster biogenesis. This pathway maintains the activity of the Fe-S cluster-containing ribosome recycling factor ABCE1, whose impaired function results in movement of ribosomes into 3' UTRs, where they displace exon junction complexes, abrogating NMD. Importantly, these effects extend beyond NMD substrates, with ABCE1 activity required to maintain the accessibility of 3' UTRs to diverse regulators, including microRNAs and RNA binding proteins. Because of the sensitivity of the Fe-S cluster of ABCE1 to iron availability and reactive oxygen species, these findings reveal an unanticipated vulnerability of 3' UTR-directed regulation to lysosomal dysfunction, iron deficiency, and oxidative stress.
    Keywords:  3' UTR; 3' untranslated region; ABCE1; iron homeostasis; iron-sulfur cluster; lysosome; nonsense-mediated decay; post-transcriptional regulation
  3. Nat Commun. 2020 Jul 17. 11(1): 3612
    Wang Y, Gunewardena S, Li F, Matye DJ, Chen C, Chao X, Jung T, Zhang Y, Czerwiński M, Ni HM, Ding WX, Li T.
      Bile acid synthesis plays a key role in regulating whole body cholesterol homeostasis. Transcriptional factor EB (TFEB) is a nutrient and stress-sensing transcriptional factor that promotes lysosomal biogenesis. Here we report a role of TFEB in regulating hepatic bile acid synthesis. We show that TFEB induces cholesterol 7α-hydroxylase (CYP7A1) in human hepatocytes and mouse livers and prevents hepatic cholesterol accumulation and hypercholesterolemia in Western diet-fed mice. Furthermore, we find that cholesterol-induced lysosomal stress feed-forward activates TFEB via promoting TFEB nuclear translocation, while bile acid-induced fibroblast growth factor 19 (FGF19), acting via mTOR/ERK signaling and TFEB phosphorylation, feedback inhibits TFEB nuclear translocation in hepatocytes. Consistently, blocking intestinal bile acid uptake by an apical sodium-bile acid transporter (ASBT) inhibitor decreases ileal FGF15, enhances hepatic TFEB nuclear localization and improves cholesterol homeostasis in Western diet-fed mice. This study has identified a TFEB-mediated gut-liver signaling axis that regulates hepatic cholesterol and bile acid homeostasis.
  4. Front Cell Dev Biol. 2020 ;8 510
    Banerjee S, Kane PM.
      Luminal pH and the distinctive distribution of phosphatidylinositol phosphate (PIP) lipids are central identifying features of organelles in all eukaryotic cells that are also critical for organelle function. V-ATPases are conserved proton pumps that populate and acidify multiple organelles of the secretory and the endocytic pathway. Complete loss of V-ATPase activity causes embryonic lethality in higher animals and conditional lethality in yeast, while partial loss of V-ATPase function is associated with multiple disease states. On the other hand, many cancer cells increase their virulence by upregulating V-ATPase expression and activity. The pH of individual organelles is tightly controlled and essential for function, but the mechanisms for compartment-specific pH regulation are not completely understood. There is substantial evidence indicating that the PIP content of membranes influences organelle pH. We present recent evidence that PIPs interact directly with subunit isoforms of the V-ATPase to dictate localization of V-ATPase subpopulations and participate in their regulation. In yeast cells, which have only one set of organelle-specific V-ATPase subunit isoforms, the Golgi-enriched lipid PI(4)P binds to the cytosolic domain of the Golgi-enriched a-subunit isoform Stv1, and loss of PI(4)P binding results in mislocalization of Stv1-containing V-ATPases from the Golgi to the vacuole/lysosome. In contrast, levels of the vacuole/lysosome-enriched signaling lipid PI(3,5)P2 affect assembly and activity of V-ATPases containing the Vph1 a-subunit isoform. Mutations in the Vph1 isoform that disrupt the lipid interaction increase sensitivity to stress. These studies have decoded "zip codes" for PIP lipids in the cytosolic N-terminal domain of the a-subunit isoforms of the yeast V-ATPase, and similar interactions between PIP lipids and the V-ATPase subunit isoforms are emerging in higher eukaryotes. In addition to direct effects on the V-ATPase, PIP lipids are also likely to affect organelle pH indirectly, through interactions with other membrane transporters. We discuss direct and indirect effects of PIP lipids on organelle pH, and the functional consequences of the interplay between PIP lipid content and organelle pH.
    Keywords:  Golgi apparatus; PIKfyve; V-ATPase; acidification; endosome; lysosome; organelle; phosphatidylinositol phosphate
  5. ACS Appl Mater Interfaces. 2020 Jul 13.
    Chang RL, Pratiwi F, Chen BC, Chen P, Wu SH, Mou CY.
      Nanoparticles (NPs)-based targeted drug delivery is intended to transport therapeutically active molecules to specific cells, and particular intracellular compartments. However, there is limited knowledge regarding the complete route of NPs in this targeting scenario. In this study, simultaneously performing motion and dynamic pH sensing using single-particle tracking (SPT) leads to an alternative method of gaining insight on the mesoporous silica nanoparticle's (MSN) journey in targeting lysosome. Two different pH-sensitive dyes and a reference dye, are incorporated into mesoporous silica nanoparticles (MSNs) via co-condensation to broaden the measurable pH range (pH 4~7.5) of nanoprobe. The phosphonate, amine, and lysosomal sorting peptides (YQRLGC) are conjugated onto MSN's surface to study intracellular nano-bio interactions of two oppositely charged- and lysosome targetable-MSN, respectively. The brightness and stability of these MSNs allow their movement and dynamic pH evolution during their journey to be simultaneously monitored in real-time. Importantly, a multidimensional analysis of MSN's movement and local-pH have revealed a new model intracellular dynamic states and distributions of MSNs, previously inaccessible when using single parameters alone. A key result is that YQRLGC-conjugated MSNs took an alternative route to target lysosomes apart from the traditional one, which sped up to 4h and enhanced their targeting efficiency (up to 32%). The findings enrich our understanding of the intracellular journey of MSNs. This study offers complementary information on correlating the surface design with the full pathway of nanoparticles to achieve targeted delivery of therapeutic payload.
  6. Am J Pathol. 2020 Jul 14. pii: S0002-9440(20)30336-9. [Epub ahead of print]
    Zhou K, Dichlberger A, Ikonen E, Blom T.
      Studies of Lysosome Associated Protein Transmembrane 4B (LAPTM4B) have mainly focused on the 35 kDa isoform and its association with poor prognosis in cancers. Here, by employing a novel monoclonal antibody we found that the 24 kDa LAPTM4B isoform predominated in most, both healthy and malignant, human cells and tissues studied. LAPTM4B-24 lacks the extreme N-terminus and contrary to LAPTM4B-35, failed to promote cell migration. The endogenous LAPTM4B-24 protein was subject to rapid turnover with a t1/2 ∼1 h. The protein was degraded by both lysosomal and proteasomal pathways and its levels were increased by the availability of nutrients and lysosomal ceramide. These findings underscore the pathophysiological relevance of the LAPTM4B-24 isoform and identify it as a dynamically regulated effector in lysosomal nutrient signaling.
    Keywords:  Ceramide; LAPTM4B-24; Nutrient signaling; Protein turnover
  7. Bioorg Chem. 2020 Jun 25. pii: S0045-2068(20)31337-7. [Epub ahead of print]102 104040
    Bertman KA, Abeywickrama CS, Pang Y.
      A bright far-red emitting flavonoid derivative (FuraET) was synthesized in good yields by inserting a π extension group (i.e., furan) into the flavonoid skeleton, via using the Suzuki-Miyaura cross-coupling reaction. FuaraET exhibited optical absorption at λab ≈ 450 nm and emission λem ≈ 660 nm by recognizing as the first far-red emitting flavonoid derivative reported. FuraET exhibited a large Stokes shift (Δλ > 150 nm) high fluorescent quantum yield (φfl ≈ 0.2-0.4), and good photostability indicating excellent characteristics for an imaging probe. Live cell fluorescent confocal microscopy imaging revealed the exceptional selectivity of the FuraET towards cellular lysosomes (Mander's overlap coefficients >0.9). The observed non-alkalinizing nature and high biocompatibility (LC50 > 50 µM) suggested that FuraET can a reliable lysosome marker for live cell imaging experiments. Our further study also indicated that FuraET may likely internalized into hydrophobic regions of the cellular lysosomes in contrast to acidic lysosomal lumen.
    Keywords:  Biocompatibility; Flavonoid derivative; Fluorescence confocal microscopy; Fluorescent probes; Large Stokes shift; Lysosome probes
  8. Cancers (Basel). 2020 Jul 13. pii: E1877. [Epub ahead of print]12(7):
    Zeng C, Riad A, Mach RH.
      The sigma-2 receptor was originally defined pharmacologically and recently identified as TMEM97. TMEM97 has been validated as a biomarker of proliferative status and the radioligand of TMEM97, [18F]ISO-1, has been developed and validated as a PET imaging biomarker of proliferative status of tumors and as a predictor of the cancer therapy response. [18F]ISO-1 PET imaging should be useful to guide treatment for cancer patients. TMEM97 is a membrane-bound protein and localizes in multiple subcellular organelles including endoplasmic reticulum and lysosomes. TMEM97 plays distinct roles in cancer. It is reported that TMEM97 is upregulated in some tumors but downregulated in other tumors and it is required for cell proliferation in certain tumor cells. TMEM97 plays important roles in cholesterol homeostasis. TMEM97 expression is regulated by cholesterol-regulating signals such as sterol depletion and SREBP expression levels. TMEM97 regulates cholesterol trafficking processes such as low density lipoprotein (LDL) uptake by forming complexes with PGRMC1 and low density lipoprotein receptor (LDLR), as well as cholesterol transport out of lysosome by interacting with and regulating NPC1 protein. Understanding molecular functions of TMEM97 in proliferation and cholesterol metabolism will be important to develop strategies to diagnose and treat cancer and cholesterol disorders using a rich collection of TMEM97 radiotracers and ligands.
    Keywords:  LDLR; MAC30; PGRMC1; TMEM97; [18F]ISO-1; cancer; cholesterol; sigma-2 receptors
  9. PLoS Pathog. 2020 Jul 13. 16(7): e1008220
    Kehl A, Göser V, Reuter T, Liss V, Franke M, John C, Richter CP, Deiwick J, Hensel M.
      The intracellular lifestyle of Salmonella enterica is characterized by the formation of a replication-permissive membrane-bound niche, the Salmonella-containing vacuole (SCV). As a further consequence of the massive remodeling of the host cell endosomal system, intracellular Salmonella establish a unique network of various Salmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, the Salmonella-induced filaments (SIF), is rather well-defined. However, the corresponding host cell proteins are still poorly understood. To identify host factors required for the formation SIF, we performed a sub-genomic RNAi screen. The analyses comprised high-resolution live cell imaging to score effects on SIF induction, dynamics and morphology. The hits of our functional RNAi screen comprise: i) The late endo-/lysosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, consisting of STX7, STX8, VTI1B, and VAMP7 or VAMP8, which is, in conjunction with RAB7 and the homotypic fusion and protein sorting (HOPS) tethering complex, a complete vesicle fusion machinery. ii) Novel interactions with the early secretory GTPases RAB1A and RAB1B, providing a potential link to coat protein complex I (COPI) vesicles and reinforcing recently identified ties to the endoplasmic reticulum. iii) New connections to the late secretory pathway and/or the recycling endosome via the GTPases RAB3A, RAB8A, and RAB8B and the SNAREs VAMP2, VAMP3, and VAMP4. iv) An unprecedented involvement of clathrin-coated structures. The resulting set of hits allowed us to characterize completely new host factor interactions, and to strengthen observations from several previous studies.
  10. Adv Pharm Bull. 2020 Jul;10(3): 452-457
    Keshavarz M, Farrokhi MR, Amirinezhad Fard E, Mehdipour M.
      Purpose: Memantine is an approved drug for the treatment of Alzheimer's disease (AD). Autophagy, lysosome dysfunction, and sigma receptors have possible roles in the pathophysiology of AD. Therefore, we aimed to investigate the contribution of sigma receptors and lysosome inhibition to the neuroprotective effects of memantine against amyloid-beta (Aβ)-induced neurotoxicity in SH-SY5Y cells. Methods: We determined the neuroprotective effects of memantine (2.5 µM), dizocilpine (MK801, as a selective N-methyl-D-aspartate (NMDA) receptor antagonist) (5 μM) against Aβ25- 35 (2 μg/μL)-induced neurotoxicity. We used chloroquine (10, 20, and 40 μM) as a lysosome inhibitor and BD-1063 (1, 10, and 30 μM) as a selective sigma receptor antagonist. The MTT assay was used to measure the neurotoxicity in the SH-SY5Y cells. Data were analyzed using the one-way ANOVA. Results: Memantine (2.5 µM), dizocilpine (5 µM), chloroquine (10 and 20 µM) and BD-1063 (1, 10 and 30 µM) decreased the neurotoxic effects of Aβ on the SH-SY5Y cells. However, chloroquine (40 µM) increased the neurotoxic effects of Aβ. Cell viability in the cells treated with memantine + Aβ + chloroquine (10, 20, and 40 μM) was significantly lower than the memantine + Aβ-treated group. Moreover, cell viability in the memantine + Aβ group was higher than the memantine + Aβ + BD-1063 (10 and 30 μM) groups. Conclusion: The lysosomal and sigma receptors may contribute to the neuroprotective mechanism of memantine and other NMDA receptor antagonists. Moreover, the restoration of lysosomes function and the modulation of sigma receptors are potential targets in the treatment of AD.
    Keywords:  Amyloid beta-Peptides; Lysosomes; Memantine; Neuroprotection; Sigma receptors
  11. Biochimie. 2020 Jul 10. pii: S0300-9084(20)30159-0. [Epub ahead of print]
    Rabia M, Leuzy V, Soulage C, Durand A, Fourmaux B, Errazuriz-Cerda E, Köffel R, Draeger A, Colosetti P, Jalabert A, Di Filippo M, Villard-Garon A, Bergerot C, Luquain-Costaz C, Moulin P, Rome S, Delton I, Hullin-Matsuda F.
      Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid specifically enriched in the late endosome-lysosome compartment playing a crucial role for the fate of endocytosed components. Due to its presence in extracellular fluids during diseases associated with endolysosomal dysfunction, it is considered as a possible biomarker of disorders such as genetic lysosomal storage diseases and cationic amphiphilic drug-induced phospholipidosis. However, there is no true validation of this biomarker in human studies, nor a clear identification of the carrier of this endolysosome-specific lipid in biofluids. The present study demonstrates that in absence of any sign of renal failure, BMP, especially all docosahexaenoyl containing species, are significantly increased in the urine of patients treated with the antiarrhythmic drug amiodarone. Such urinary BMP increase could reflect a generalized drug-induced perturbation of the endolysosome compartment as observed in vitro with amiodarone-treated human macrophages. Noteworthy, BMP was associated with extracellular vesicles (EVs) isolated from human urines and extracellular medium of human embryonic kidney HEK293 cells and co-localizing with classical EV protein markers CD63 and ALIX. In the context of drug-induced endolysosomal dysfunction, increased BMP-rich EV release could be useful to remove excess of undigested material. This first human pilot study not only reveals BMP as a urinary biomarker of amiodarone-induced endolysosomal dysfunction, but also highlights its utility to prove the endosomal origin of EVs, also named as exosomes. This peculiar lipid already known as a canonical late endosome-lysosome marker, may be thus considered as a new lipid marker of urinary exosomes.
    Keywords:  Bis(monoacylglycero)phosphate; Docosahexaenoic acid; Exosomes; Extracellular vesicles; Lysobisphosphatidic acid; Lysosomal storage diseases
  12. Cells. 2020 Jul 13. pii: E1679. [Epub ahead of print]9(7):
    Yadati T, Houben T, Bitorina A, Shiri-Sverdlov R.
      Cathepsins are the most abundant lysosomal proteases that are mainly found in acidic endo/lysosomal compartments where they play a vital role in intracellular protein degradation, energy metabolism, and immune responses among a host of other functions. The discovery that cathepsins are secreted and remain functionally active outside of the lysosome has caused a paradigm shift. Contemporary research has unraveled many versatile functions of cathepsins in extralysosomal locations including cytosol and extracellular space. Nevertheless, extracellular cathepsins are majorly upregulated in pathological states and are implicated in a wide range of diseases including cancer and cardiovascular diseases. Taking advantage of the differential expression of the cathepsins during pathological conditions, much research is focused on using cathepsins as diagnostic markers and therapeutic targets. A tailored therapeutic approach using selective cathepsin inhibitors is constantly emerging to be safe and efficient. Moreover, recent development of proteomic-based approaches for the identification of novel physiological substrates offers a major opportunity to understand the mechanism of cathepsin action. In this review, we summarize the available evidence regarding the role of cathepsins in health and disease, discuss their potential as biomarkers of disease progression, and shed light on the potential of extracellular cathepsin inhibitors as safe therapeutic tools.
    Keywords:  Lysosomes; cathepsins; site-specific functions; targeted-drug delivery; translocation
  13. Trends Pharmacol Sci. 2020 Aug;pii: S0165-6147(20)30138-3. [Epub ahead of print]41(8): 582-594
    Jin X, Zhang Y, Alharbi A, Hanbashi A, Alhoshani A, Parrington J.
      Two-pore channels (TPCs) are cation-permeable channels located on endolysosomal membranes and important mediators of intracellular Ca2+ signalling. TPCs are involved in various pathophysiological processes, including cell growth and development, metabolism, and cancer progression. Most studies of TPCs have used TPC-/- cell or whole-animal models, or Ned-19, an indirect inhibitor. The TPC activation mechanism remains controversial, which has made it difficult to develop selective modulators. Recent studies of TPC structure and their interactomes are aiding the development of direct pharmacological modulators. This process is still in its infancy, but will facilitate future research and TPC targeting for therapeutical purposes. Here, we review the progress of current research into TPCs, including recent insights into their structures, functional roles, mechanisms of activation, and pharmacological modulators.
    Keywords:  NAADP; calcium; cell signalling; endolysosomes; interactome; two-pore channels
  14. Adv Exp Med Biol. 2020 ;1207 87-102
    Ren H, Wang G.
      Lysosomal storage disorders (LSDs) are one of the most common human genetic metabolic diseases caused by gene mutations. Up to now, more than 70 LSDs have been identified and mainly divided into five categories. LSDs are mainly caused by defects in the function of enzymes or lysosomal-related proteins in lysosomes, which causes progressive accumulation of undigested macromolecules within the cell and results in stress and dysfunction in cells, tissues and organs. LSDs can result in multiple systemic damages, including the nervous system, skeletal system and reticuloendothelial system, especially in the early stages of the disease. The central nervous system is severely affected. Lysosome is the final degradative organelles for autophagy by which macromolecules and damaged cellular components and organelles are degraded. Impairment in autophagy is a central and common mechanism underlying many LSDs. The modulation of autophagy has been considered as novel therapeutic approach for LSDs.
    Keywords:  Autophagic flux; Autophagy; Combination therapy; Lysosomal storage disorders; Lysosome
  15. J Cell Mol Med. 2020 Jul 15.
    Choi SI, Woo JH, Kim EK.
      Granular corneal dystrophy type 2 (GCD2) is the most common form of transforming growth factor β-induced (TGFBI) gene-linked corneal dystrophy and is pathologically characterized by the corneal deposition of mutant-TGFBIp. The defective autophagic degradation of pathogenic mutant-TGFBIp has been shown in GCD2; however, its exact mechanisms are unknown. To address this, we investigated lysosomal functions using corneal fibroblasts. Levels of cathepsins K and L (CTSK and CTSL) were significantly decreased in GCD2 cells, but of cathepsins B and D (CTSB and CTSD) did not change. The maturation of the pro-enzymes to their active forms (CTSB, CTSK and CTSL) was inhibited in GCD2 cells. CTSL enzymes directly degraded both LC3 (autophagosomes marker) and mutant-TGFBIp. Exogenous CTSL expression dramatically reduced mutant-TGFBIp in GCD2 cells, but not TGFBIp in WT cells. An increased lysosomal pH and clustered lysosomal perinuclear position were found in GCD2 cells. Transcription factor EB (TFEB) levels were significantly reduced in GCD2 cells, compared to WT. Notably, exogenous TFEB expression improved mutant-TGFBIp clearance and lysosomal abnormalities in GCD2 cells. Taken together, lysosomal dysfunction in the corneal fibroblasts underlies the pathogenesis of GCD2, and TFEB has a therapeutic potential in the treatment of GCD2.
    Keywords:  LC3 degradation; TGFBIp; autophagy; cathepsin; corneal fibroblasts; granular corneal dystrophy type 2; lysosomal pH
  16. Mol Ther Methods Clin Dev. 2020 Sep 11. 18 199-214
    Meena NK, Ralston E, Raben N, Puertollano R.
      Pompe disease, a deficiency of glycogen-degrading lysosomal acid alpha-glucosidase (GAA), is a disabling multisystemic illness that invariably affects skeletal muscle in all patients. The patients still carry a heavy burden of the disease, despite the currently available enzyme replacement therapy. We have previously shown that progressive entrapment of glycogen in the lysosome in muscle sets in motion a whole series of "extra-lysosomal" events including defective autophagy and disruption of a variety of signaling pathways. Here, we report that metabolic abnormalities and energy deficit also contribute to the complexity of the pathogenic cascade. A decrease in the metabolites of the glycolytic pathway and a shift to lipids as the energy source are observed in the diseased muscle. We now demonstrate in a pre-clinical study that a recently developed replacement enzyme (recombinant human GAA; AT-GAA; Amicus Therapeutics) with much improved lysosome-targeting properties reversed or significantly improved all aspects of the disease pathogenesis, an outcome not observed with the current standard of care. The therapy was initiated in GAA-deficient mice with fully developed muscle pathology but without obvious clinical symptoms; this point deserves consideration.
    Keywords:  Pompe disease; acid alpha glucosidase; autophagy; enzyme replacement therapy; glycogen; lysosomal targeting; mTORC1/AMPK signaling; metabolome; muscle
  17. Cureus. 2020 Jun 08. 12(6): e8517
    Khosa S, Mishra SK.
      Charcot-Marie-Tooth neuropathy type 1 (CMT1) is an inherited demyelinating neuropathy characterized by distal muscle weakness and atrophy. Charcot-Marie-Tooth disease type 1C (CMT1C) is a rare form of CMT1 caused by mutations in the lipopolysaccharide-induced tumor necrosis factor (LITAF) or small integral membrane protein of the lysosome/late endosome (SIMPLE) gene. Phenotypically, CMT1C is characterized by sensory loss and slow conduction velocity, and is typically slowly progressive and often associated with pes cavus foot deformity and bilateral foot drop. A 42-year-old female presented with a 10-year history of slowly progressive bilateral calf pain and cramps. After multiple electromyography/nerve conduction studies (EMG/NCS) and genetic testing, the patient was revealed to have CMT1C with a heterozygous pathogenic variant, c.334G>A (p.Gly112Ser). However, the presentation of the patient's CMT1C phenotype was unusual compared to patients with similar diagnosis in a previous study, including a normal sensory exam with the exception of high arches and mildly reduced vibratory sense. Additionally, the patient's teenage son already started showing symptoms of CMT1C despite the fact that the onset of the disease typically occurs at an older age. This particular case further highlights the idea that the phenotype related to CMT1C may have a wide spectrum of disease severity.
    Keywords:  charcot-marie-tooth; demyelinating diseases; genetic mutation; neurology; pes cavus; sensorimotor neuropathy
  18. Int J Mol Sci. 2020 Jul 14. pii: E4966. [Epub ahead of print]21(14):
    Blumenreich S, Barav OB, Jenkins BJ, Futerman AH.
      The lysosome is a central player in the cell, acting as a clearing house for macromolecular degradation, but also plays a critical role in a variety of additional metabolic and regulatory processes. The lysosome has recently attracted the attention of neurobiologists and neurologists since a number of neurological diseases involve a lysosomal component. Among these is Parkinson's disease (PD). While heterozygous and homozygous mutations in GBA1 are the highest genetic risk factor for PD, studies performed over the past decade have suggested that lysosomal loss of function is likely involved in PD pathology, since a significant percent of PD patients have a mutation in one or more genes that cause a lysosomal storage disease (LSD). Although the mechanistic connection between the lysosome and PD remains somewhat enigmatic, significant evidence is accumulating that lysosomal dysfunction plays a central role in PD pathophysiology. Thus, lysosomal dysfunction, resulting from mutations in lysosomal genes, may enhance the accumulation of α-synuclein in the brain, which may result in the earlier development of PD.
    Keywords:  Gaucher disease; Parkinson’s disease; lysosomal storage diseases; α-synuclein
  19. Cureus. 2020 Jun 07. 12(6): e8487
    Gayed A, Schott VA, Meltzer L.
      Mucopolysaccharidoses (MPS) are rare genetic lysosomal storage disorders caused by a deficiency of enzymes that catalyze the breakdown of glycosaminoglycans. MPS-III, also known as Sanfilippo syndrome, is caused by a deficiency of one of four enzymes that catalyze heparan sulfate proteoglycan degradation. MPS-IIIA results from a deficiency of heparan sulfatase. The natural history of MPS-IIIA is marked by progressive neurodegeneration. A nine-year-old boy with developmental delay presented with progressive three-month functional decline culminating in emergency department presentation for lethargy and immobility. Laboratory workup revealed hepatic and renal failure, coagulopathy, pancytopenia, hypernatremia, and uremia requiring emergent dialysis. He developed hyperkalemia during the second month of hospitalization, the workup of which led to a diagnosis of hyperreninemic hypoaldosteronism with normal cortisol. Blood chemistry consistent with renal hypoperfusion prompted exploration of adrenal ischemia, specifically affecting the zona glomerulosa and sparing the zona fasciculata, to explain low aldosterone with normal cortisol. Heparan sulfate (HS) normally acts as a storage site for basic fibroblast growth factor (bFGF), a paracrine stimulator of aldosterone, but accumulates in MPS-IIIA due to deficiency of heparan sulfatase. If bFGF is sequestered in HS deposits in MPS-III, then paracrine signaling is reduced, accounting for the state of hypoaldosteronism. To our knowledge, this is the first reported case of hyperreninemic hypoaldosteronism caused by an MPS disorder.
    Keywords:  adrenal cortex; heparan sulfate sulfatase; hyperkalemia; hypoaldosteronism; mucopolysaccharidosis iii; zona glomerulosa
  20. Liver Int. 2020 Jul 13.
    Malinová V, Balwani M, Sharma R, Arnoux JB, Kane J, Whitley CB, Marulkar S, Abel F.
      BACKGROUND AND AIMS: Lysosomal acid lipase deficiency is characterized by hepatomegaly and dyslipidemia, which can lead to liver cirrhosis and premature atherosclerosis. Sebelipase alfa is an approved recombinant human lysosomal acid lipase. In an open-label extension study of adults with lysosomal acid lipase deficiency (LAL-CL04), sebelipase alfa treatment for 1 year reduced serum transaminase levels and liver fat content and improved serum lipid levels.METHODS: Final data from LAL-CL04 are reported herein for patients who received sebelipase alfa infusions (1.0 or 3.0 mg/kg every other week) for up to 5 years.
    RESULTS: Of 8 patients enrolled, 7 received sebelipase alfa for 224-260 weeks; 1 was lost to follow-up. Median baseline levels of alanine aminotransferase and aspartate aminotransferase (81.5 and 50.0 U/L, respectively) were decreased through the end-of-study visit (54.0 and 34.0 U/L). Median low-density lipoprotein cholesterol decreased from 113 to 78 mg/dL, total cholesterol decreased from 171 to 132 mg/dL, and high-density lipoprotein cholesterol increased from 37 to 42 mg/dL. Most treatment-emergent adverse events were nonserious (99%), mild/moderate (98%), and unrelated to sebelipase alfa (87%); no patient discontinued due to treatment-emergent adverse events. One patient had 2 serious treatment-emergent adverse events (cholecystitis and cholelithiasis; assessed as unlikely related to sebelipase alfa). Two patients had 20 nonserious infusion-associated reactions in weeks 6-38; all were manageable. One patient tested positive for anti-drug antibodies (single occurrence).
    CONCLUSIONS: Sebelipase alfa was well tolerated and improved serum transaminase and lipid levels for up to 5 years in adults with lysosomal acid lipase deficiency.
    Keywords:  enzyme replacement therapy; lipids; liver; lysosomal storage diseases; transaminases
  21. ACS Med Chem Lett. 2020 Jul 09. 11(7): 1377-1385
    Olarte-Avellaneda S, Cepeda Del Castillo J, Rojas-Rodriguez AF, Sánchez O, Rodríguez-López A, Suárez García DA, Pulido LMS, Alméciga-Díaz CJ.
      Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding for the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal accumulation of keratan sulfate (KS) and chondroitin-6-sulfate. In this study, we identified and characterized bromocriptine (BC) as a novel PC for MPS IVA. BC was identified through virtual screening and predicted to be docked within the active cavity of GALNS in a similar conformation to that observed for KS. BC interacted with similar residues to those predicted for natural GALNS substrates. In vitro inhibitory assay showed that BC at 50 μM reduced GALNS activity up to 30%. However, the activity of hrGALNS produced in HEK293 cells was increased up to 1.48-fold. BC increased GALNS activity and reduced lysosomal mass in MPS IVA fibroblasts in a mutation-dependent manner. Overall, these results show the potential of BC as a novel PC for MPS IVA and contribute to the consolidation of PCs as a potential therapy for this disease.
  22. Int J Mol Sci. 2020 Jul 13. pii: E4953. [Epub ahead of print]21(14):
    Blaess M, Kaiser L, Sauer M, Csuk R, Deigner HP.
      In line with SARS and MERS, the SARS-CoV-2/COVID-19 pandemic is one of the largest challenges in medicine and health care worldwide. SARS-CoV-2 infection/COVID-19 provides numerous therapeutic targets, each of them promising, but not leading to the success of therapy to date. Neither an antiviral nor an immunomodulatory therapy in patients with SARS-CoV-2 infection/COVID-19 or pre-exposure prophylaxis against SARS-CoV-2 has proved to be effective. In this review, we try to close the gap and point out the likely relationships among lysosomotropism, increasing lysosomal pH, SARS-CoV-2 infection, and disease process, and we deduce an approach for the treatment and prophylaxis of COVID-19, and cytokine release syndrome (CRS)/cytokine storm triggered by bacteria or viruses. Lysosomotropic compounds affect prominent inflammatory messengers (e.g., IL-1B, CCL4, CCL20, and IL-6), cathepsin-L-dependent viral entry of host cells, and products of lysosomal enzymes that promote endothelial stress response in systemic inflammation. As supported by recent clinical data, patients who have already taken lysosomotropic drugs for other pre-existing conditions likely benefit from this treatment in the COVID-19 pandemic. The early administration of a combination of antivirals such as remdesivir and lysosomotropic drugs, such as the antibiotics teicoplanin or dalbavancin, seems to be able to prevent SARS-CoV-2 infection and transition to COVID-19.
    Keywords:  COVID-19; SARS-CoV-2; approved active compounds; cathepsin L; cytokine release syndrome; cytokine storm; lysosome; lysosomotropic compounds; lysosomotropism; viral host cell entry
  23. Cell Rep. 2020 Jul 14. pii: S2211-1247(20)30886-X. [Epub ahead of print]32(2): 107905
    Kozik P, Gros M, Itzhak DN, Joannas L, Heurtebise-Chrétien S, Krawczyk PA, Rodríguez-Silvestre P, Alloatti A, Magalhaes JG, Del Nery E, Borner GHH, Amigorena S.
      Cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumor immune responses. Yet, key steps involved in trafficking of antigens taken up by DCs remain incompletely understood. Here, we screen 700 US Food and Drug Administration (FDA)-approved drugs and identify 37 enhancers of antigen import from endolysosomes into the cytosol. To reveal their mechanism of action, we generate proteomic organellar maps of control and drug-treated DCs (focusing on two compounds, prazosin and tamoxifen). By combining organellar mapping, quantitative proteomics, and microscopy, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release. Enhancing antigen import facilitates cross-presentation of soluble and cell-associated antigens. Systemic administration of prazosin leads to reduced growth of MC38 tumors and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumor immune responses and efficacy of checkpoint blockers.
    Keywords:  cross-presentation; dendritic cells; dynamic organellar maps; immunotherapy; lysosomes; organellar proteomics; small molecule screening
  24. Am J Respir Cell Mol Biol. 2020 Jul 15.
    O'Leary EM, Tian Y, Nigdelioglu R, Witt LJ, Cetin-Atalay R, Meliton AY, Woods PS, Kimmig LM, Sun KA, Gökalp GA, Mutlu GM, Hamanaka RB.
      Idiopathic pulmonary fibrosis is a fatal disease characterized by the TGF-β-dependent differentiation of lung fibroblasts into myofibroblasts, leading to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by myofibroblasts requires de novo synthesis of glycine, the most abundant amino acid found in collagen protein. TGF-β upregulates the expression of the enzymes of the de novo serine/glycine synthesis pathway in lung fibroblasts; however, the transcriptional and signaling regulators of this pathway remain incompletely understood. Here we demonstrate that TGF-β promotes accumulation of Activating Transcription Factor 4 (ATF4) which is required for increased expression of the serine/glycine synthesis pathway enzymes in response to TGF-β. We found that induction of the Integrated Stress Response (ISR) contributes to TGF-β-induced ATF4 activity; however, the primary driver of ATF4 downstream of TGF-β is activation of the Mechanistic Target of Rapamycin Complex 1 (mTORC1). TGF-β activates the PI3-kinase-Akt-mTOR pathway, and inhibition of PI3-kinase prevents activation of downstream signaling and induction of ATF4. Using a panel of mTOR inhibitors, we found that ATF4 activation is dependent on mTORC1, independent of mTORC2. Rapamycin, which incompletely and allosterically inhibits mTORC1 had no effect on TGF-β-mediated induction of ATF4; however, Rapalink-1, which specifically targets the kinase domain of mTORC1 completely inhibited ATF4 induction and metabolic reprogramming downstream of TGF-β. Our results provide insight into the mechanisms of metabolic reprogramming in myofibroblasts and clarify contradictory published findings on the role of mTOR inhibition in myofibroblast differentiation.
    Keywords:  Fibrosis; Glycolysis; Metabolism; Mitochondria
  25. J Biochem. 2020 Jul 11. pii: mvaa076. [Epub ahead of print]
    Nada S, Okada M.
      Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mTORC1. However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signaling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signaling.
    Keywords:  Rag; Ragulator; Rheb; lysosome; mTORC1; p18
  26. Front Mol Biosci. 2020 ;7 117
    An E, Friend K.
      During translation elongation, the ribosome serially adds amino acids to a growing polypeptide over many rounds of catalysis. The ribosome remains bound to mRNAs over these multiple catalytic cycles, requiring high processivity. Despite its importance to translation, relatively little is known about how mRNA sequences or signaling pathways might enhance or reduce ribosome processivity. Here, we describe a metric for ribosome processivity, the ribosome density index (RDI), which is readily calculated from ribosomal profiling data. We show that ribosome processivity is not strongly influenced by open-reading frame (ORF) length or codon optimality. However, we do observe that ribosome processivity exists in two phases and that the early phase of ribosome processivity is enhanced by mTORC1, a key translational regulator. By showing that ribosome processivity is regulated, our findings suggest an additional layer of control that the cell can exert to govern gene expression.
    Keywords:  mTOR; ribosome density index; ribosome processivity; ribosome profiling; translation elongation
  27. Oncotarget. 2020 Jul 07. 11(27): 2597-2610
    VanCleave A, Palmer M, Fang F, Torres H, Rodezno T, Li Q, Fuglsby K, Evans C, Afeworki Y, Ross A, Rao P, Leiferman P, Zheng S, Houghton P, Tao J.
      Outcomes have not improved for metastatic osteosarcoma for several decades. In part, this failure to develop better therapies stems from a lack of understanding of osteosarcoma biology, given the rarity of the disease and the high genetic heterogeneity at the time of diagnosis. We report here the successful establishment of a new human osteosarcoma cell line, COS-33, from a patient-derived xenograft and demonstrate retention of the biological features of the original tumor. We found high mTOR signaling activity in the cultured cells, which were sensitive to a small molecule inhibitor, rapamycin, a suppressor of the mTOR pathway. Suppressed mTOR signaling after treatment with rapamycin was confirmed by decreased phosphorylation of the S6 ribosomal protein. Increasing concentrations of rapamycin progressively inhibited cell proliferation in vitro. We observed significant inhibitory effects of the drug on cell migration, invasion, and colony formation in the cultured cells. Furthermore, we found that only a strong osteogenic inducer, bone morphogenetic protein-2, promoted the cells to differentiate into mature mineralizing osteoblasts, indicating that the COS-33 cell line may have impaired osteoblast differentiation. Grafted COS-33 cells exhibited features typical of osteosarcoma, such as production of osteoid and tumorigenicity in vivo. In addition, we revealed that the COS-33 cell line retained a complex karyotype, a homozygous deletion of the TP53 gene, and typical histological features from its original tumor. Our novel cellular model may provide a valuable platform for studying the etiology and molecular pathogenesis of osteosarcoma as well as for testing novel drugs for future genome-informed targeted therapy.
    Keywords:  COS-33; TP53; mTOR; osteogenic differentiation; osteosarcoma
  28. Front Oncol. 2020 ;10 1012
    La Manna F, De Menna M, Patel N, Karkampouna S, De Filippo M, Klima I, Kloen P, Beimers L, Thalmann GN, Pelger RCM, Jacinto E, Kruithof-de Julio M.
      Bone metastasis is the leading cause of prostate cancer (PCa) mortality, frequently marking the progression to castration-resistant PCa. Dysregulation of the androgen receptor pathway is a common feature of castration-resistant PCa, frequently appearing in association with mTOR pathway deregulations. Advanced PCa is also characterized by increased tumor heterogeneity and cancer stem cell (CSC) frequency. CSC-targeted therapy is currently being explored in advanced PCa, with the aim of reducing cancer clonal divergence and preventing disease progression. In this study, we compared the molecular pathways enriched in a set of bone metastasis from breast and prostate cancer from snap-frozen tissue. To further model PCa drug resistance mechanisms, we used two patient-derived xenografts (PDX) models of bone-metastatic PCa, BM18, and LAPC9. We developed in vitro organoids assay and ex vivo tumor slice drug assays to investigate the effects of mTOR- and CSC-targeting compounds. We found that both PDXs could be effectively targeted by treatment with the bivalent mTORC1/2 inhibitor Rapalink-1. Exposure of LAPC9 to Rapalink-1 but not to the CSC-targeting drug disulfiram blocked mTORC1/2 signaling, diminished expression of metabolic enzymes involved in glutamine and lipid metabolism and reduced the fraction of CD44+ and ALDEFluorhigh cells, in vitro. Mice treated with Rapalink-1 showed a significantly delayed tumor growth compared to control and cells recovered from the tumors of treated animals showed a marked decrease of CD44 expression. Taken together these results highlight the increased dependence of advanced PCa on the mTOR pathway, supporting the development of a targeted approach for advanced, bone metastatic PCa.
    Keywords:  ALDH; PDX; bone metastasis; disulfiram; mTOR; prostate cancer
  29. FEBS Lett. 2020 Jul 17.
    Upadhyaya S, Agrawal S, Gorakshakar A, Rao BJ.
      Target Of Rapamycin (TOR) kinase is a sensor as well as a central integrator of internal and external metabolic cues. However, in algae and in higher plants, the components of TOR kinase-signaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in Chlamydomonas reinhardtii using the phosphorylation status of its putative downstream target, CrS6K. Using this assay, we probe the modulation of cellular TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy and nitrogen (N)-starvation. Importantly, we uncover that excess acetate in the medium leads to high cellular ROS levels, triggering autophagy and a concomitant drop in TOR kinase activity in a dose-dependent manner, thus leading to a N-starvation-like cellular phenotype, even when nitrogen is present.
    Keywords:  Acetate; Autophagy; Chlamydomonas S6 Kinase; Hexokinase1; Lipid accumulation; N starvation; TOR kinase activity
  30. Nature. 2020 Jul 15.
    Caffa I, Spagnolo V, Vernieri C, Valdemarin F, Becherini P, Wei M, Brandhorst S, Zucal C, Driehuis E, Ferrando L, Piacente F, Tagliafico A, Cilli M, Mastracci L, Vellone VG, Piazza S, Cremonini AL, Gradaschi R, Mantero C, Passalacqua M, Ballestrero A, Zoppoli G, Cea M, Arrighi A, Odetti P, Monacelli F, Salvadori G, Cortellino S, Clevers H, De Braud F, Sukkar SG, Provenzani A, Longo VD, Nencioni A.
      Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.
  31. PLoS Biol. 2020 Jul 17. 18(7): e3000778
    Xiao GY, Schmid SL.
      The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression.
  32. Protein Cell. 2020 Jul 14.
    Xiao J, Xiong Y, Yang LT, Wang JQ, Zhou ZM, Dong LW, Shi XJ, Zhao X, Luo J, Song BL.
      Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B'/B and C'/C. POST1 promotes the generation of the functional S1P-C'/C from S1P-B'/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.
    Keywords:  SREBP; activating transcription factor 6; mannose-6-phosphate; proteolytic activation; site-1 protease; unfolded protein response
  33. Front Cell Dev Biol. 2020 ;8 496
    Hermann J, Bender M, Schumacher D, Woo MS, Shaposhnykov A, Rosenkranz SC, Kuryshev V, Meier C, Guse AH, Friese MA, Freichel M, Tsvilovskyy V.
      Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.
    Keywords:  Ca2+ homeostasis; NAADP; glutamate; hippocampal neurons; neuronal excitotoxicity
  34. ACS Omega. 2020 Jul 07. 5(26): 15911-15921
    Li ZY, Shi YL, Liang GX, Yang J, Zhuang SK, Lin JB, Ghodbane A, Tam MS, Liang ZJ, Zha ZG, Zhang HT.
      Glucose metabolism is an essential process for energy production and cell survival for both normal and abnormal cellular metabolism. Several glucose transporter/solute carrier 2A (GLUT/SLC2A) superfamily members, including glucose transporter 1 (GLUT1), have been shown to mediate the cellular uptake of glucose in diverse cell types. GLUT1-mediated glucose uptake is a transient and rapid process; thus, the real-time monitoring of GLUT1 trafficking is pivotal for a better understanding of GLUT1 expression and GLUT1-dependent glucose uptake. In the present study, we established a rapid and effective method to visualize the trafficking of GLUT1 between the plasma membrane (PM) and endolysosomal system in live cells using an mCherry-EGFP-GLUT1 tandem fluorescence tracing system. We found that GLUT1 localized at the PM exhibited both red (mCherry) and green (EGFP) fluorescence (yellow when overlapping). However, a significant increase in red punctate fluorescence (mCherry is resistant to acidic pH), but not green fluorescence (EGFP is quenched by acidic pH), was observed upon glucose deprivation, indicating that the mCherry-EGFP-GLUT1 functional protein was trafficked to the acidic endolysosomal system. Besides, we were able to calculate the relative ratio of mCherry to EGFP by quantification of the translocation coefficient, which can be used as a readout for GLUT1 internalization and subsequent lysosomal degradation. Two mutants, mCherry-EGFP-GLUT1-S226D and mCherry-EGFP-GLUT1-ΔC4, were also constructed, which indirectly confirmed the specificity of mCherry-EGFP-GLUT1 for monitoring GLUT1 trafficking. By using a series of endosomal (Rab5, Rab7, and Rab11) and lysosomal markers, we were able to define a model of GLUT1 trafficking in live cells in which upon glucose deprivation, GLUT1 dissociates from the PM and experiences a pH gradient from 6.8-6.1 in the early endosomes to 6.0-4.8 in the late endosomes and finally pH 4.5 in lysosomes, which is appropriate for degradation. In addition, our proof-of-concept study indicated that the pmCherry-EGFP-GLUT1 tracing system can accurately reflect endogenous changes in GLUT1 in response to treatment with the small molecule, andrographolide. Since targeting GLUT1 expression and GLUT1-dependent glucose metabolism is a promising therapeutic strategy for diverse types of cancers and certain other glucose addiction diseases, our study herein indicates that pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening.
  35. Aging Cell. 2020 Jul 13. e13171
    Coryell PR, Goraya SK, Griffin KA, Redick MA, Sisk SR, Purvis JE.
      The tumor suppressor protein p16INK4a (p16) is a well-established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the protein's intracellular localization and degradation. The autophagy-lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stress-response proteins and has also been shown to attenuate age-related pathologies, but it is unclear whether p16 is involved in this pathway. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time-lapse fluorescence microscopy using an endogenous p16-mCherry reporter revealed that serum starvation, etoposide, and hydrogen peroxide stimulate autophagy and drive p16 recruitment to acidic cytoplasmic vesicles within 4 hr. Blocking lysosomal proteases with leupeptin and ammonium chloride resulted in the accumulation of p16 within lysosomes and increased total p16 levels suggesting that p16 is degraded by this pathway. Furthermore, autophagy blockers chloroquine and bafilomycin A1 caused p16 aggregation within stalled vesicles containing autophagosome marker LC3. Increase of p16 within these vesicles coincided with the accumulation of LC3-II. Knockdown of autophagosome chaperone p62 attenuated the formation of p16 aggregates in lysosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and age-related diseases.
    Keywords:  Ink4a; autophagy; lysosomes; p16
  36. Adv Exp Med Biol. 2020 ;1207 21-51
    Lu J, Wu M, Yue Z.
      Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by motor system dysfunction. The etiology of PD has been linked with aging, environmental toxins and genetic mutation, while molecular pathogenesis of PD includes various factors, such as impaired protein homeostasis, oxidative stress, mitochondria dysfunction, synaptic transmission impairment, calcium homeostasis imbalance, prion-like α-synuclein transmission and neuron inflammation. Autophagy is a conserved bulk degradation process to maintain cellular homeostasis. Impairment of autophagy has been reported to be involved in the pathogenesis of PD. Coding proteins of several PD-related genes, such as SNCA, LRRK2, GBA, ATP13A2, VPS35 and FBXO7, are implicated in or affected by autophagy process. Furthermore, various pathogenic events during PD directly or indirectly interfere with the autophagy pathway, and dysregulation of autophagy has been observed in different neurotoxic PD models. Autophagy has been regarded as a potential therapeutic target for PD treatment. Indeed, modulations of autophagy-regulated genes (BECN1 and TFEB) expression exerted neuroprotection against PD models, and various autophagy regulators, such as rapamycin, trehalose, lysosome modulators and other small molecule autophagy inducers, have displayed neuroprotective effects in experimental PD models. Taken together, autophagy dysfunction has been implicated in the pathogenesis of PD, and pharmacological modulation of autophagy may be a new therapeutic strategy for the PD treatment.
    Keywords:  Autophagy; Parkinson disease; α-synuclein
  37. Aging (Albany NY). 2020 Jul 18. 12
    Zhu Y, Wang R, Chen W, Chen Q, Zhou J.
      BACKGROUND: Autophagy, a highly conserved cellular catabolic process by which the eukaryotic cells deliver autophagosomes engulfing cellular proteins and organelles to lysosomes for degradation, is critical for maintaining cellular homeostasis in response to various signals and nutrient stresses. The dysregulation of autophagy has been noted in the pathogenesis of cancers. Our study aims to investigate the prognosis-predicting value of autophagy-related genes (ARG) in hepatocellular carcinoma (HCC).RESULTS: The signature was constructed based on eight ARGs, which stratified HCC patients into high- and low-risk groups in terms of overall survival (OS) (Hazard Ratio, HR=4.641, 95% Confidential Interval, CI, 3.365-5.917, P=0.000). The ARG signature is an independent prognostic indicator for HCC patients (HR = 1.286, 95% CI, 1.194-1.385; P < 0.001). The area under the receiver operating characteristic (ROC) curve (AUC) for 5-year survival is 0.765.
    CONCLUSION: This study provides a potential prognostic signature for predicting the prognosis of HCC patients and molecular insights into the significance of autophagy in HCC.
    METHODS: Sixty-two differentially expressed ARGs and the clinical characteristics and basic information of the 369 enrolled HCC patients were retrieved from The Cancer Genome Atlas (TCGA) database. the Cox proportional hazard regression analysis was adopted to identify survival-related ARGs, based on which a prognosis predicting signature was constructed.
    Keywords:  The Cancer Genome Atlas; autophagy-related genes; hepatocellular carcinoma; prognosis
  38. FASEB J. 2020 Jul 14.
    Yang J, Liu H, Han S, Fu Z, Wang J, Chen Y, Wang L.
      Autophagy is an important mechanism for cellular homeostasis and survival during pathologic stress conditions in the kidney, such as ischemia-reperfusion (IR) injury. In this study, renal IR was induced in female C57BL/6 mice after melatonin administration. Renal function, histological damage, inflammatory infiltration, cytokine production, oxidative stress, antioxidant capacity, autophagy changing, apoptosis levels, and autophagy-associated intracellular signaling pathway were assessed to evaluate the impact of antecedent melatonin treatment on IR-induced renal injury. The administration of melatonin resulted in significantly preserved renal function, and the protective effect was associated with ameliorated oxidative stress, limited pro-inflammatory cytokine production, and neutrophil and macrophage infiltration. Moreover, autophagic flux was increased after melatonin administration while the apoptosis levels were decreased in the melatonin-pretreated mice. Using TAK-242 and CRX-527, we confirmed that MyD88-dependent TLR4 and MEK/ERK/mTORC1 signaling participated in melatonin-induced autophagy in IR mice. Collectively, our results provide novel evidence that antecedent melatonin treatment provides protection for the kidney against IR injury by enhancing autophagy, as regulated by the TLR4/MyD88/MEK/ERK/mTORC1 signaling pathway. Therefore, melatonin preconditioning offers a potential therapeutic approach to prevent renal IR injury related to various clinical conditions.
    Keywords:  autophagy; ischemia-reperfusion injury; melatonin